InteractiveFly: GeneBrief

supernumerary limbs : Biological Overview | Regulation | Developmental Biology / Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

An important mechanism for regulating protein abundance in eukaryotes is the ubiquitin (Ub)-proteasome degradation pathway. The protein Supernumerary limbs (Slmb or more familiarly Slimb) is a subunit of a multi-protein complex that targets proteins for degradation by the ubiquitin-proteasome pathway. Slimb is an important regulator of several developmental pathways, in particular the Wingless, Hedgehog and Dorsal pathways. These developmental roles will be dealt with later, after the ubiquitin-proteosome pathway has been examined. To understand how Slimb functions, it helps to look at its yeast homolog, Cdc4c, because the involvement of this Slimb homolog in regulating the cell cycle in yeast is well understood. The pathway is rather complex, involving many proteins, five of which will be dealt with in some detail here. Briefly, the five protein targets of the pathway in yeast are: (1) cell cycle regulators such as Sic1p, a cyclin dependent kinase inhibitor (see Drosophila Dacapo; (2) the E2 ubiquitin conjugating enzyme Cdc34 that attaches ubiquitin to proteins, targeting them for destruction; (3) Cdc53, a scaffolding protein that binds Cdc34 and other components into a complex; (4) Skp1, an all purpose adaptor protein that holds together Cdc34 and various Slimb homologs, and (5) a dual domain protein, the Slimb homolog (Cdc4, for example), containing an F-box that attaches to Skp1, and a WD40 domain that attaches to the Cdc35 target (Sic1p, for example). A complex composed of Cdc53, Skp1, and an F-box protein (SCF complex) performs what is termed the E3 function, that is, activation of Cdc34 (Mathias, 1999 and references therein).

Ub is a member of a family of conserved polypeptides that are covalently attached to protein substrates. Multiple rounds of modification create a poly(Ub) chain on the substrate that targets the substrate for degradation by the proteasome. The transfer of free Ub onto a protein substrate is a multistep process. E1 activates free Ub at the expense of ATP. Ub is then transferred to an E2 (or ubiquitin protein-conjugating enzyme). Based on sequence comparison, yeast has 11 E2s: it is believed that each E2 is responsible for ubiquitinating distinct substrates. Although a free E2 enzyme may directly transfer Ub onto a substrate in a purified system, this reaction is promoted by additional proteins referred to as E3s or ubiquitin protein ligases. Some E3s act as intermediary Ub carriers in the transfer of Ub from E2 to substrate. Other E3s act as adapters, tethering E2 to E2's substrates. It turns out that a variety of structurally distinct E3 proteins each serve to regulate the interaction between E2 proteins and various distinct substrates (Mathias, 1999 and references therein).

In yeast, the ubiquitin-proteasome degradation pathway regulates two major cell cycle events: entry into S phase and entry into anaphase. Cell cycle progression is mediated by the activity of the cyclin-dependent kinase (CDK) Cdc28p (see Drosophila cdc2). The activation state and specificity of Cdc28p are determined by cyclins and CDK inhibitors. Association with cyclins Cln1 to Cln3 activates Cdc28p during G1, while the mitotic cyclins, Clb1 to Clb6 (see Drosophila CyclinA and CyclinB, are required for the S through M phases. Proteins regulating mitosis, including mitotic cyclins, are targeted for degradation by a cell cycle-regulated E3 complex, the anaphase-promoting complex. During G1, the CDK inhibitor Sic1p acts to inhibit CDK-Clb complex formation and prevent the initiation of S phase. Entry into S phase requires degradation of Sic1p by the ubiquitin-proteasome pathway. Multiple components of an E2-E3 complex are necessary to target Sic1p for degradation (Mathias, 1999 and references therein).

Cells lacking the gene encoding the E2 enzyme Cdc34p remain in G1, develop multiple elongated buds, and fail to separate duplicated spindle pole bodies. This phenotype is consistent with failure to activate the Clb-CDK complexes because of the inability to degrade Sic1p. Mutations in two other genes, CDC4 and CDC53, cause phenotypes indistinguishable from mutations in CDC34. Cdc53p is a member of the so-called 'cullin' family of proteins, characterized by the presence of WD-40 repeats and a second motif known as the F-box. The entry into S phase requires CDC34 to act in concert with CDC4 and CDC53. Indeed, CDC34, CDC4, and CDC53 gene products form a complex in vivo and that complex formation is necessary for S phase entry. A fourth gene, SKP1 encodes yet another member of this complex, and recombinant Cdc34p, together with Cdc4p, Cdc53p, and Skp1p (produced in insect cells) ubiquitinate Sic1p in vitro. Thus, the Cdc4p-Cdc53p-Skp1p complex is an E3 for Cdc34p (Mathias, 1999 and references therein).

In addition to Cdc4p, two other F-box-containing proteins in yeast, Met30p and Grr1p, have been proposed to interact with Skp1p and Cdc53p to form complexes referred as SCFs. Like Cdc4p, both Met30p (50) and Grr1p (9) contain repetitive domains, WD-40 repeats, and leucine-rich repeats, respectively (20), which potentially interact with distinct Cdc34p substrates. Thus, a family of potential E3 complexes has been identified that is distinguished by a component that has been proposed to recognize distinct substrates. It has been suggested that each SCF complex contains only one F-box protein. Thus, Cdc4p, Met30p, and Grr1p interact with a common set of components: Skp1p and Cdc53p (Mathias, 1999 and references therein).

Members of the Hedgehog (Hh) and Wnt/Wingless (Wg) families of secreted proteins control many aspects of growth and patterning during animal development. Hh signal transduction leads to increased stability of the transcription factor Cubitus interruptus (Ci), whereas Wg signal transduction causes increased stability of Armadillo (Arm/beta-catenin), a possible co-factor for the transcriptional regulator Lef1/TCF, known as Pangolin. slimb, negatively regulates both of these signal transduction pathways. Loss of slimb function results in a cell-autonomous accumulation of high levels of both Ci and Arm, and the ectopic expression of both Hh- and Wg- responsive genes. Clones of slimb1 cells in the leg or wing disc ectopically express dpp or wg when they arise in the anterior (but not the posterior) compartments of these discs. Anterior clones reorganize normal limb pattern, creating supernumerary 'double-anterior' limbs. Slimb, like PKA, is a negative regulator that normally prevents activity of the Hh signal transduction pathway in the absence of ligand. slimb mutant cells that arise in the presumptive wing blade ectopically express Scute and differentiate ectopic sensory bristles instead of epidermal hairs on the surface of the wing blade. Both phenotypes are strictly autonomous to the mutant cells, as is the case when the Wg signal transduction pathway is constitutively activated, but not when Wg is ectopically expressed. It is proposed that Slimb normally targets Ci and Arm for processing or degradation by the ubiquitin/proteasome pathway, and that Hh and Wg regulate gene expression, at least in part, by inducing changes in Ci and Arm, which protect both Ci and Arm from Slimb-mediated proteolysis (Jiang, 1998).

In Drosophila, Hh transduces its signal via Cubitus interruptus (Ci), a transcription factor present in two forms: a full-length activator and a carboxy-terminally truncated repressor that is derived from the full-length form by proteolytic processing. The proteolytic processing of Ci is promoted by the activities of Protein kinase A (PKA) and Slimb, whereas it is inhibited by Hh. PKA inhibits the activity of the full-length Ci in addition to its role in regulating Ci proteolysis. Whereas Ci processing is blocked in both PKA and slimb mutant cells, the accumulated full-length Ci becomes activated only in PKA but not in slimb mutant cells. Moreover, PKA inhibits an uncleavable activator form of Ci. These observations suggest that PKA regulates the activity of the full-length Ci independent of its proteolytic processing. Evidence exists that PKA regulates both the proteolytic processing and transcriptional activity of Ci by directly phosphorylating Ci. It is proposed that phosphorylation of Ci by PKA has two separable roles: (1) it blocks the transcription activity of the full-length activator form of Ci, and (2) it targets Ci for Slimb-mediated proteolytic processing to generate the truncated form that functions as a repressor (G. Wang, 1999).

A recent study by Methot (1999) demonstrates the existence of distinct activator and repressor forms of Ci. These two forms play separable roles in Drosophila limb development by regulating different sets of Hh target genes. In the developing wing, the expression of patched and engrailed appears to be exclusively controlled by the activator form of Ci whereas decapentaplegic expression is governed by both the activator and repressor forms. Interestingly, preventing Ci proteolysis with an uncleavable form of Ci is not sufficient to convert Ci into a constitutive activator, suggesting that the full-length activator form of Ci encounters additional regulatory block(s) that need to be alleviated by Hh signaling (Methot, 1999). Evidence is provided that PKA activity exerts such a block. Initial evidence that PKA regulates the activator form of Ci comes from a close examination of PKA and slimb mutant phenotypes. In slimb mutant cells, Ci processing is nearly abolished, and, as a consequence, full-length Ci accumulates. However, the expression of ptc-lacZ and en is not induced, suggesting that the full-length form of Ci that accumulates in slimb mutant cells is transcriptionally silent. In contrast, PKA mutant cells express ptc-lacZ and en even though they accumulate full-length Ci at levels comparable to slimb mutant cells. This suggests that the full-length Ci that accumulates in PKA mutant cells is transcriptionally active. Furthermore, slimb mutant cells with reduced PKA activity ectopically express ptc-lacZ, arguing that the lack of Ci activity in slimb mutant cells is due to an inhibitory role for PKA rather than a positive requirement for Slimb in the Hh signaling pathway. Further evidence that PKA regulates the activator form of Ci independent of its processing come from the gain-of-function studies. The ectopic expression of ptc-lacZ induced by the uncleavable activator form of Ci (Ci^U) is suppressed by overexpression of a constitutively active form of PKA (mC*) (G. Wang, 1999).

In support of the view that Ci is a direct target for PKA in regulating Hh signaling, it was found that a modified form of Ci with three PKA phosphorylation consensus sites mutated is not processed but exhibits constitutive activity when expressed in the developing wings. Although these observations suggest that PKA antagonizes Hh signaling by directly phosphorylating Ci and targeting it for proteolysis, they do not to address whether phosphorylation of Ci by PKA also regulates the activity of the full-length activator form of Ci. The low levels of constitutive activity exhibited by the PKA phosphorylation-deficient form of Ci could be secondary to the lack of Ci processing, which results in a dramatic increase in the levels of the full-length activator form of Ci, since it has been shown that overexpression of a full-length wild type form of Ci can activate ptc expression in wing discs. To define the role of PKA phosphorylation in regulating the activity of the full-length Ci, advantage was taken of the uncleavable activator form of Ci (Ci^U). Mutating multiple PKA phosphorylation sites in Ci^U dramatically alters its transcriptional activity and renders it constitutively active. This observation suggests that the activity of Ci^U is normally blocked by PKA phosphorylation, even though its processing is impaired. This result provides compelling evidence that PKA phosphorylation of Ci inhibits the activator form of Ci, independent of its role in promoting Ci processing. Taken together, these results suggest the following working model for the inhibitory function of PKA in the Hh pathway. It is proposed that PKA phosphorylation of Ci in its carboxy-terminal region has two separable roles: (1) it blocks the activity of the full-length activator form of Ci, and (2) it targets the full-length Ci for Slimb-mediated proteolysis to generate the truncated repressor form of Ci. Such a dual regulation ensures that only the repressor form of Ci is active in the absence of any Hh signaling. This model accounts for the difference between PKA and slimb mutant phenotypes. In slimb mutant cells, Ci is not processed to the repressor form but accumulates in an inactive phosphorylated form, and, as a consequence, dpp is derepressed at low levels but ptc and en are not activated. In PKA mutant cells, however, Ci accumulates in an unphosphorylated or hypophosphorylated active form, and, as a consequence, ptc and en are activated (G. Wang, 1999).

How phosphorylation of Ci regulates its activity and proteolytic processing remains to be explored. It has been proposed that Su(fu) attenuates Hh signaling activity by blocking a maturation step that converts Ci into a short-lived nuclear transcriptional activator. Analyses of slimb Su(fu) double mutant and slimb Su(fu) PKA triple mutant phenotypes suggest that inhibition of Ci activity by PKA is independent of Su(fu). When Ci processing is blocked, removing Su(fu) only partially stimulates the activity of the full-length Ci whereas simultaneously removing Su(fu) function and reducing PKA activity leads to virtually full activation of Ci. These observations suggest that PKA and Su(fu) act in parallel through independent mechanisms to regulate the activity of the full-length Ci. In slimb Su(fu) double mutant cells, the majority of unprocessed full-length Ci appears to be transformed into a labile nuclear form, and yet the activity of this nuclear form of Ci seems to be inhibited by PKA. This implies that PKA might inhibit Ci at a step after it enters the nucleus. For example, phosphorylation of Ci by PKA might prevent the formation of an active Ci transcription complex or might attenuate its ability to bind DNA. Another possible mechanism by which PKA exerts its influence on Ci is to regulate its nuclear trafficking. It has been shown recently that Hh signaling increases the nuclear import of full-length Ci. As PKA and Hh act antagonistically, it is possible that PKA phosphorylation of Ci might tether the full-length Ci in the cytoplasm in the absence of Hh signaling (G. Wang, 1999).

Su(fu), Cos2, and the Ser/Thr kinase Fused form a multiprotein complex with Ci and the complex associates with microtubules in a manner regulated by Hh. It has been proposed that the assembly of the microtubule-associated Ci complex is critical for inhibiting Ci activity, possibly by tethering Ci in the cytoplasm. The relationship between PKA phosphorylation and the formation of Ci complex is not known. It is not clear whether they are two independent processes or whether one step might regulate the other. The nearly identical phenotypes caused by loss of PKA or Cos2 function in limb development suggest that these two regulatory events might be intimately related. For example, Cos2 might target Ci for efficient phosphorylation by PKA, allowing PKA to exert its negative regulation on Ci. Alternatively, phosphorylation of Ci by PKA might regulate the complex formation, allowing Cos2 to exert its influence on Ci. Further genetic and biochemical studies are required to resolve this important issue (G. Wang, 1999).

It has been proposed that phosphorylation of Ci by PKA allows Slimb to bind Ci and target it for ubiquitin/proteasome-mediated proteolysis. The epistatic relationship between PKA and Slimb defined by this study is consistent with this hypothesis. Moreover, it has been shown recently that proteasome is involved in Ci proteolytic processing. However, no evidence has been obtained to indicate that Ci is ubiquitinated. It is possible that the polyubiquitin chains added to Ci might be unstable and thus might escape detection. Alternatively, the proteolytic processing of Ci might not be directly mediated by ubiquitination, and Slimb might regulate Ci processing indirectly. For example, the so-called SCF (Skp1, Cdc53, and F-box) ubiquitin ligase complex (SCF^Slimb) might promote the ubiquitination and degradation of an inhibitor of a protease that cleaves Ci (G. Wang, 1999).

Another important question that remains largely unanswered is how Hh antagonizes PKA. The structural similarity between Smo and G protein-coupled seven-transmembrane receptors suggests that Hh signaling might antagonize PKA by down-regulating its cAMP dependent kinase activity. However, the observations that a constitutively active cAMP independent form of PKA (mC*) can rescue PKA mutant phenotypes without perturbing normal Hh signaling both in embryos and in imaginal discs strongly argue against this possibility. The finding that high but not low levels of mC* are able to override Hh signaling is more consistent with a model in which Hh and PKA act competitively and antagonistically on Ci. For example, Hh may activate a phosphatase that removes the phosphates added to Ci by PKA. In support of this view, pharmacological evidence suggests that Hh stimulates target gene expression via a PP2A-like phosphatase in tissue culture cells. However, there is no genetic evidence for the involvement of a phosphatase in the Hh pathway (G. Wang, 1999).

In vertebrates, Hh signaling is mediated by three members of the Gli family of transcription factors: Gli1, Gli2, and Gli3. Like Ci, all three Gli proteins contain multiple PKA phosphorylation consensus sites at conserved positions, so they are likely to be direct targets for PKA regulation in the vertebrate Hh signaling pathway. Among the three Gli proteins, Gli3 is both structurally and functionally related to Ci. Gli3 has been implicated to have both activator and repressor function depending on the developmental contexts. Moreover, PKA appears to promote Gli3 processing to generate a putative repressor form. Thus, the mechanism by which PKA targets Ci for Slimb-mediated processing may well be conserved from invertebrates to vertebrates. Gli1 and Gli2 appear to function mainly as positive regulators in the vertebrate Hh signaling pathway. Unlike Gli3 and Ci, Gli1 and Gli2 do not undergo PKA-dependent processing, however, their activities are likely to be regulated by PKA. For example, it has been shown that overexpression of a constitutive active form of PKA represses the transcriptional activity of Gli1 in mammalian culture cells. Thus, whereas the mechanism by which PKA regulates Ci processing may only apply to Gli3, the processing-independent inhibitory mechanism defined by this study may well apply to all three Gli proteins and is likely to be a more general mechanism by which PKA negatively regulates Hh signaling (G. Wang, 1999 and references therein).

Fat-regulated adaptor protein Dlish binds the growth suppressor Expanded and controls its stability and ubiquitination

The Drosophila protocadherin Fat controls organ size through the Hippo pathway, but the biochemical links to the Hippo pathway components are still poorly defined. Previous work has identified Dlish, an SH3 domain protein that physically interacts with Fat and the type XX myosin Dachs, and has shown that Fat's regulation of Dlish levels and activity helps limit Dachs-mediated inhibition of Hippo pathway activity. This study characterizes a parallel growth control pathway downstream of Fat and Dlish. Using immunoprecipitation and mass spectrometry to search for Dlish partners, Dlish was found to binds the FERM domain growth repressor Expanded (Ex); Dlish SH3 domains directly bind sites in the Ex C terminus. It was further shown that, in vivo, Dlish reduces the subapical accumulation of Ex, and that loss of Dlish blocks the destabilization of Ex caused by loss of Fat. Moreover, Dlish can bind the F-box E3 ubiquitin ligase Slimb and promote Slimb-mediated ubiquitination of Expanded in vitro. Both the in vitro and in vivo effects of Dlish on Ex require Slimb, strongly suggesting that Dlish destabilizes Ex by helping recruit Slimb-containing E3 ubiquitin ligase complexes to Ex (Wang, 2019).

The intracellular domain (ICD) of the giant Drosophila protocadherin Fat reduces cell proliferation in imaginal disc tissues by regulating the Hippo pathway, an effect potentiated by heterophilic binding between Fat and the protocadherin Dachsous (Ds). The Fat ICD increases the activity of NDR family kinase Warts, the Drosophila homolog of vertebrate LATS and the final effector kinase in the Hippo pathway, and decreases the activity of the Warts target Yorkie (Yki), the Drosophila homolog of the vertebrate YAP and TAZ transcriptional coactivators. Active Warts phosphorylates and inhibits Yki by increasing the binding between Yki and its cytoplasmic tethers. In the absence of Fat, Warts activity is reduced, increasing the proportion of Yki that enters the nucleus with its TEAD-family cofactor Scalloped (Sd), driving transcription and overgrowth of imaginal disc epithelia (Wang, 2019).

While Fat is one of the best-studied transmembrane regulators of the Hippo pathway, the biochemical pathways that link Fat's ICD to changes in Warts and Yki activity have not been fully elucidated. The portions of Fat's ICD that suppress growth lack obvious catalytic or protein-binding motifs and, until recently, binding partners. However, recent work indicates that Fat's ICD binds to the cytoplasmic SH3-domain protein Dlish (also known as Vamana), reducing Dlish levels and activity, and thereby regulating a Dlish-binding partner, the atypical type XX myosin Dachs (Zhang, 2016; Misra, 2016) (see Model of inputs from the Fat, Ds, and Crumbs ICDs into the Hippo growth control pathway). Fat, Dlish, Dachs, and Warts are all concentrated at the subapical cell cortex of disc epithelial cells near their adherens junctions, although some subapical Warts is also concentrated at nonjunction sites, and for both Dlish and Dachs this depends on the formation of a Dlish-Dachs complex. When Fat is lost, the subapical levels of Dlish and Dachs greatly increase, an effect specific to the Fat branch of the Hippo pathway. The increased Dachs binds and inhibits Warts by altering its conformation and reducing its levels, thereby inducing Yki-mediated overgrowth (Wang, 2019).

Previous evidence suggested that the increased Dlish of fat mutants stimulates growth only by increasing subapical Dachs, rather than through any direct effect on Warts or Yki. Unlike Dachs, Dlish does not bind Warts. And while Dlish is necessary and sufficient for the localization and activity of wild-type Dachs, the overgrowth induced by a membrane-targeted Dachs construct is not reduced by the loss of Dlish; concentrating Dachs at the membrane is sufficient to bypass Dlish. Instead, Dlish provides a physical link between Dachs and the Fat ICD, along with the DHHC palmitoyltransferase Approximated (App), which can bind Fat and Dachs and palmitoylate Dlish. Loss of either Dlish or App disrupts Fat's ability to regulate Dachs levels and Yki activity. In the simplest view, the Fat ICD binds and inhibits Dlish and App, reducing their ability to regulate Dachs; when Fat is lost, App and palmitoylated Dlish are free to bind Dachs and concentrate it near the subapical cell membrane where it inhibits Warts. Dachs is also needed to concentrate the Dlish/Dachs complex in the cortex, likely by binding to the actin cytoskeleton or other scaffolds; thus, the reduction of Dachs by the Fat-tethered ubiquitin ligase Fbxl7 provides a parallel route for regulating Dlish and Dachs activity. The Ds ICD also binds and recruits Dachs and Dlish, but it is uncertain how strongly the endogenous Ds ICD affects the Hippo pathway (Wang, 2019).

While Dachs plays a major role in growth control through its ability to inhibit Warts, this study shows that this is not the sole mediator of Dlish activity. Rather, Dlish also regulates a parallel pathway mediated by the growth-inhibiting FERM domain protein Expanded (Ex). Ex regulates the Hippo pathway at multiple levels that are distinct from the Dachs-mediated alterations in Warts conformation. Ex binds Hippo, Warts, and the pathway modulators Merlin and Kibra and stimulates the phosphorylation and activity of Warts; Ex can also bypass Warts by binding and inhibiting Yki (Wang, 2019).

Indeed, Fat was originally linked to the Hippo pathway, not through Dachs, but through Ex: loss of Fat decreases Ex levels in the subapical cell cortex. The Ex decrease is particularly striking as it is at odds with the increased Yki-driven ex transcription caused by loss of Fat, indicating that the effect is posttranscriptional; the effect is also specific for the Fat branch of the Hippo pathway, as increasing Yki activity through other branches increases both ex transcription and Ex protein levels as part of a negative feedback loop (Wang, 2019).

This study shows that mimicking the effects of Fat loss by increasing Dlish levels has a growth-inducing activity that is independent of the Dachs myosin, and thus Dachs-mediated inhibition of Warts.Two of the three SH3 domains of Dlish bind directly to multiple sites in Ex, Dlish decreases Ex protein levels in wing imaginal discs independently of ex transcription, and that without Dlish the loss of Fat no longer reduces Ex protein levels. Previous studies showed that Ex levels are reduced by ubiquitination mediated by the Ex-binding F-box E3 ubiquitin ligase Slimb and the Skp-Cullin-F-box (SCF) complex, a process stimulated by the Ex-binding transmembrane protein Crumbs. This study will confirm and extend previous finding that Dlish binds to Slimb and shows that Dlish stimulates the Slimb-dependent ubiquitination of Ex in vitro (Wang, 2019).

REGULATION

Protein Interactions

Signal-induced phosphorylation of IkappaBalpha (Drosophila homolog: Cactus) targets this inhibitor of NF-kappaB for ubiquitination and subsequent degradation, thus allowing NF-kappaB to enter the nucleus to turn on its target genes. An IkappaB-ubiquitin (Ub) ligase complex has been identified that contains the F-box/WD40-repeat protein, beta-TrCP, a vertebrate homolog of Drosophila Slimb. beta-TrCP binds to IkappaBalpha only when the latter is specifically phosphorylated by an IkappaB kinase complex. Moreover, immunopurified beta-TrCP ubiquitinates phosphorylated IkappaBalpha at specific lysines in the presence of Ub-activating (E1) and -conjugating (Ubch5) enzymes. A beta-TrCP mutant lacking the F-box inhibits the signal-induced degradation of IkappaBalpha and subsequent activation of NF-kappaB-dependent transcription. Furthermore, Drosophila embryos deficient in slimb fail to activate twist and snail, two genes known to be regulated by the NF-kappaB homolog, Dorsal. These biochemical and genetic data strongly suggest that Slimb/beta-TrCP is the specificity determinant for the signal-induced ubiquitination of IkappaBalpha (Spencer, 1999).

The Hedgehog signal transduction pathway is involved in diverse patterning events in many organisms. In Drosophila, Hedgehog signaling regulates transcription of target genes by modifying the activity of the DNA-binding protein Cubitus interruptus (Ci). Hedgehog signaling inhibits proteolytic cleavage of full-length Ci (Ci-155) to Ci-75, a form that represses some target genes, and also converts the full-length form to a potent transcriptional activator. Reduction of Protein kinase A (PKA) activity also leads to accumulation of full-length Ci and to ectopic expression of Hedgehog target genes, prompting the hypothesis that PKA might normally promote cleavage to Ci-75 by directly phosphorylating Ci-155. A mutant form of Ci lacking five potential PKA phosphorylation sites (Ci5m) is not detectably cleaved to Ci-75 in Drosophila embryos. Moreover, changes in PKA activity dramatically alters levels of full-length wild-type Ci in embryos and imaginal discs, but does not significantly alter full-length Ci5m levels. These results are corroborated by showing that Ci5m is more active than wild-type Ci at inducing ectopic transcription of the Hh target gene wingless in embryos and that inhibition of PKA enhances induction of wingless by wild-type Ci but not by Ci5m. It is therefore proposed that PKA phosphorylation of Ci is required for the proteolysis of Ci-155 to Ci-75 in vivo. It is also showm that the activity of Ci5m remains Hedgehog responsive if expressed at low levels, providing further evidence that the full-length form of Ci undergoes a Hedgehog-dependent activation step (Price, 1999).

How does PKA phosphorylation of Ci-155 lead to its proteolysis? Loss of cos2 activity in wing disc clones induces high levels of Ci-155, suggesting that the integrity of the multiprotein cytoplasmic complex that contains Ci or the association of this complex with microtubules may be necessary in order for proteolysis to occur. It is possible that Ci phosphorylation also affects proteolysis by altering these interactions. A more direct role for PKA phosphorylation of Ci has been proposed based on the sequence and properties of the Slimb protein, which affects the conversion of Ci-155 to Ci-75. Slimb belongs to a family of F-box/ WD40-repeat proteins implicated in binding to and targeting phosphorylated molecules for ubiquitin-mediated degradation. It was recently shown that the vertebrate Slimb homolog, beta-TRCP, targets IkappaB and beta-catenin for ubiquitin-mediated degradation by binding specifically to a phosphorylated motif (DSGXXS, where both serines must be phosphorylated) present in both proteins. Whether Slimb participates in such a direct manner in Ci proteolysis is not clear. Slimb has not been shown to bind to Ci, and Ci proteolysis has not been shown to involve ubiquitination; Ci proteolysis is also unusual in being incomplete, leaving a stable 75 kDa product. Sequences around three Ci sites show some extended similarity to each other but are quite different from the IkappaB and beta-catenin consensus. It will be interesting to determine if Slimb, or another F-box protein, can bind directly to these regions of Ci when phosphorylated by PKA. Since Slimb recognition requires phosphorylation at multiple residues and the PKA site consensus in Ci contains additional serines, it is worth considering that the activity of another protein kinase in addition to PKA may also contribute to the regulation of Ci proteolysis (Price, 1999 and references).

In Drosophila, signaling by the protein Hedgehog (Hh) alters the activity of the transcription factor Cubitus interruptus (Ci) by inhibiting the proteolysis of full-length Ci (Ci-155) to its shortened Ci-75 form. Ci-75 is found largely in the nucleus and is thought to be a transcriptional repressor, whereas there is evidence to indicate that Ci-155 may be a transcriptional activator. However, Ci-155 is detected only in the cytoplasm, where it is associated with the protein kinase Fused (Fu), with Suppressor of Fused [Su(fu)], and with the microtubule-binding protein Costal-2. It is not clear how Ci-155 might become a nuclear activator. Mutations in Su(fu) cause an increase in the expression of Hh-target genes in a dose-dependent manner while simultaneously reducing Ci-155 concentration by some mechanism other than proteolysis to Ci-75. Conversely, eliminating Fu kinase activity reduces Hh-target gene expression while increasing Ci-155 concentration. It is proposed that Fu kinase activity is required for Hh to stimulate the maturation of Ci-155 into a short-lived nuclear transcriptional activator and that Su(fu) opposes this maturation step through a stoichiometric interaction with Ci-155 (Ohlmeyer, 1999).

Hh signaling thus elicits several changes that are required to convert Ci into an effective transcriptional activator. Hh spares Ci-155 from Protein kinase A- and Slimb-dependent protolysis to Ci-75, perhaps by modifying the phosphorylation status of Ci, and promotes dissociation of the Ci-155 complex from microtubules. It is proposed that in wing discs some of this 'primed' Ci-155 is not associated with Su(fu) and can activate dpp and ptc transcription but not anterior en expression. Most of the primed Ci-155 in wing discs and perhaps all of the primed Ci-155 in embryos is inactive while it is in complex with Su(fu) and signaling by Hh and Fused kinase are necessary for Ci-155 to become a transcriptional activator. This active form of Ci appears to be unstable and so is not detectable in the nuclei of cells responding to Hh. The lower levels of Ci-155 that are found in wing discs close to the source of Hh, as compared with levels in more distant regions of the Hh-signaling domain, may be explained by this model if more Hh is required to stimulate the Fu-kinase-dependent step in Ci activation than to protect Ci-155 from proteolytic degradation to Ci-75. This dosage dependence may account for the restricted range of engrailed induction relative to dpp and ptc in wing discs and the single-cell range of Hh signaling in embryonic ectoderm (Ohlmeyer, 1999).

Many proteins are targeted to proteasome degradation by a family of E3 ubiquitin ligases, termed SCF complexes, that link substrate proteins to an E2 ubiquitin-conjugating enzyme. SCFs are composed of three core proteins-Skp1, Cdc53/Cull, Rbx1/Hrt1-and a substrate specific F-box protein. The closest homologs to the human components of the SCF(betaTrCP) complex and the E2 ubiquitin-conjugating enzyme UbcH5 have been identified in Drosophila. Putative Drosophila SCF core subunits skpA and Rbx1 both interact directly with Cu11 and the F-box protein Slimb. The direct interaction of UbcH5 related protein UbcD1 with Cul1 and Slimb is also reported. In addition, a functional complementation test performed on a Saccharomyces cerevisiae Hrt1p-deficient mutant shows that Drosophila Rbx1 is able to restore the yeast cells viability. These results suggest that Rbx1, SkpA, Cullin1, and Slimb proteins are components of a Drosophila SCF complex that functions in combination with the ubiquitin conjugating enzyme UbcD1 (Bocca, 2001).

Transcription factor Ci mediates Hedgehog (Hh) signaling to determine the anterior/posterior (A/P) compartment of Drosophila wing disc. While Hh-inducible genes are expressed in A compartment cells abutting the A/P border, it is unclear how the boundaries of this region are established. Here, a Ci binding protein, Debra, has been identified that is expressed at relatively high levels in the band abutting the border of the Hh-responsive A compartment region. Debra mediates the polyubiquitination of full-length Ci, which then leads to its lysosomal degradation. Debra is localized in the multivesicular body, suggesting that the polyubiquitination of Ci directs its sorting into lysosome. Thus, Debra defines the border of the Hh-responsive region in the A compartment by inducing the lysosomal degradation of Ci (Dai, 2003).

Both the PKA phosphorylation sites and the processing site of the Ci protein are required for its Dbr-induced degradation. In addition, these sites are required for the proteasome-dependent processing of Ci-155, which also involves Slimb. Thus, Ci-155 levels are regulated by two separate degradative processes. That both processes share common regulatory elements suggests that it is likely that the events leading to the lysosomal degradation and proteasome processing of Ci-155 occur in parallel. Since Slimb contains an F box/WD40 repeat, and its vertebrate homolog is a component of the SCF ubiquitin ligase complex, Slimb is likely to act as an E3 ligase in transferring the ubiquitin moiety to Ci. In the absence of Dbr, Slimb induces the proteolytic processing of Ci-155 to Ci-75 via the proteasome, possibly by mediating limited Ci-155 ubiquitination that then serves as a proteolytic processing signal. When Dbr exists, Slimb cooperates to induce the full ubiquitination of Ci-155 that targets it for lysosomal degradation via MVBs. Dbr does not induce Ci-75 degradation. Slimb binds to both the N- and C-terminal regions of Ci-155. It may be that the binding of Slimb to Ci-75, which lacks the C-terminal region of Ci-155, is too weak to induce the ubiquitination of Ci-75, resulting in the maintenance of this form of Ci in the cell (Dai, 2003).

The F-box protein Slimb controls the levels of clock proteins Period and Timeless

The Drosophila circadian clock is driven by daily fluctuations of the proteins Period and Timeless, which associate in a complex and negatively regulate the transcription of their own genes. Protein phosphorylation has a central role in this feedback loop, by controlling Per stability in both cytoplasmic and nuclear compartments as well as Per/Tim nuclear transfer. However, the pathways regulating degradation of phosphorylated Per and Tim are unknown. The product of the slimb (slmb) gene -- a member of the F-box/WD40 protein family of the ubiquitin ligase SCF complex that targets phosphorylated proteins for degradation -- is shown to be an essential component of the Drosophila circadian clock. slmb mutants are behaviorally arrhythmic, and can be rescued by targeted expression of Slmb in the clock neurons. In constant darkness, highly phosphorylated forms of the Per and Tim proteins are constitutively present in the mutants, indicating that the control of their cyclic degradation is impaired. Because levels of Per and Tim oscillate in slmb mutants maintained in light:dark conditions, light- and clock-controlled degradation of Per and Tim do not rely on the same mechanisms (Grima, 2002).

To test whether the SCF-mediated ubiquitin proteasome pathway is involved in the control of Per and Tim oscillations, circadian rhythms were examined of flies defective for genes encoding F-box proteins that are known to target phosphorylated substrates for degradation. The slimb (slmb) gene, which encodes an F-box/WD40 protein regulating transcription factors' levels in the wingless and hedgehog signaling pathways was examined. slmb⁸ mutants that normally die as early larvae were brought to adulthood by providing the slmb gene product throughout development under the control of a heat-shock promoter. The rescued HS-slmb slmb⁸ adult flies, hereafter referred to as slmb^m mutants, were then tested for their locomotor activity rhythms in both light:dark (LD) and constant darkness (DD) conditions. slmb^m mutants were completely arrhythmic in DD, whereas the heterozygous genotype displayed wild-type rhythms. The absence of anatomical defects of the PDF-expressing ventral lateral neurons (LNvs), which control the behavioral rhythms, strongly argues against a developmental origin of the mutants' rhythm defect. Furthermore, targeted slmb expression using well characterized LNvs-specific gal4 drivers restores near wild-type activity rhythms, whereas similarly targeted overexpression in a wild-type background lengthens the circadian period, indicating a cell-autonomous role of the slmb gene in circadian rhythmicity. In LD conditions, slmb^m mutants did not display the light-off anticipation of activity that characterizes a functional clock, whereas it was observed in the flies expressing slmb under the LNvs-specific gal1118 driver. These experiments identify the F-box/WD40 protein Slmb as an essential component of the Drosophila brain clock (Grima, 2002).

To understand how Slmb might affect the circadian oscillator, slmb^m mutants were analyzed for Per and Tim oscillations in the head. In wild-type flies maintained in LD cycles, Per and Tim proteins accumulate and are progressively phosphorylated during night time, with Tim disappearing at the end of the night whereas hyper-phosphorylated Per persists for a few hours in the morning. A similar temporal pattern persists in DD, and is required to sustain behavioral rhythmicity. In contrast, highly phosphorylated Per and Tim are present at all circadian times in slmb^m mutants kept in DD, although low-amplitude oscillations of the hypo-phosphorylated forms indicate a weak residual activity of the molecular clock. In agreement with the persistence of weak protein cycling in slmb^m heads, levels of per and tim transcripts displayed low-amplitude oscillations. Per immunoreactivity was examined in the LNvs that control behavioral rhythms. At circadian time (CT) 0 and CT 12, which correspond to the peak and trough of Per labelling in w flies at 20°C, slmb^m mutants showed low levels of Per immunoreactivity, indicating that the oscillations of the proteins levels are also abolished in the clock cells. To determine whether Slmb acts at the protein level or through a transcriptional control, per was constitutively overexpressed through a transgene. High-molecular-mass Per proteins were observed to accumulate in head extracts of slmb^m but not of wild-type flies carrying GMR-gal4 and UAS-per transgenes that drive strong Per expression in the eye. Altogether, these data indicate that Slmb is involved in the control of phosphorylated Per levels (Grima, 2002).

In LD conditions, Per and Tim degradation in the morning is driven by both the circadian cycle and by light. Light-induced Tim degradation involves ubiquitinylation of the protein, and is blocked by proteasome inhibitors. To test whether Slmb is involved in the light-induced degradation pathway of the clock proteins, Per and Tim levels were assayed in slmb^m flies kept in LD conditions. In contrast to constant darkness, robust oscillations of Per and Tim amounts were observed in LD, with both proteins accumulating during the night and showing a strong day-time decrease. This shows that light-induced Per and Tim degradation does not occur through the same slmb-dependent mechanism as their circadian-cycle-controlled degradation in constant darkness. In addition, the absence of light-off anticipation in the slmb^m activity profiles suggests that the mutants' altered temporal regulation of phosphorylated Per and Tim does not allow rhythmic outputs to be driven, although protein levels clearly cycle (Grima, 2002).

Clock-dependent Per and Tim degradation occurs at the end of the circadian cycle, and relieves the transcriptional repression that the proteins exert on their own genes. Per degradation has also been proposed to take place during the rising phase of the protein levels in the early night, and to be responsible for the shift (of 5 h) between per messenger RNA and Per protein peaks. In order to determine whether Slmb levels vary during a circadian cycle and may therefore affect Per and Tim only during a limited time window, anti-Slmb antibodies were raised and the Slmb protein was followed in head extracts at different circadian times. A strongly reacting protein, as well as a faintly reacting one slightly above, were detected at a relative molecular mass of 45,000 (M_r 45K) in wild-type flies, and did not show any oscillations of their levels over a 24-h time course. Similarly, slmb mRNA did not show any cycling. Slmb therefore appears not to be circadianly regulated, and could therefore act on different steps of the cycle (Grima, 2002).

Both early- and late-night Per degradation steps appear to depend upon Per phosphorylation, which requires the casein kinase I encoded by the double-time (dbt) gene. To find out how Slmb could affect Per and Tim phosphorylation, Tests were performed to see whether Dbt, and Shaggy (Sgg), that has been shown to phosphorylate Tim, are affected in slmb^m mutants. No alterations of the level or the mobility of these kinases were detected in slmb^m head extracts. Next, whether Slmb could associate with the Per protein was examined, by searching for Per-Slmb interactions in co-immunoprecipitation experiments on head extracts. The Slmb protein was found to be co-precipitated by anti-Per antibodies, and anti-Slmb can precipitate Per in wild-type flies collected at CT 0. Similar results were obtained with pooled extracts. In addition, Slmb co-precipitates with Dbt . Because Per, but not Dbt, is profoundly affected in slmb^m mutants, these results support Per rather than Dbt as a Slmb target for ubiquitinylation, and suggest that the three proteins constitute a complex. Slmb was co-immunoprecipitated by anti-Per antibodies in tim⁰ flies, indicating that Per-Slmb complexes can form in the absence of Tim. Although twice as much extract was used for tim⁰ flies to compensate for the low Per levels in this genotype, the amount of immunoprecipitated Slmb suggests that the absence of Tim may favor Per-Slmb complexes. These results fit well with Slmb being involved in the control of unbound Per, either during its cytoplasmic accumulation at the beginning of the protein cycle or during its nuclear degradation at the end. To test whether the formation of Per-Slmb complexes is circadianly controlled, co-immunoprecipitations were performed at the beginning of the night when Per is mostly hypo-phosphorylated, or at the end of the night when Per is highly phosphorylated. All time points showed comparable levels of Per-Slmb complexes, and several forms of Per were immunoprecipitated by the anti-Slmb antibodies (compare CT 1 and CT 13). This indicates that differently phosphorylated Per molecules can be committed to Per-Slmb complexes (Grima, 2002).

Possible explanations for the accumulation of highly phosphorylated Per in slmb^m mutants would be that partially phosphorylated Per is the relevant Slmb substrate for degradation, or that Slmb targets some Per kinase that is bound to Per. The presence of highly phosphorylated Per in slmb^m indicates that Slmb is required for the control of phosphorylated Per accumulation in the early night. Moreover, Slmb overexpression in the LNvs results in a lengthening of the circadian period. In agreement with the behavioral data, Slmb overexpression slows down the oscillations of Per immunoreactivity in these cells, which showed a ~6 hour delay compared to wild-type controls after two days. These data can be explained by high levels of cytoplasmic Slmb inducing too much degradation of cytoplasmic Per, thus further delaying the night accumulation of the protein, whereas high levels of nuclear Slmb would rather precipitate the fall of the Per protein and shorten the circadian period. It is therefore thought that Slmb is, at least, involved in the control of cytoplasmic Per accumulation in the early night (Grima, 2002).

The presence of low-mobility Tim proteins at all circadian times in slmb^m mutants indicates that the accumulation of phosphorylated Tim is also Slmb-dependent. Remarkably, the Tim kinase Sgg controls the Slmb-dependent proteolysis of Cubitus interruptus and degradation of Armadillo. The results suggest that phosphorylated Tim could be a Slmb target or that Tim is phosphorylated by a Slmb-dependent kinase. Because Tim is hypo-phosphorylated in per⁰ flies, it is also possible that the accumulation of hyper-phosphorylated Per in slmb^m influences Tim phosphorylation (Grima, 2002).

Although protein degradation is commonly believed to have a major role in the control of the oscillations of clock proteins, the present work is the first to implicate a characterized component of the ubiquitin proteasome pathway. Because cycling of phosphorylated Per proteins also occurs in the mammalian clock, it would be interesting to determine whether the Slmb mammalian homolog ß-Trcp is involved in the control of phosphorylated Per levels. F-box proteins have been shown to be important at the G1/S transition of the cell cycle, by targeting phosphorylated cyclins and inhibitors of cyclin kinases for degradation by the proteasome. This study therefore suggests that the cell-cycle and the circadian-clock machineries share mechanisms to control the oscillations of phosphorylated proteins (Grima, 2002).

Protein phosphorylation has a key role in modulating the stabilities of circadian clock proteins in a manner specific to the time of day. A conserved feature of animal clocks is that Period (Per) proteins undergo daily rhythms in phosphorylation and levels, events that are crucial for normal clock progression. Casein kinase Iepsilon (CKIepsilon) has a prominent role in regulating the phosphorylation and abundance of Per proteins in animals. This was first shown in Drosophila with the characterization of Doubletime (Dbt), a homolog of vertebrate casein kinase Iepsilon. However, it has not been clear how Dbt regulates the levels of Per. Using a cell culture system, this study shows that Dbt promotes the progressive phosphorylation of Per, leading to the rapid degradation of hyperphosphorylated isoforms by the ubiquitin–proteasome pathway. Slimb, an F-box/WD40-repeat protein functioning in the ubiquitin–proteasome pathway interacts preferentially with phosphorylated Per and stimulates its degradation. Overexpression of slimb or expression in clock cells of a dominant-negative version of slimb disrupts normal rhythmic activity in flies. These findings suggest that hyperphosphorylated Per is targeted to the proteasome by interactions with Slimb (Ko, 2002).

NEMO/NLK phosphorylates PERIOD to initiate a time-delay phosphorylation circuit that sets circadian clock speed

The speed of circadian clocks in animals is tightly linked to complex phosphorylation programs that drive daily cycles in the levels of Period (Per) proteins. Using Drosophila, a time-delay circuit based on hierarchical phosphorylation was identified that controls the daily downswing in Per abundance. Phosphorylation by the Nemo/Nlk kinase at the 'per-short' phospho cluster domain on Per stimulates phosphorylation by Doubletime (Dbt/Ck1delta/epsilon) at several nearby sites. This multisite phosphorylation operates in a spatially oriented and graded manner to delay progressive phosphorylation by Dbt at other more distal sites on Per, including those required for recognition by the F box protein Slimb/β-TrCP and proteasomal degradation. Highly phosphorylated Per has a more open structure, suggesting that progressive increases in global phosphorylation contribute to the timing mechanism by slowly increasing Per susceptibility to degradation. These findings identify Nemo as a clock kinase and demonstrate that long-range interactions between functionally distinct phospho-clusters collaborate to set clock speed (Chiu, 2011).

This study shows that the per-short domain functions as a discrete hierarchical phospho-cluster that delays Dbt-mediated phosphorylation at the Slimb recognition site on Per, providing new insights into how clock protein phosphorylation contributes to circadian timing mechanisms. The cumulative effect of this delay circuit is to slow down the pace of the clock by ~8 hr. It is proposed that Dbt functions in a stepwise manner to phosphorylate clusters on Per that have distinct biochemical functions and effects on the rate of Per degradation, e.g., elements such as the per-short phospho-cluster that delays Per degradation and those such as the Slimb-binding site and global phosphorylation that enhance instability. Nmo plays a major role in the relative timing of Dbt activity at these different elements because it stimulates multisite phosphorylation at the per-short delay cluster by Dbt, which slows down the ability of Dbt to phosphorylate instability elements. Thus, a large portion of the phosphorylation events dictating when in a daily cycle Per is targeted for rapid degradation is not directly linked to binding ofSlimb per se. The current findings demonstrate that presumptive long-range interactions between distinct positively and negatively acting phospho-clusters collaborate to set clock speed and helps to explain why mutations in clock protein phosphorylation sites and/or the kinases that phosphorylate them can yield both fast and slow clocks (Chiu, 2011).

A proposed mechanism for the function of the per-short domain is supported by the congruence between in vitro biochemical studies based on purified recombinant Per protein from cultured S2 cells and in vivo changes in the pace of behavioral rhythms using transgenic models. This suggests that a primary biochemical effect of the per-short domain on clock speed in the fly is via modulating the rate of Dbt-mediated phosphorylation at the Slimb phospho-degron on Per. The physiological role of T583 phosphorylation is not clear, as mutating this site does not have detectable effects on the binding of Per to Slimb in S2 cells. In this regard, it is interesting to note that the original per-short domain was identified as encompassing aa 585-601 of Per (Baylies, 1992). Thus, it is likely that the 8 hr per-short delay circuit is governed by the dynamics underlying the phosphorylated status of three sites (i.e., S596, S589, and S585) (Chiu, 2011).

At present, it is not clear how phosphorylation in the per-short cluster slows down subsequent phosphorylation by Dbt at Ser47 and other sites. Inactivating the per-short cluster leads to increases in the rate of Dbt-mediated phosphorylation at not only the N terminus, but also the C terminus of Per, suggesting that it is a major control center for regulating the relative efficiency of Dbt phosphorylation at many sites on Per. It is suggested that the per-short phospho- cluster acts as a transient 'temporal trap' for Dbt. Once the sites in the per-short domain are phosphorylated by Dbt, this somehow allows it to continue its normal rate of phosphorylation at other phospho-clusters. Although speculative, progressive increases in phosphorylation at some of these other phospho-clusters might generate time-dependent local/overall conformational changes in Per, possibly via electrostatic repulsion, eventually leading to a more open Per structure that is more accessible to phosphorylation by Dbt at the Slimb-binding site and/or a more efficient substrate for degradation. Thus, the rapid degradation of Per during the early day is likely due to a combination of synchronous increases in the phospho-occupancy of Ser47 and overall phosphorylation of Per. Other factors such as protein phosphatases and the action of Timeless also play major roles in regulating the speed of the Per phosphorylation program (Chiu, 2011).

How might phosphorylation at S596 enhance phosphorylation at S589 and S585 by Dbt? Phosphorylation by the CK1 kinase family is generally enhanced by priming. However, phosphorylation at the per-short domain by Dbt does not follow the consensus priming-dependent recognition motif for the CK1 family of kinases (i.e., S/Tp-X-X-S/T, wherein S/Tp refers to the primed site, X is any amino acid, and the italicized residues the CK1 target site, as the S596 priming site is located C terminal to the Dbt sites. Thus, it is likely that phosphorylation of S596 by Nmo stimulates Dbt phosphorylation at the per-short region in a nonpriming-dependent manner (Chiu, 2011).

Ongoing studies are aimed at understanding the biochemical events underlying the ability of phosphorylation at S596 to enhance phosphorylation by Dbt in the per-short region. The discovery of a delay phospho-circuit also sheds light on why mutations in different phosphorylation sites on Per or Frq proteins, although affecting stability, can speed up or slow down the clock. The current findings also offer a logical explanation for why mutations that lower the kinase activity of CKI, which overall is expected to slow down the rate of PER degradation, can yield fast clocks. For example, although other mechanisms have been offered to explain the short-period phenotypes that are observed for the CKI3tau mutation in hamsters and a CKIdelta mutation associated with familial advanced sleep phase syndrome (FASPS) in humans, it is possible that phosphorylation at a per-short type delay cluster is preferentially compromised by the mutant kinase, which could appear as a substrate-specific gain-of-function mutation (Chiu, 2011).

Negatively acting phospho-clusters are likely to be a general feature of the timing mechanisms regulating the daily abundance cycles of clock proteins such as Pers in animals and Frq in Neurospora. However, other regulatory modules that operate in a phase-specific manner must participate to generate an ~24 hr oscillator. Most conspicuously, clock speed is intimately linked to the Per and Frq abundance cycles necessitating daily phases of de novo synthesis to replenish the pools of previously degraded proteins.Asrecently shown, the transcriptional negative feedback aspect of Per regulating Clk-Cyc-mediated transcription is also a component of the period-setting mechanism in Drosophila. Therefore, the ~24 hr Per abundance cycle is based on a combination of 'time constraints' that are generated using different regulatory modules. It is proposed that the per short- based timer mainly functions once Per has accumulated and begins participating in transcriptional repression, controlling Per abundance once it is disengaged from the dynamics of its cognate mRNA by setting in motion a series of sequential phosphorylation events that are calibrated to stimulate Per degradation in the nucleus at the appropriate time in a daily cycle, enabling the next round of circadian gene expression. In this context, it is interesting to note that a prior study analyzing the per-short domain suggested that it functions with a nearby 'perSD' domain to increase the transcriptional repressor function of Per. It is possible that the same phosphorylation events leading to Per degradation also function to increase its potency within the repressor complex (Chiu, 2011).

These studies also identify Nemo as a clock kinase. Nemo is the founding member of the evolutionarily conserved Nemo-like kinase (Nlk) family of proline-directed serine/threonine kinases closely related to mitogen-activated protein kinases (MAPK). It was originally characterized in Drosophila as required for planar cell polarity during eye development and is now known to function in many pathways. Nmo/Nlk is localized in the nucleus and is another factor in the circadian clock that also functions in the Wnt/Wg-signaling pathway, such as CKI3/Dbt, β-TrCP/Slimb, and GSK-3β/Sgg. It will be of interest to determine whether Nlk functions in the mammalian clock. Intriguingly, the phosphorylation sites on Per are largely clustered, and several of them have the same spatial arrangement as the per-short cluster, with a predicted pro-directed kinase site at the C-terminal end of the phospho-cluster. This suggests that Nmo and/or other pro-directed kinases serve as control points to activate spatially and perhaps functionally distinct phospho-clusters. Indeed, it has recently been shown that phosphorylation at Ser661 of Per by an as yet unidentified pro-directed kinase primes further phosphorylation by Sgg at Ser657 to regulate the timing of Per nuclear entry in key pacemaker neurons (Chiu, 2011).

In summary, a central aspect of circadian clocks is the presence of one or more clock proteins that provide a dual function by behaving as phospho-based timers and linking its timer role to gene expression by operating in a phase-specific manner to recruit repressor complexes that inhibit central clock transcription factors. These studies suggest that a major part of the timing mechanism underlying these phospho-clock proteins is based on spatially and functionally discrete phospho-clusters that interact to impose calibrated and sequentially ordered biochemical time constraints. In the case of Per, the per-short phospho-cluster functions as a central timing module by slowing down the ability of Dbt to phosphorylate instability elements regulating Per degradation and, hence, when Per repressor activity is terminated and the next round of circadian gene expression begins (Chiu, 2011).

Involvement of an SCF^Slmb complex in timely elimination of E2F upon initiation of DNA replication in Drosophila

Cul1 is a core component of the evolutionarily conserved SCF-type ubiquitin ligases that target specific proteins for destruction. SCF action contributes to cell cycle progression but few of the key targets of its action have been identified. Expression of the mouse Cul1 (mCul1) in the larval wing disc of Drosophila has a dominant negative effect. It reduces, but does not eliminate, the function of SCF complexes, promotes accumulation of Cubitus interruptus (a target of SCF action), triggers apoptosis, and causes a small wing phenotype. A screen for mutations that dominantly modify this phenotype showed effective suppression upon reduction of E2F function, suggesting that compromised downregulation of E2F contributes to the phenotype. Partial inactivation of Cul1 delays the abrupt loss of E2F immunofluorescence beyond its normal point of downregulation at the onset of S phase. Additional screens showed that mild reduction in function of the F-box encoding gene slimb enhances the mCul1 overexpression phenotype. Cell cycle modulation of E2F levels is virtually absent in slimb mutant cells in which slimb function is severely reduced. This implicates Slimb, a known targeting subunit of SCF, in E2F downregulation. In addition, Slimb and E2F interacted in vitro in a phosphorylation-dependent manner. Thus the G1/S transcription factor E2F is an SCFSlmb target in Drosophila. These results argue that the SCF^Slmb ubiquitin ligase directs E2F destruction in S phase (Hériché, 2003).

This study provides evidence indicating a role for an SCF^Slmb complex in elimination of E2F during S phase in Drosophila. Mouse Cul1 overexpression has a dominant negative effect on SCF function in the wing imaginal disc resulting in apoptotic cell death leading to a small wing phenotype in adult flies. Thus, SCF activity normally suppresses cell death. The cell death that occurs upon reduction of Cul1 function is suppressed when E2F function is reduced by mutation. This genetic interaction might be due to action of E2F and Cul1 in parallel pathways, one suppressing and one enhancing cell death, or they might act in opposite directions in the same pathway. Because SCF complexes are involved in controlling protein degradation, the hypothesis is favored that Cul1 promotes the destruction of E2F and that the inappropriate persistence of E2F leads to cell death. Consistent with this idea, E2F overproduction can induce apoptosis. Furthermore, cell cycle specific destruction of E2F is suggested by the finding that E2F protein becomes undetectable in the synchronized S phase cells of the morphogenetic furrow of the eye imaginal disc. Double labelling analysis extended this finding to the asynchronously cycling cells of the wing imaginal disc and demonstrated that E2F is rapidly degraded prior to significant DNA replication. In contrast, it was found that E2F is detected in a fraction of S phase cells when SCF function is reduced by mCul1. This shows that normal E2F downregulation is delayed or slowed by reduction in SCF function (Hériché, 2003).

Although the dominant negative action of mCul1 in Drosophila was unanticipated, it offers a fortuitously convenient tool for the analysis of SCF function. The Drosophila Cul1 mutant is less useful because maternal contributions of Cul1 are so large that homozygous mutant animals develop to pupariation. Additionally, unlike the spatially restricted expression of mCul1, the strong Cul1 mutant alleles result in lethality. The stage and generality of the defects make analysis of the Cul1 mutant phenotype particularly difficult. In contrast, the induced expression of mCul1 in the wing disc does not compromise viability and it gives a distinctive graded phenotype well suited for the study of genetic modification. While the basis for the dominant negative effect of mCul1 remains unknown, it is important to recognize that inactivation of the endogenous Cul1 is incomplete so that a reduced level of Cul1 activity persists. One likely explanation for the negative effect is that mCul1 overexpression leads to sequestration of some limiting SCF components into weakly active complexes. This hypothesis is consistent with the fact that mCul1 retains some positive function in flies as revealed by its ability to prolong survival of dCul1 mutant flies (J.-K. Hériché and P.H. O'Farrell, unpublished data) (Hériché, 2003).

The essentially complete suppression of the mCul1 phenotype by reduction of the genetic dose of E2F suggests a remarkable level of specificity, in which, among the many targets of Cul1 action, the destruction of E2F appears to be particularly important. Similarly, limitations of Cul1 function in other organisms also uncovered the disproportionate importance of particular substrates. For example, Cul1 mutations in mice result in the accumulation of the SCF substrate cyclin E but not of p27, another well characterized SCF substrate. Among all of the substrates targeted for degradation by SCF in S. cerevisiae, it is the failure to degrade Sic1 that underlies the G1 arrest in cdc53 mutants. Thus, different substrates of the SCF appear to have particularly high dependence on SCF activity in different biological contexts. The experimental context using mCul1 expression in the wing disc is thought to be particularly effective in exposing the involvement of SCF in E2F destruction (Hériché, 2003).

Since SCF complexes function in conjunction with a variety of F-box proteins that act as specificity factors, mutations in individual F-box proteins ought to affect particular subsets of SCF substrates. In an extensive screen for loci that modify the reduction of function phenotype for Cul1, the gene encoding the F-box protein Slimb as a modifier was identified, but no contributions of other F-box encoding genes to the phenotype were detected. This implicates Slimb in the action of SCF on E2F. Analysis of E2F levels reveals that cell cycle oscillations in E2F levels were absent when Slimb function was severely reduced. Note that the severity of this E2F destruction phenotype in comparison to the mild defect in cell cycle programming of E2F destruction upon mCul1 expression is entirely consistent with the fact that the slimb mutant gives a stronger loss of function than the reduction of function imposed by mCul1 overexpression. The absence of cell cycle oscillation in E2F presence in the slimb mutant suggests that SCF^Slmb is responsible for targeting E2F for S phase destruction. If this destruction is the consequence of direct action of SCF^Slmb on E2F, the F-box protein would be expected to interact with E2F. This prediction was confirmed by pull-down experiments. Furthermore, it is demonstrated that the interaction between Slimb and E2F is dependent on phosphorylation, as expected for the interaction of F-box proteins with their substrates. The S phase specificity of E2F destruction is regulated by a targeting phosphorylation event, but this level of regulation has yet to be investigated (Hériché, 2003).

Since E2F is a positive regulator of the G1/S transition, it is not clear why S phase destruction of E2F is required. However, it is noted that slimb mutant cells do not replicate DNA normally and the replication defect is correlated with an increase in E2F in individual cells. This observation is consistent with observations indicating that E2F can limit DNA replication both in mammalian cells and in flies. This enigmatic feature of E2F regulation suggests that its continued presence has a negative effect on DNA replication and indicates that there is still much to learn about E2F roles in cell cycle regulation (Hériché, 2003).

SCF^Slmb appears to influence other cell cycle events, perhaps by actions on other substrates, or perhaps as a result of events that are secondary to its influence on E2F destruction. For example, a slimb allele shows defects in centrosome duplication control, and slimb null cells undergo apoptosis. Both effects can be explained by a failure to inactivate E2F, since E2F promotes centrosome duplication in mammalian cells and E2F can induce apoptosis. A link between Slimb function and the RB/E2F pathway of cell proliferation control is also suggested by the genetic interaction between the C. elegans slimb ortholog lin-23 and the RBF ortholog lin-35 in which lin-35 function limits the severity of the loss of lin-23 . Since RBF is an important modifier of E2F activity, perhaps this interaction is a reflection of the action of both Lin-23 and Lin-35 on E2F. However, in the Drosophila system there was no modification of the mCul1 overexpression phenotype by mutations in either the RBF or DP gene, two known partners of E2F. This result suggests that these factors are not limiting factors in this situation or that E2F acts independently of RBF and DP. The latter hypothesis is the most likely explanation for the lack of an RBF interaction for the following reason. The E2F/RBF complex has to be disrupted for E2F to drive cells into S phase and E2F degradation occurs after the G1/S phase transition, which only happens after E2F has been released from its association with RBF. According to this view, it would be predicted that the mCul1-induced phenotype should be specifically sensitive to factors impinging on the elimination of E2F activity during S phase, and factors involved in the unknown processes by which persistence of E2F has negative outcome. Consequently, this experimental system may provide an avenue for the genetic dissection of this mysterious facet of E2F function (Hériché, 2003).

Thus, to explore potential roles for Cul1 in cell cycle control in D. melanogaster, mouse Cul1 (mCul1) was overexpressed in the wing imaginal disc. This overexpression has a dominant negative effect leading to a reduction in SCF function. The resulting small wing phenotype was used in a modifier screen to identify mutations in cell cycle genes capable of dominantly modifying the mCul1-induced phenotype. E2F loss-of-function mutations were the strongest modifiers since they completely suppress the phenotype. The reduction in SCF function associated with mCul1 overexpression also correlates with a failure to downregulate E2F normally in S phase. Additionally, mutations in slimb that reduce the function of the F-box protein Slimb enhance the phenotype and lead to persistence of E2F in S phase cells. Finally, this F-box protein interacts with E2F in vitro. These results indicate that an SCF^Slmb complex is involved in regulating E2F activity during S phase (Hériché, 2003).

Phosphorylation by double-time/CKIepsilon and CKIalpha targets cubitus interruptus for Slimb/beta-TRCP-mediated proteolytic processing

Hedgehog (Hh) proteins govern animal development by regulating the Gli/Ci family of transcription factors. In Drosophila, Hh signaling blocks proteolytic processing of full-length Ci to generate a truncated repressor form. Ci processing requires sequential phosphorylation by PKA, GSK3, and a casein kinase I (CKI) family member(s). This study shows that Double-time (DBT)/CKIε and CKIα act in conjunction to promote Ci processing. CKI phosphorylates Ci at three clusters of serine residues primed by PKA and GSK3 phosphorylation of other residues. CKI phosphorylation of Ci confers binding to the F-box protein Slimb/β-TRCP, the substrate recognition component of the SCF^{Slimb/β-TRCP} ubiquitin ligase required for Ci processing. CKI phosphorylation sites act cooperatively to promote Ci processing in vivo. Substitution of Ci phosphorylation clusters with a canonical Slimb/β-TRCP recognition motif found in β-catenin renders Slimb/β-TRCP binding and Ci processing independent of CKI. It is proposed that phosphorylation of Ci by CKI creates multiple Slimb/β-TRCP binding sites that act cooperatively to recruit SCF^{Slimb/β-TRCP} (Jia, 2005).

Regulation of Ci/Gli processing is a key regulatory step in the Hh signal transduction pathway; however, the underlying mechanism is still not fully understood. This study provides evidence that two CKI isoforms, DBT/CKIε and CKIα, act additively to promote Ci processing. It was found that CKI phosphorylates multiple Ser residues arranged in three clusters in the C-terminal half of Ci, and that CKI can phosphorylate sites primed by PKA or GSK3 phosphorylation. In addition, DBT/CKIε and CKIα are required for Ci phosphorylation in vivo. CKI sites in different phosphorylation clusters act cooperatively to promote Ci processing in vivo. More importantly, Slimb/β-TRCP was shown to directly bind CKI-phosphorylated Ci through its WD40 repeats. Finally, substitution of multiple CKI sites with a Slimb/β-TRCP binding motif found in β-catenin renders Ci processing independent of CKI. Based on these and other observations, it is proposed that PKA- and GSK3-primed CKI phosphorylation of Ci creates docking sites for Slimb/β-TRCP that recruit SCF^{Slimb/β-TRCP} to regulate Ci processing (Jia, 2005).

This study employed dominant-negative kinase, genetic mutations, and heritable RNAi knockdown to investigate the role of two CKI isoforms in Ci processing in vivo. Overexpression of a dominant-negative DBT/CKIε (DN-DBT) caused cell-autonomous accumulation of Ci155 and ectopic dpp expression, suggesting that interference with DBT/CKIε activity impairs Ci processing. As a further support, it was found that A compartment dbt/dco mutant cells accumulate high levels of Ci155. The phenotypes associated with dbt/dco mutations differ depending on the alleles used. The hypomorphic allele, dco³, does not seem to affect Ci processing, although it does affect cell growth and proliferation. By contrast, more severe alleles, including dco^P103 and dco^le88, affect Ci processing. The lack of Hh-related phenotypes associated with the weak allele of dbt/dco is likely due to compensation by other CKI isoforms. This may explain why RNAi knockdown of DBT/CKIε does not affect Hh signaling in cultured cells, since RNAi knockdown usually does not completely eliminate the function of the targeted genes, and hence often resembles hypomorphic genetic mutations. Alternatively, other CKI isoforms might be expressed in cultured cells at higher levels than in imaginal discs, so that they can compensate for the complete loss of DBT/CKIε in cultured cells (Jia, 2005).

To investigate the role of CKIα in Ci processing, the heritable RNAi approach was used, and two CKIα RNAi constructs were generated: CRS and CRL. CRL knocks down CKIα more effectively than CRS, likely due to its larger targeting sequence; however, it also knocks down DBT/CKIε. In contrast, CRS appears to be more specific for CKIα. Expressing CRL in wing discs induces high levels of Ci155 accumulation and ectopic dpp expression. In contrast, expressing CRS resulted only in a modest increase in Ci155 without inducing ectopic dpp expression. However, expressing CRS in DBT/CKIε hypomorphic (dco³/dco^le88) wing discs completely blocked Ci processing, as evident by the accumulation of high levels of Ci155 and ectopic dpp expression in these discs. These data suggest that CKIα and CKIε play partially redundant roles in Ci processing, and that they act additively to provide optimal CKI kinase activity required for efficient Ci phosphorylation and processing. Consistent with this notion, CKIα and CKIε bind equally well to Cos2. This is in contrast to what has been proposed for the Wnt pathway, where CKIε and CKIα appear to play opposing roles and act on distinct protein substrates. Since CKI sites are conserved in Gli proteins, it awaits to be determined whether CKIε or CKIα or both are involved in Gli regulation (Jia, 2005).

Using an in vitro kinase assay, two types of CKI phosphorylation events were uncovered: one primed by PKA and the other by GSK3 phosphorylation. CKI phosphorylation sites are arranged in three clusters. Whereas cluster 1 contains only PKA-primed CKI sites, both cluster 2 and 3 contain PKA- and GSK3-primed CKI sites. Using an in vivo functional assay, it was demonstrated that both PKA- and GSK3-primed CKI sites are involved in Ci processing. For example, the two types of CKI sites in cluster 2 appear to have overlapping function; mutations in either one only partially blocked Ci processing, whereas mutations in both completely blocked Ci processing (Jia, 2005).

CKI sites in different phosphorylation clusters appear to act cooperatively to promote Ci processing. Strikingly, mutating the two CKI sites in cluster 1 (Ci^SA12) completely abolishes Ci processing. Similarly, mutating all the CKI sites in cluster 2 also abolishes Ci processing. A dosage-sensitive interaction was observed between two phosphorylation clusters. For example, partial loss of function of both cluster 2 and cluster 3 nearly abolish Ci processing. Based on these and other observations, it is proposed that each phosphorylation cluster acts as a functional module, and Ci processing requires cooperative action among the three modules (Jia, 2005).

Ci lacks the canonical Slimb/β-TRCP binding motif (DSGXXS) found in other SCF^{Slimb/β-TRCP} substrates such as β-catenin and Iκ-B, inviting speculation that Ci phosphorylation could recruit a protein(s) other than Slimb/β-TRCP and that the involvement of SCF^{Slimb/β-TRCP} in Ci processing could be indirect. This study assessed whether hyperphosphorylation of Ci directly recruits Slimb/β-TRCP. It was found that a GST-Ci fusion protein binds Slimb/β-TRCP efficiently after it is phosphorylated by CKI, following primed phosphorylation by the other kinases. In addition, binding of GST-Ci to Slimb is compromised when a subset of CKI sites was mutated to Ala. These observations support the hypothesis that phosphorylation of Ci at CKI sites confers Slimb/β-TRCP binding. The in vivo relevance of Slimb/β-TRCP binding was demonstrated by the finding that a single canonical Slimb/β-TRCP binding site can substitute for the three phosphorylation clusters to promote Ci processing. Strikingly, Ci variants bearing the DSGXXS motif can undergo processing even when CKI activity is blocked. These observations suggest that the major function of CKI in Ci processing is to recruit SCF^{Slimb/β-TRCP} by phosphorylating Ci at multiple Ser residues that function as docking sites for Slimb/β-TRCP (Jia, 2005).

The recently solved crystal structure of the β-TRCP/β-catenin phospho-peptide complex reveals that the two phospho-Ser and the aspartate residues in the DSGXXS motif make critical contacts with several basic residues from the WD40 repeats of β-TRCP that form a single substrate binding pocket. Although none of the three phosphorylation clusters in Ci contains a DSGXXS motif, they all contain related sequences. For example, cluster 1 contains DSQNSTAS, cluster 2 contains SSQSS and SSQVSS, and cluster 3 contains SSQMS. It is proposed that these phospho-Ser motifs represent low-affinity or suboptimal sites for Slimb/β-TRCP recognition, and optimal binding of Slimb/β-TRCP to Ci is achieved by cooperative binding among multiple low-affinity sites. The high local concentration of binding sites greatly increases the probability of interaction so that Ci is unable to diffuse away from Slimb/β-TRCP before rebinding occurs. Hence, Ci becomes kinetically trapped in close proximity to Slimb/β-TRCP once the binding is engaged. Alternatively, phosphorylation of Ci could recruit a cofactor that binds cooperatively with Slimb/β-TRCP to hyperphosphorylated Ci. Both models can explain the observed high cooperativity among multiple phosphorylation clusters in Ci processing (Jia, 2005).

The ability to bind a single high-affinity site or multiple low-affinity sites appears to be a general feature for the SCF family of ubiquitin ligases. Another well-characterized SCF complex, SCF^CDC4, can bind certain substrates such as Cyclin E through a single high-affinity site and other substrates such as Sic1 through multiple low-affinity sites. In the case of Sic1, phosphorylation at multiple sites appears to set a threshold for kinase activity that converts a smooth temporal gradient of kinase activity into a switch-like response for degradation of Sic1 and onset of S phase. In the case of Ci/Gli regulation, first, the requirement for hyperphosphorylation may render Ci processing highly dependent on the activity of individual kinases and hence highly sensitive to Hh, since low levels of Hh suffice to block Ci processing although such levels of Hh may only cause a small reduction in Ci phosphorylation levels. Second, cooperativity among multiple phosphorylation sites may convert a smooth spatial Hh activity gradient into a sharp response for Ci processing, since a small drop in the level of Ci phosphorylation could result in a dramatic reduction in Ci processing and hence the level of Ci75. Third, employing multiple phosphorylation events may allow the levels of Ci phosphorylation to be fine-tuned by different thresholds of Hh signaling activity, leading to differential regulation of Ci processing and activity, as the activity of Ci155 appears to be regulated by phosphorylation independent of its processing. Finally, employing multiple kinases to regulate Ci/Gli may provide opportunities for crosstalk between the Hh and other signaling pathways in certain developmental contexts (Jia, 2005).

Processing of the Drosophila hedgehog signaling effector Ci-155 to the repressor Ci-75 is mediated by direct binding to the SCF component Slimb

Signaling by extracellular Hedgehog (Hh) molecules is crucial for the correct allocation of cell fates and patterns of cell proliferation in humans and other organisms. Responses to Hh are universally mediated by regulating the activity and the proteolysis of the Gli family of transcriptional activators such that they induce target genes only in the presence of Hh. In the absence of Hh, the sole Drosophila Gli homolog, Cubitus interruptus (Ci), undergoes partial proteolysis to Ci-75, which represses key Hh target genes. This processing requires phosphorylation of full-length Ci (Ci-155) by protein kinase A (PKA), casein kinase 1 (CK1), and glycogen synthase kinase 3 (GSK3), as well as the activity of Slimb. Slimb is homologous to vertebrate ß-TRCP1, which binds as part of an SCF (Skp1/Cullin1/F-box) complex to a defined phosphopeptide motif to target proteins for ubiquitination and subsequent proteolysis. Phosphorylation of Ci at the specific PKA, GSK-3, and CK1 sites required in vivo for partial proteolysis stimulates binding to Slimb in vitro. Furthermore, a consensus Slimb/ß-TRCP1 binding site from another protein can substitute for phosphorylated residues of Ci-155 to direct conversion to Ci-75 in vivo. From this, it is concluded that Slimb binds directly to phosphorylated Ci-155 to initiate processing to Ci-75. The phosphorylated motifs in Ci that are recognized by Slimb have been explored and some evidence is provided that silencing of Ci-155 by phosphorylation may involve more than binding to Slimb (Smelkinson, 2006).

The mechanism and consequences of Hh signaling have been studied extensively in the developing Drosophila wing imaginal disc, where Hh, secreted from posterior compartment cells, induces a strip of nearby, responsive anterior cells (AP border cells) to express a small set of target genes, including decapentaplegic (dpp), that subsequently pattern the developing wing. In anterior cells far from Hh, Ci-155 is processed slowly to Ci-75, which crucially represses potential Hh target genes, including hh itself and dpp, to ensure that they are not ectopically expressed. Even low-level Hh signaling at the AP border blocks Ci-75 production, thereby also increasing the concentration of Ci-155. Hh further activates Ci-155 in a dose-dependent manner by facilitating its nuclear accumulation and potentially also by modifying its binding partners in the nucleus. Because formation of Ci-75 requires both Ci-155 phosphorylation and the activity of Slimb, it was proposed that Slimb might promote partial proteolysis of Ci-155 by directly binding to phosphorylated Ci-155 and catalyzing its ubiquitination. However, despite some support for this hypothesis, Ci-155 contains no obvious consensus binding site for Slimb/β-TRCP1: there are only two well-studied examples where proteasomal degradation of a ubiquitinated protein is incomplete (NF-KappaB precursors, p100 and p105), and ubiquitinated Ci-155 has not been detected when Ci-155 is stabilized by inhibiting the proteasome (Smelkinson, 2006).

To determine whether Slimb can bind to Ci in a manner dependent on phosphorylation, a purified GST fusion protein was used that includes the key phosphorylation sites of Ci. This GST-Ci protein undergoes a significant mobility shift in SDS polyacrylamide gels when phosphorylated by PKA and CK1 together and an even greater shift if GSK3 is also included. GST-Ci binds more avidly than GST alone to ³⁵S-labeled full-length Slimb produced by in vitro translation in a reticulocyte lysate and to HA-tagged full-length Slimb from crude extracts of transiently transfected Drosophila Kc cells. This binding is reproducibly increased by using PKA together with CK1, and to a much greater extent by using all three protein kinases to phosphorylate GST-Ci prior to the binding assay. The synergistic contribution of GSK3 was clearest in the HA-Slimb binding assay, and so this assay was used to investigate further the characteristics of Ci binding to Slimb (Smelkinson, 2006).

Three PKA sites ('P1-3'), the neighboring three PKA-primed CK1 sites ('C1-3') and the two adjacent PKA-primed GSK3 sites ('G2,3') are required for Ci-155 to be converted to Ci-75 in Drosophila embryos and wing discs. When the serine residues at these PKA, CK1, or GSK3 sites were replaced with alanines, significant stimulation of binding of GST-Ci to HA-Slimb was no longer seen by any combination of PKA, CK1, and GSK3. Thus, strong binding of HA-Slimb to a Ci fragment in vitro requires the same protein kinases and the same phosphorylation sites that are required in vivo to convert Ci-155 to Ci-75 (Smelkinson, 2006).

Whether a defined, minimal Slimb binding site from another protein could direct processing of Ci-155 to Ci-75 was tested. β-catenin is a prototypical substrate for the β-TRCP1 SCF complex, in which a dually phosphorylated motif (DpSGIHpS, where pS stands for phosphoserine) is the critical recognition element for binding. This motif is conserved in Drosophila β-catenin (Armadillo), and Armadillo proteolysis depends on both this sequence and Slimb activity. The motif is also expected to serve as a direct binding site for Slimb, the Drosophila homolog of β-TRCP1. Tests revealed that this consensus Slimb/β-TRCP1 binding site, engineered into Ci, is functional and directs Slimb binding in vitro with an apparent avidity similar to that seen for fully phosphorylated wild-type Ci (Smelkinson, 2006).

Binding assays were used to search for the direct Slimb recognition elements in Ci because no established Slimb/β-TRCP consensus binding sites are apparent in the sequence of Ci or Gli proteins. Each of the three PKA sites in Ci is required for detectable processing to Ci-75 in Kc tissue-culture cells; therefore, whether each site contributes to Slimb binding in vitro was tested. Replacement of all three PKA-primed CK1 sites (and the two predicted CK1-primed CK1 sites) with acidic residues abolished any stimulation of HA-Slimb binding by phosphorylation of GST-Ci (Smelkinson, 2006).

It cannot be readily determined whether the PKA sites and PKA-primed CK1 sites are directly recognized by Slimb. However, the evident contribution of each PKA site to Slimb binding implies that each must nucleate at least one direct Slimb binding site. Surrounding the three PKA sites there are two types of motifs that are related to previously recognized or postulated Slimb/β-TRCP binding motifs (DSGXXS, DSGXXXS, TSGXXS, EEGXXS, DDGXXD, and DSGXXL. (1) The six-amino-acid motif (D/pS)(pS/pT)(Q/Y)XX(pS/pT) might be created in three places if phosphorylation occurs, as is suspected, at some nonconsensus sites. These motifs most closely resemble the β-TRCP1 binding site postulated for p100 (DpSAYGpS), but the presence of glutamine or tyrosine at the third position in place of glycine in the ideal consensus would be expected to reduce binding by more than an alanine substitution. (2) Many five-amino-acid motifs are created in which two acidic residues (DpS, pSpS, or pSpT) are separated from a phosphorylated residue (pS or pT) by only two amino acid residues. Four of these eight motifs include a glutamine at the third position. However, this residue does not appear to be instrumental in Slimb binding because substitution with alanine has little effect. On the basis of the crystal structure of β-TRCP1, it is hard to predict the affinity of the designated five-amino-acid motifs for Slimb, but it is likely to be lower than for any of the motifs cited above. Regardless of which precise motifs contribute most significantly to Slimb binding, it is clear from the mutational analysis that Ci must be highly phosphorylated over a region spanning almost sixty amino acid residues to generate several suboptimal binding sites that must collaborate to provide physiologically significant affinity for Slimb. This follows a precedent established for degradation of the yeast cell-cycle regulator Sic1 by binding of the SCF_CDC4 complex to multiple low-affinity phosphodegrons. The requirement for extensive Ci phosphorylation could account for the essential role of the scaffolding protein Cos2 in facilitating phosphorylation and for the relatively slow conversion of Ci-155 to Ci-75 seen in vivo (Smelkinson, 2006).

In summary, processing of Ci-155 to Ci-75 is initiated by direct binding of Slimb to Ci-155 molecules that have been extensively phosphorylated by PKA, GSK3, and CK1. This presumably leads to ubiquitination of Ci-155 and its partial proteolysis by the proteasome, generating a transcriptional repressor that plays a key developmental role in cells that are not exposed to Hh. Whether phosphorylation also prevents Ci-155 from activating transcription through an additional mechanism remains to be explored, as does the mechanism by which proteolysis of Ci-155 is limited to preserve its N-terminal domains as Ci-75 (Smelkinson, 2006).

Regulation of Ci-SCF^Slimb binding, Ci proteolysis, and hedgehog pathway activity by Ci phosphorylation

Hedgehog (Hh) proteins signal by inhibiting the proteolytic processing of Ci/Gli family transcription factors and by increasing Ci/Gli-specific activity. When Hh is absent, phosphorylation of Ci/Gli triggers binding to SCF ubiquitin ligase complexes and consequent proteolysis. This study shows that multiple successively phosphorylated CK1 sites on Ci create an atypical extended binding site for the SCF substrate recognition component Slimb. GSK3 enhances binding primarily through a nearby region of Ci, which might contact an SCF component other than Slimb. Studies of Ci variants with altered CK1 and GSK3 sites suggest that the large number of phosphorylation sites that direct SCF^Slimb binding confers a sensitive and graded proteolytic response to Hh, which collaborates with changes in Ci-specific activity to elicit a morphogenetic response. When Ci proteolysis is compromised, its specific activity is limited principally by Su(fu), and not by Cos2 cytoplasmic tethering or PKA phosphorylation (Smelkinson, 2007).

The central task of Hh signal transduction in Drosophila is to regulate the activity of the transcription factor Ci. This is accomplished by regulating the levels of Ci-155 activator and Ci-75 repressor and the specific activity of Ci-155. To examine the regulation of Ci-155-specific activity in isolation, a Ci variant (Ci-S849A) was developed that escapes PKA-dependent proteolysis but has a minimally altered pattern of PKA-initiated phosphorylation. At 29°C, Ci-S849A was expressed in wing discs (via C765-GAL4) at roughly the same level as endogenous Ci, but at 20°C, the levels of Ci-S849A were distinctly lower. At 29°C, Ci-S849A induced the Hh target gene reporter ptc-lacZ in A cells that are not stimulated by Hh. Since ptc induction requires Ci-155 activator and is not accomplished simply by loss of Ci-75 repressor, it is concluded that elevated levels of Ci-155 can suffice to confer some activator function. At 20°C, Ci-S849A induced ptc-lacZ in A cells only when Su(fu) was removed. By contrast, Ci-S849A activity was not detectably increased by loss of either PKA activity or Cos2 activity in P smo mutant clones, where Hh signaling is blocked. Thus, Su(fu) is the principal component that limits Ci-155-specific activity when Ci-155 is protected from PKA-dependent proteolysis, whereas PKA and Cos2 normally limit Ci-155 activity simply by promoting its proteolysisby promoting its proteolysis.

The prior assertion that PKA limits Ci-155-specific activity by direct phosphorylation was based on the use of an inappropriate reagent (Ci-U), which was mistakenly thought to be inert to PKA-dependent proteolysis. A similar role for Cos2 was previously inferred principally from the observation that Ci-155 accumulated more rapidly in the nuclei of anterior Leptomycin B-treated wing disc cells when those cells lack . It appears that the inferred cytoplasmic retention of Ci-155 by Cos2 contributes very little quantitatively to limiting the activity of stabilized Ci-155. That conclusion is supported by the observation that loss of Cos2 function did not enhance the weak induction of En seen in pka mutant clones of otherwise WT wing discs. The remaining, long-standing observations suggesting roles for PKA and Cos2 in limiting Ci-155-specific activity are the different degrees of Hh target gene induction in pka (strongest), cos2 (intermediate), and slimb (weakest) mutant wing disc clones. It is suggested that this might result from different degrees of disruption of PKA-dependent proteolysis in these clones. This suggestion is consistent with the proposed role of Cos2 in facilitating Ci-155 phosphorylation and with the observation that ptc-lacZ can be induced in slimb mutant clones when PKA activity is halved (Smelkinson, 2007).

Both GST-Ci association with SCF^Slimb and Ci-155 proteolysis depend on two phosphorylated regions of Ci. The first region provides an essential Slimb binding site that can be created by five successive CK1 phosphorylations primed initially by PKA site 1. Of the four phosphorylated residues within the motif (₈₄₄SpTpYYGSpMQSp₈₅₂) that interacts directly with Slimb, at least one (S849) is essential for binding, and two others (S844 and S852) enhance binding (T845 is essential, but priming and binding functions have not been separated). The second critical phosphorylated region of Ci includes two GSK3 sites (S884 and S888) that are primed by PKA site 3. This region enhances, but is not sufficient for, binding to SCF^Slimb (Smelkinson, 2007).

The requirement for multiple successive phosphorylations by PKA, CK1, and GSK3 to create a high-affinity SCF^Slimb binding domain on Ci-155 has two important consequences. First, it demands a special mechanism for facilitating Ci phosphorylation that is met by Cos2. Second, it provides a mechanism through which a small change in Ci-155 phosphorylation, induced for example by limited dissociation of protein kinases from Cos2, can be translated into a substantial inhibition of Ci-155 proteolysis. The sharp increase in Ci-155 levels at the anterior limit of Hh signaling territory shows that a low dose of Hh does indeed severely curtail PKA-dependent Ci-155 proteolysis. Hh could inhibit proteolytic processing of Ci variants driven by either PKA-primed GSK3 sites (Ci-SL) or PKA-primed CK1 sites (Ci-G2,3E and Ci-Y846G), but complete inhibition was observed only for the latter pair. This suggests that the sensitive response of Ci proteolysis to Hh depends principally on CK1 (Smelkinson, 2007).

How does inhibition of Ci-155 proteolysis affect Ci-155 activity? Previously, the properties of Ci-U and slimb mutant clones were taken as evidence that inhibition of proteolysis does not suffice for Ci activation. Since Ci-U is subject to PKA-dependent proteolysis and PKA does not affect the specific activity of Ci-155, instead the properties of Ci-S849A and pka mutant clones were relied upon to conclude that complete inhibition of PKA-dependent proteolysis does suffice to induce the Hh target gene ptc. This, in turn, suggests that the high Ci-155 levels anterior to the stripe of elevated ptc expression at the AP border of wing discs result from substantial, but incomplete, inhibition of Ci-155 proteolysis (Smelkinson, 2007).

It has generally been assumed that inhibition of Ci-155 proteolysis is uniformly strong throughout the AP border and that the activation of ptc and en in nested domains is due solely to changes in Ci-155-specific activity elicited by increasing levels of Hh. However, several factors suggest that there may also be a significant gradient of residual PKA-dependent proteolysis at the AP border that contributes to morphogen action (Smelkinson, 2007).

First, the precise degree of substantially inhibited Ci-155 proteolysis can determine whether Hh target genes are induced or not. This is evident from differences in ptc-lacZ induction among proteolytically impaired Ci variants and between pka and slimb mutant clones (Smelkinson, 2007).

Second, Su(fu) is the principal regulator of Ci-155 activity when Ci-155 levels are elevated, yet Hh instructs an almost unchanged morphogenetic response in the absence of Su(fu). It is likely that a proteolytic gradient is critical under these conditions, although it is also possible that Cos2 assumes a more significant role in regulating Ci-155-specific activity when Su(fu) is absent (Smelkinson, 2007).

Third, it was found that the loss of any one of four phosphoserines that contribute to Ci-Slimb binding (S844, S852, S884, and S888) diminishes, but does not abolish, Slimb binding. For S888A (G3A) this results in elevated Ci-155 levels and an increased activity, but residual proteolysis is clearly evident from the generation of sufficient Ci-75 to repress hh-lacZ. Thus, dispersion of direct Slimb binding determinants among several phosphorylatable residues provides a mechanism for Hh to elicit graded inhibition of Ci-155 proteolysis. It is speculated that in response to high levels of Hh, most Ci-155 molecules will not bind to SCF^Slimb at all because they lack at least two of the six key phosphorylated residues, whereas a large proportion of Ci-155 molecules may bind SCF^Slimb with intermediate affinity in response to low or intermediate Hh levels because they lack only one critical phosphoserine (Smelkinson, 2007).

Fourth, regulation of Ci-155-specific activity depends on Ci-155 levels. Thus, Ci-155 is only activated by loss of Su(fu) when Ci-155 levels are elevated by Hh or appropriate mutations, presumably because other stoichiometric binding partners such as Cos2 act redundantly with Su(fu) when their Ci-155 binding capacity is not saturated. The release of Ci-155 from repressive partners would therefore be expected to be progressively facilitated as the relative levels of Ci-155 increase. This would allow increasing Hh levels to enhance Ci-155 activity through synergistic effects on Ci-155 levels and Ci-155-specific activity (Smelkinson, 2007).

The archetypal β-TRCP/Slimb substrates, β-catenin and IKB, contain a single, dually phosphorylated, high-affinity binding site (DSpGxxSp) that triggers rapid substrate proteolysis. The primary direct Slimb binding motif that has been defined (SpTpYYGSpMQSp) in Ci differs notably by the presence of Tyr instead of Gly at the third position, by the inclusion of a fourth electronegative residue at its C terminus, and by binding with lower affinity, permitting additional influences on Ci-SCF^Slimb association. The fourth electronegative residue (pS852) likely interacts with at least one of two positively charged Slimb surface residues (R333 and R353) based on their potential proximity and reduced GST-Ci binding to the R333A/R353A Slimb variant (Smelkinson, 2007).

Most known β-TRCP substrates include phosphorylated or acidic residues that are two to four residues C-terminal to the standard six amino acid binding motif (DSpGXXSp), but their contribution to binding has not generally been assessed. Even β-catenin includes such phosphorylated residues that are known to have an essential priming role but have not been tested rigorously for direct interactions with β-TRCP. The variant β-TRCP binding motif (EEGFGSpSSp) of mammalian Wee1A presents a notable exception, in which a β-TRCP Arg residue equivalent to R353 of Slimb interacts with the phosphoserines at position 6 and 8 of this motif. This suggests that positive surface residues of β-TRCP/Slimb may commonly stabilize association with extended binding motifs. It is speculated that extended β-TRCP/Slimb binding motifs are likely to be especially important and prevalent in substrates lacking Gly at the third position because it was found that R333 or R353 (or both) of Slimb promotes binding to Ci-WT, but not to Ci-SL, and pS852 contributes significantly to Slimb binding in Ci-WT, but not in Ci-Y846G (Smelkinson, 2007).

Vertebrate Gli homologs of Ci also have a residue other than Gly at the third position (generally Ala) and a potentially phosphorylated Ser at the C terminus of a putative extended β-TRCP binding motif of 9 to 11 residues (SSAYx(x)SRRSS). Both a second Wee1A binding motif (DSAFQEPDS) and a β-TRCP binding motif of the p100 precursor of NFKB p52 (DSAYGSQSVE) also lack a Gly residue at the third position and include residues beyond the six amino acid core motif that might, by analogy to Ci, potentiate binding (Smelkinson, 2007).

Studies of Ci provide a clear precedent for the use of an extended β-TRCP/Slimb binding motif to translate regulated substrate phosphorylation into regulated proteolysis. However, it was also found that Slimb binding cannot be predicted by focusing on only the interactions of charged residues. Thus, fully phosphorylated Ci includes two sequences with a distribution of charged residues similar to that of the primary SCF^Slimb binding site (₈₃₇DSpQNSpTpASpTp and ₈₅₈SpSpQVSpSpIPTp compared with ₈₄₄SpTpYYGSpMQSp), but those sites neither suffice for Slimb binding (in Ci-S849A) nor enhance binding significantly in vitro (as revealed by D837A, T842A, S858A, S859A, and Ds2 variants). This probably reflects significant binding contributions of nonpolar residues in positions 3-5 of an extended Slimb/β-TRCP binding motif, as suggested by the presence of Tyr or Phe at position 4 of the functional motifs of Ci, Gli, p100, and Wee1A (Smelkinson, 2007).

Several F-box proteins that use WD40 repeats to bind substrate (FBW proteins) also include a dimerization domain that directs assembly of higher-order SCF complexes. Some substrates of these SCF complexes (for example, Cyclin E for Fbw7 and Wee1A for β-TRCP) contain more than one phosphorylated region capable of interacting with the same WD40 binding surface of the FBW protein. This raises the question of whether the cooperative function of two or more such regions depends on SCF dimerization and simultaneous interaction with two FBW subunits of a dimeric complex (Smelkinson, 2007).

This study found that Ci also contains two phosphorylated regions that contribute to SCF association, and Slimb molecules can bind to each other within higher-order functional SCF complexes. It was also found that Slimb self-association enhanced binding to GST-Ci relative to GST-Ci-SL, which contains a single DSGxxS motif; however, it was not required for both phosphorylated regions of GST-Ci to stimulate binding. Hence, simultaneous binding to separate Slimb monomers within a larger complex can be excluded as a requisite mechanism for cooperativity between the two phosphorylated regions of Ci. This is consistent with recent structural studies that predict a wider separation of WD40 binding surfaces within an SCF dimer than can be spanned readily by the two critical phosphorylated regions of a single Ci molecule (Smelkinson, 2007).

Does the region preceding PKA site 3 of Ci bind directly to the FBW component (Slimb) of an SCF complex as for Cyclin E and Wee1A? That model was proposed for Gli-2/3 proteins, which include a recognizable, potentially extended, variant Slimb-binding motif (DSYDPISTDAS). The analogously positioned sequence in Drosophila (SFYDPISPGCS) retains the YDPIS sequence and the two GSK3 sites at position 7 and 11 (underlined) but lacks a Ser at position 2 (italics), which is required in Gli-2/3 for normal β-TRCP association and proteolysis. Also, Ala substitution of the first Ser in the Drosophila motif (together with three other Ser residues) had only a minor effect on Slimb binding in vitro and Ci-155 proteolysis in vivo. Thus, this region of Ci does not have a clearly recognizable and demonstrably functional, conventional β-TRCP/Slimb binding site. It is, nevertheless, conceivable that the conserved elements of the putative β-TRCP binding motif of Gli-2/3 might provide a very weak direct interaction with the WD40 domain of Slimb that is sufficient to enhance SCF^Slimb association (Smelkinson, 2007).

However, this study also found that the GSK3 enhancement of Ci-Slimb binding conferred by the GSK3 sites preceding PKA site 3 was lost if Slimb lacked an F-box domain and consequent direct association with SkpA and its SCF complex partners. This result is interpreted with some caution because Slimb-ΔF also bound less well than wild-type Slimb to a canonical β-catenin substrate and to GST-Ci that was phosphorylated only at its primary Slimb binding site. Nevertheless, the result suggests that the region of Ci immediately preceding PKA site 3 might augment SCF association by binding directly to an SCF component other than Slimb (Smelkinson, 2007).

Whether GSK3 stimulates Ci binding to SCF^Slimb via a direct interaction with Slimb, an unprecedented interaction with another SCF component, or a conformational effect on the primary Slimb binding site of Ci, the Ci-G3A transgene reveals that the stimulation conferred by GSK3 phosphorylation is critical for efficient Ci-155 proteolysis and for Hh pathway silencing. Since Slimb self-association enhanced GST-Ci, but not GST-Ci-SL, binding in vitro, it is suspected that this may also be important for Ci-155 proteolysis in vivo. It is not known if SCF^Slimb dimerization (or oligomerization) is regulated, but the different modes of association of SCF^Slimb with Ci and β-catenin certainly provide several opportunities for SCF regulatory mechanisms or mutations to affect the Hh pathway without altering the Wnt/β-catenin pathway (Smelkinson, 2007).

The phospho-occupancy of an atypical Slimb-binding site on Period that is phosphorylated by Doubletime controls the pace of the clock

A common feature of animal circadian clocks is the progressive phosphorylation of Period (Per) proteins, which is highly dependent on casein kinase Idelta/epsilon (CKIdelta/epsilon, termed Doubletime [Dbt] in Drosophila), and ultimately leads to the rapid degradation of hyperphosphorylated isoforms via a mechanism involving the F-box protein, beta-TrCP (Slimb in Drosophila). This study use the Drosophila model system shows that a key step in controlling the speed of the clock is phosphorylation of an N-terminal Ser (S47) by DBT, which collaborates with other nearby phosphorylated residues to generate a high-affinity atypical Slimb-binding site on Per. Dbt-dependent increases in the phospho-occupancy of S47 are temporally gated, dependent on the centrally located Dbt docking site on Per and partially counterbalanced by protein phosphatase activity. It is proposed that the gradual Dbt-mediated phosphorylation of a nonconsensus Slimb-binding site establishes a temporal threshold for when in a daily cycle the majority of Per proteins are tagged for rapid degradation. Surprisingly, most of the hyperphosphorylation is unrelated to direct effects on Per stability. This study also used mass spectrometry to map phosphorylation sites on Per, leading to the identification of a number of 'phospho-clusters' that explain several of the classic per mutants (Chiu, 2008).

To better understand the physiological role of phosphorylation in regulating PER stability, Drosophila was used as a model system. Using a range of strategies, including mutational analysis, mass spectrometry and phospho-specific antibodies S47 was identified as a key phospho-determinant regulating the efficiency of SLIMB binding to dPER. By evaluating the behavior of dPER mutants whereby amino acid 47 is constitutively 'nonphosphorylated' (S47A) or 'phosphorylated' (S47D), it was shown that the phospho-occupancy of S47 is a key biochemical throttle adjusting the pace of the clock. However, phosphorylation of S47 occurs within an atypical SLIMB-binding site. Additional DBT-dependent phosphorylated residues, which likely include one or more nearby Ser residues at amino acid 44/45 and possibly others within the first 100 amino acids, collaborate with pS47 to generate a high-affinity SLIMB-binding region on dPER. As such, the affinity of SLIMB for dPER is proportional to the degree of phospho-occupancy within an extended phosphorylation network centered on S47 that as a unit yields a graded response in the affinity of SLIMB-dPER interactions. Attaining a high proportion of dPER molecules that are phosphorylated at S47 and other key sites mediating SLIMB binding is progressive and occurs several hours after DBT stably interacts with dPER via the centrally located dPDBD, likely because DBT 'activity' is counterbalanced by TIM and protein phosphatases. It is proposed that the relatively slow assembly of a high-affinity SLIMB-binding site on dPER is at least partly 'designed' to extend the time that dPER acts as a transcriptional repressor, critical in generating transcriptional feedback loops with daily time frames. Finally, this mass spectrometry analysis identify 'hot spots' for phosphorylation, indicates that the majority of dPER phosphorylation is unrelated to direct effects on stability and sheds new insights into the underpinnings of several previously characterized mutants, including the classic per^S allele (Chiu, 2008).

Early studies identified DpSGPhiX_1+npS (pS, phosphorylated Ser; Phi, any hydrophobic amino acid; X, any amino acid) as the consensus motif for recognition by the β-TrCP/SLIMB F-box protein (Fuchs. 2004). Phosphorylation at both sites in this six amino acid consensus generally leads to a high-affinity β-TrCP/SLIMB-binding site. Indeed, the three negatively charged residues (Asp/Glu and two phospho-S/T residues) are important binding contacts underlying β-TrCP/substrate interactions (Wu, 2003). Furthermore, it is thought that the presence of an Asp/Glu at position 2 of the canonical binding domain can circumvent the need for a phospho-S/T at that position, as is the case for Wee1A (Watanabe, 2004). However, accumulating evidence indicates that β-TrCP-binding sites can deviate from this consensus. For example, recent work on the Ci/Gli family of transcription factors suggests a novel class of degenerate and weaker β-TrCP-binding sites that extend beyond the standard six-amino-acid binding motif, especially for those missing a Gly at the third position (Smelkinson, 2007). It was suggested that for these extended β-TrCP/SLIMB-binding motifs, significant contributions are made by the local presence of nonpolar residues, such as those found in the motifs for Ci, Gli, and Wee1A. Additional phosphorylation events at nearby regions are also thought to enhance the inherently weak binding affinities of extended β-TrCP-binding sites, enabling a more graded response compared with the standard sequence (Chiu, 2008).

The major SLIMB-dependent phospho-degron identified in this study [⁴⁴p*Sp*SGpSSGYGG⁵²; where p* = possible phosphorylation] seems to include signature elements found in both the standard and extended β-TrCP-binding motifs. A rather unique feature of the SLIMB-binding domain on dPER is that it includes two SSG repeats. S47 is phosphorylated, and based on mutational analysis and mass spectrometry, it is almost certain that either S44 and/or S45 are phosphorylated. A physiological role for S45 is further indicated by the per^SLIH mutant (S45Y) that exhibits long periods, which based on the findings is likely due to reduced dPER-SLIMB interactions. Although changing S48 to Ala phenocopied the S47A mutation, the S48D mutation did not enhance binding to SLIMB, as was the case for S47D. Together with results showing that S48A did not modulate phosphorylation of S47, the data strongly suggest that S48 has a non-phosphorylation-dependent role as a crucial structural element. Mass spectrometry identified two phospho-residues in a dPER peptide from amino acids 40-48. Thus, there might only be two negatively charged residues in the major SLIMB-binding site on dPER. It is possible that the presence of a SSG tandem and a Tyr at position 50 can compensate for the lack of a third acidic residue normally found in β-TrCP-binding sites. It is also highly probable that other, yet to be identified, DBT-dependent phosphorylation sites besides those within the atypical SLIMB-binding site identified in this study contribute to enhancing SLIMB-dPER interactions (Chiu, 2008).

The presence of numerous suboptimal phospho-determinants is thought to generate a graded response in the binding efficiencies of F-box proteins to substrates. The general molecular framework is that progressive increases in the phospho-occupancy of multiple phosphorylated residues eventually reaches a threshold value that drives sufficient F-box protein/substrate interactions to yield desired outcomes. As such, regulating the kinetics of phosphorylation within the phospho-network mediating F-box recognition is a key determinant in the timing of substrate degradation. In the case of animal PER proteins, they undergo progressive increases in global phosphorylation that occur over an ~10-h time frame, whereby highly phosphorylated isoforms are associated with a rapid decline in levels (Chiu, 2008).

What accounts for the hours-long kinetics underlying the gradual increases in phosphorylation of S47 and likely many other DBT-dependent sites on dPER? Based on the in vitro ability of DBT to phosphorylate S47 despite phosphatase treatment of dPER, it is not believed that hierarchical phosphorylation based on prior priming is a major component in regulating the timing of when S47 is phosphorylated in vivo. Rather, the findings strongly suggest that the gradual build-up in the phospho-occupancy of S47 and other sites is at least partly based on a dynamic balance between DBT-mediated phosphorylation and the opposing activities of TIM and protein phosphatases. In agreement, blocking phosphatase activity strongly enhanced the abundance of phosphorylated S47. Recent evidence suggests that the ability of TIM to stabilize dPER might be by acting as a bridge that facilitates the targeting of protein phosphatase activity toward dPER (Fang, 2007). Indeed, it is likely that the strong protective function of TIM on dPER partially overrides the destabilizing effects of the S47D mutant as it attains peak levels comparable with those of wild-type dPER. Following this line of reasoning, it is suggested that a major reason for the advanced dper RNA and protein cycles in the S47D mutant is that as TIM levels decline in the late night the 'released' dPER(S47D) protein is no longer protected (or less so) and undergoes accelerated nuclear clearance, leading to an earlier disengagement from transcriptional repression, which advances the subsequent dper RNA and protein cycles. Likewise, while this manuscript was under review a recent report showed that the CKIepsilon tau mutation, which shortens rhythms in mice, has an 'asymmetrical' effect on PER protein stability, preferentially accelerating nuclear clearance and hence advancing the molecular oscillations underpinning the clockworks (Meng, 2008). Thus, although differential phosphorylation plays a major role in setting the intrinsic stabilites of PER proteins, additional variables, such as phase-specific protein-protein interactions, are critical in the 'readout' from these phospho-signals (Chiu, 2008).

Many of the DBT-dependent phosphorylation sites that were identified using mass spectrometry do not lie within optimal CKI sites, suggesting that inefficient phosphorylation by DBT might also contribute to the overall rate of progressive increases in dPER phosphorylation. It is also possible that the strong binding of DBT to the centrally located dPDBD, while increasing the local concentration of DBT, could function as a slow 'time-release capsule' whereby the disengagement of DBT is first required prior to phosphorylation of dPER residues at more distantly located sites (Chiu, 2008).

Although the phosphorylation requirements and in vivo significance of regions on mPER1 and mPER2 that interact with β-TrCP are not known, it is likely to also be based on noncanonical β-TrCP-binding sites. In addition, hyperphosphorylation of mammalian PER proteins requires a centrally located CKI-binding site. Therefore, mammalian PER proteins, especially mPER1 and mPER2, are likely to be targeted by β-TrCP to the 26S proteasome in a manner similar to that described here for dPER. This type of mechanism might also apply to other clock proteins such as Frequency (Frq) in Neurospora that undergoes daily changes in phosphorylation and stability that are remarkably similar to those observed for PER proteins. In addition, the phosphorylated state of FRQ is regulated by casein kinases, protein phosphatases, and the rapid degradation of highly phosphorylated isoforms is mediated by the F-box protein FWD1, a homolog of β-TrCP (Chiu, 2008).

An interesting feature of the distribution in phosphorylation sites on dPER that were identified using mass spectrometry is that they seem to concentrate in clusters, suggesting the presence of'phospho-modules' with different functions. Most of these clusters appear anchored by proline-directed phosphorylation sites, which are phosphorylated by endogenous kinases expressed in S2 cells. Of note, one such cluster is located in the dPER 'short domain' (T585-T610). Mutations in this region result in animals with short periods. In fact, the mutated residues of two classic per mutants that have short periods, per^S (S589N, 19-h period) and per^T (G593D, 16-h period), are right in the heart of this cluster. S589 is phosphorylated in a DBT-dependent manner; and G593, when mutated, may affect phosphorylation at nearby S589 and/or S596. Although the per^S mutants was isolated more than 35 years ago, the current results provide the first biochemical understanding for the short period phenotype, suggesting that phosphorylation events in the 'short domain,' some of which are DBT-dependent, may collaboratively function to slow down the clock. It is now becoming apparent that phosphorylation at different sites on PER proteins can result in differential effects on the pace of the clockworks, whereby some lead to faster clocks while others slow it down. The presence of phosphorylated residues with opposing outcomes on the speed of the clock can explain why mutations in CKIepsilon/delta/DBT can yield a variety of period-altering phenotypes from short to long, despite the fact that overall enzymatic activity is generally reduced (Chiu, 2008).

A rather unanticipated finding is that the majority of dPER phosphorylation is unrelated to direct effects on stability. This is supported by the lack of detectable SLIMB binding to a dPER fragment only missing the first 100 amino acids despite extensive phosphorylation as inferred from being the region underlying the majority of phosphorylation-dependent electrophoretic mobility shifts and confirmed by mass spectrometric analysis. Other lines of evidence also imply that a significant amount of multiphosphorylation is not linked to direct effects on PER stability. For example, abolishing phosphorylation at many centrally located sites on mPER3 does not attenuate CKI-mediated in vitro interactions with β-TrCP. Also, a trans-dominant version of CKII reduced global hyperphosphorylation of dPER without major effects on its levels (Chiu, 2008).

Thus, there are likely to be at least two functionally distinct DBT-dependent phosphorylation programs regulating different aspects of PER metabolism and activity: one that controls β-TrCP/SLIMB binding, and another that integrates with other kinases, such as CKII, to modulate nuclear entry/accumulation and/or ability to function as a transcriptional repressor. Indeed, mass spectrometric analysis of dPER identified numerous phosphorylation sites in a putative nuclear localization site and within the CCID mediating dPER inhibition of CLK-mediated transcription. Variants of dPER missing the major DBT docking site are hypophosphorylated and weak repressors. However, the relationship between hyperphosphorylation and repressor potency is not clear, since the DBT docking site on dPER also functions as a molecular scaffold for DBT and perhaps CKII-mediated inhibition of CLK-dependent transcription. Nonetheless, it is clear that the DBT docking site is a critical nexus for coordinating multiple phosphorylation programs. A challenge is to examine the functions of the newly identified phosphorylation sites and dissect the mechanisms by which they regulate dPER metabolism and activity (Chiu, 2008).

The SCF/Slimb ubiquitin ligase limits centrosome amplification through degradation of SAK/PLK4

Centrioles are essential for the formation of microtubule-derived structures, including cilia and centrosomes. Abnormalities in centrosome number and structure occur in many cancers and are associated with genomic instability. In most dividing animal cells, centriole formation is coordinated with DNA replication and is highly regulated such that only one daughter centriole forms close to each mother centriole. Centriole formation is triggered and dependent on a conserved kinase, SAK/PLK4. Downregulation and overexpression of SAK/PLK4 is associated with cancer in humans, mice, and flies. This study shows, in Drosophila cultured cells, that centrosome amplification is normally inhibited by degradation of SAK/PLK4, mediated by the SCF/Slimb ubiquitin ligase. This complex physically interacts with SAK/PLK4, and in its absence, SAK/PLK4 accumulates, leading to the striking formation of multiple daughter centrioles surrounding each mother. This interaction is mediated via a conserved Slimb binding motif in SAK/PLK4, mutations of which lead to centrosome amplification. This regulation is likely to be conserved, because knockout of the ortholog of Slimb, beta-Trcp1 in mice, also leads to centrosome amplification. Because the SCF/beta-Trcp complex plays an important role in cell-cycle progression, these results lead to new understanding of the control of centrosome number and how it may go awry in human disease (Cunha-Ferreira, 2009).

Substrates of the SCF/Slimb complex show a conserved degron that is recognized by Slimb. SAK/PLK4 protein has a modified Slimb recognition site in Drosophila (DSGIIT; position 293) and humans (DSGHAT; position 285). In previous studies, human SAK/PLK4 was shown to be ubiquitinated and removal of a region encompassing amino acids 272–311 led to stabilization of the human SAK/PLK4, although no analysis was performed on centrosome number. The conserved Slimb recognition site was mutated to DAGIIA in Drosophila SAK/PLK4, which is called SAK-ND (nondegradable). This mutation abolished SAK's ability to interact with Slimb and led to a decrease in SAK's ubiquitinated species. Moreover, SAK-ND is more stable in comparison to its WT counterpart. Thus, this mutation allowed testing of the biological significance of SAK/PLK4 degradation by the SCF/Slimb complex. Upon expression of the mutated SAK/PLK4 construct, a very similar phenotype was observed to the one registered after Slimb RNAi, i.e, rosette-like structures. These structures contained centriole percursors or elongating centrioles. Those results suggest a common centriole-amplification phenotype observed after Slimb RNAi and after expression of the SAK-ND mutant. The observation of at least five centrioles clustered at spindle poles in mitosis suggests that some of the supranumerary centrioles formed in one cycle after impairment of SAK/PLK4 degradation by the SCF/Slimb complex become proper microtubule-organizing centers. The formation of supranumerary centrosomes was quantified at the light microscope level. Upon transient low-level expression of the SAK-ND construct fused to GFP, a statistical significant increase in centrosome number was systematically observed in transfected cells compared to cells expressing low levels of the WT SAK fused to GFP. Indeed, expression of low levels of SAK-ND led to a 2-fold increase in centrosome amplification and after expression of a dominant-negative form of Slimb, suggesting that high levels of SAK/PLK4 underlie those phenotypes (Cunha-Ferreira, 2009).

Together, these results show that degradation of SAK/PLK4 by the SCF/Slimb complex is critical to restrict its function, preventing centrosome amplification. Although other mechanisms for regulation of SAK/PLK4 activity may exist, proteolytic degradation is likely to be conserved across species and be relevant in the animal. The Slimb-binding phosphodegron in SAK/PLK4 is conserved in vertebrates. Moreover, knockout of the ortholog of Slimb in mice, β-Trcp1, and both SkpA and Slimb Drosophila mutants show an increase in centrosome number, as do flies that overexpress SAK/PLK4 (Cunha-Ferreira, 2009).

It has been suggested that centriole overduplication is normally prevented by a 'licensing event,' the disengagement of centrioles that occurs at the exit of mitosis, required for the next duplication cycle. Requirement for this event ensures that centrioles duplicate only once in every cell cycle. However, it was recently shown that centriole amplification can also occur through the simultaneous formation of many daughters from a single mother, as it is after overexpression of SAK/PLK4 and SAS-6. These data suggest that proteolysis mediated by SCF/Slimb plays an important role in limiting the amount of SAK/PLK4, but not SAS-6, available to trigger multiple daughter formation. When SAK/PLK4 is not degraded, multiple daughter centrioles may be generated, which led to centrosome amplification. The regulation of centriole number thus emerges as a multistep mechanism, where proteolysis controls the activity of key players, SAK/PLK4 and SAS-6 (Cunha-Ferreira, 2009).

The mammalian F-box counterpart of Slimb, β-Trcp, plays a role in the DNA damage response by halting the cycle in response to genotoxic inputs. Misregulation of β-Trcp has been observed in cancer cells in which centrosome number is often increased after stress. It is thus possible that Slimb/β-Trcp coordinates checkpoints that monitor the status of DNA replication with centrosome number. The misregulation of this F-box protein would therefore result in changes in levels of SAK/PLK4, which are associated with mitotic abnormalities and oncogenesis. Given that other substrates of the Slimb/β-Trcp F-box protein require to be phosphorylated and the current results with the SAK-ND mutant, it is likely that SAK/PLK4 also requires such a modification at the serine and threonine residues in its DSGIIT degron to mark it for degradation. Future research on the identity and regulation of the kinase that phosphorylates SAK/PLK4 and on the localization of Slimb and SAK/PLK4 phosphorylated at the DSGIIT degron should indicate how different signaling events are coordinated in the cell to prevent centrosome amplification. These results open new avenues for understanding the mechanisms underlying centrosome misregulation that are of direct relevance to human disease (Cunha-Ferreira, 2009).

The Protein Phosphatase 2A regulatory subunit Twins stabilizes Plk4 to induce centriole amplification

Centriole duplication is a tightly regulated process that must occur only once per cell cycle; otherwise, supernumerary centrioles can induce aneuploidy and tumorigenesis. Plk4 (Polo-like kinase 4) activity initiates centriole duplication and is regulated by ubiquitin-mediated proteolysis. Throughout interphase, Plk4 autophosphorylation triggers its degradation, thus preventing centriole amplification. However, Plk4 activity is required during mitosis for proper centriole duplication, but the mechanism stabilizing mitotic Plk4 is unknown. This paper shows that PP2A [Protein Phosphatase 2A(Twins)] counteracts Plk4 autophosphorylation, thus stabilizing Plk4 and promoting centriole duplication. Like Plk4, the protein level of PP2A's regulatory subunit, Twins (Tws), peaks during mitosis and is required for centriole duplication. However, untimely Tws expression stabilizes Plk4 inappropriately, inducing centriole amplification. Paradoxically, expression of tumor-promoting simian virus 40 small tumor antigen (ST), a reported PP2A inhibitor, promotes centrosome amplification by an unknown mechanism. ST actually mimics Tws function in stabilizing Plk4 and inducing centriole amplification (Brownlee, 2011).

Plk4 protein is maintained at near-undetectable levels for the majority of the cell cycle by ubiquitin-mediated proteolysis. The ubiquitin ligase SCF^Slimb is responsible for Plk4 degradation and recognizes an extensively phosphorylated degron situated immediately downstream of the kinase domain (KD; ~50 amino acids containing the Slimb-binding domain)KD. Slimb is appropriately positioned on centrioles throughout the cell cycle to promote rapid Plk4 destruction, but centrioles are not required for its activity. In any case, Plk4 degradation is critical in blocking all pathways of centriole amplification. Unlike other Polo kinase members, Plk4 is a homodimer capable of autophosphorylating its downstream regulatory element (DRE), a serine-rich region surrounding its SBD, in trans to promote Slimb binding. Autoregulation is a conserved feature of Plk4. Moreover, a RNAi screen of the fly kinome suggests that no other kinase is required for Plk4 degradation. The continuous and efficient degradation of Plk4 indicates that Plk4 is immediately active when expressed and that control of Plk4’s protein level is key to regulating its activity (Brownlee, 2011).

However, surprisingly little is known about the converse event: how Plk4 is activated. The results reveal the existence of a previously unknown facet of the regulation of centriole duplication, a process which transiently stabilizes and activates Plk4 specifically during mitosis. Serine/threonine phosphatases were investigated as possible effectors to counteract Plk4 autophosphorylation. PP2A is an excellent candidate to fulfill this role as it has important functions in mitosis and localizes to mitotic centrioles in cultured fly cells and centrosomes in dividing Caenorhabditis elegans embryos. A previous study found that the number of γ-tubulin foci in mitotic S2 cells was diminished after PP2A RNAi, but whether this resulted from a bona fide loss of centrioles or instead reflects a requirement for PP2A for centrosome maturation was not determined. Subsequently, a role for PP2A in centrosome maturation was identified in a genome-wide RNAi screen. The current results indicate that PP2A and the regulatory subunit Tws are required for centriole duplication by dephosphorylating and stabilizing Plk4. Without PP2A^Tws, Plk4 cannot be stabilized, and centrioles fail to duplicate. PP2A is also required for centriole assembly in C. elegans embryos but functions downstream in the centriole assembly process (Kitagawa, 2011; Song, 2011). Although the catalytic and structural PP2A subunits are abundant, regulatory subunits are needed for intracellular targeting and recognition of a myriad of substrates. Tws overexpression is sufficient to stabilize Plk4 in a dose-dependent manner, causing centriole amplification and multipolar spindle formation. Like Plk4, Tws protein levels are low during interphase but rise and peak during mitosis. Accordingly, the results suggest that PP2A^Tws stabilizes mitotic Plk4 by counteracting Plk4 autophosphorylation, enabling cells to switch Plk4 activity (and thus centriole duplication) on and off. This mechanism is inherently highly sensitive to the presence of Tws, a rate-limiting component. Moreover, this is likely a conserved mechanism because overexpression of human Tws also stabilizes fly Plk4 in S2 cells. Clearly, an important goal for future studies is to establish whether the regulation of Tws levels and cell cycle control are linked. In addition, the results suggest that up-regulation of Tws could be a means to amplify centrioles in multiciliated cells and that increased Tws activity could be a condition found in cancerous cells (Brownlee, 2011).

Centrosome amplification is a hallmark of cancer and is also observed upon expression of DNA tumor virus proteins, which include SV40 ST, human papillomavirus E7, human T cell leukemia virus type-1 Tax, hepatitis B virus oncoprotein X, and human adenovirus E1A. However, mechanisms for centrosome amplification by viral oncoproteins are not known. SV40 ST has been found to directly bind the highly conserved Drosophila catalytic and structural PP2A subunits and to induce centrosome overduplication in cultured fly cells (Kotadia, 2008). Notably, ST is a well-established PP2A inhibitor and is known to bind structural PP2A subunits, forcing endogenous PP2A regulatory subunits to be displaced and inhibiting PP2A activity. However, the current results demonstrate that ST expression does not inhibit all PP2A activities but, instead, stimulates PP2A stabilization of Plk4. This represents the first evidence that ST mimics the function of a PP2A regulatory subunit in cells. It will be important to determine whether ST targets additional PP2A substrates during tumorigenesis and whether other tumorigenic viruses (e.g., human papillomavirus and hepatitis B) known to promote centrosome amplification exploit this same mechanism. Intriguingly, human papillomavirus E7 oncoprotein binds PP2A catalytic and structural subunits and prevents PP2A from dephosphorylating Akt. Although a previous study has suggested that PP2A may function as a tumor suppressor, these findings indicate that unregulated PP2A activity leads to centriole amplification and chromosomal instability and should therefore be considered as a potential oncogenic factor (Brownlee, 2011).

Cullin-3 controls Timeless oscillations in the Drosophila circadian clock

Eukaryotic circadian clocks rely on transcriptional feedback loops. In Drosophila, the Period and Timeless proteins accumulate during the night, inhibit the activity of the Clock (Clk)/Cycle (Cyc) transcriptional complex, and are degraded in the early morning. The control of Per and Tim oscillations largely depends on post-translational mechanisms. They involve both light-dependent and light-independent pathways that rely on the phosphorylation, ubiquitination, and proteasomal degradation of the clock proteins. Slmb, which is part of a CULLIN-1-based E3 ubiquitin ligase complex, is required for the circadian degradation of phosphorylated Per. This study shows that Cullin-3 (Cul-3) is required for the circadian control of Per and Tim oscillations. Expression of either Cul-3 RNAi or dominant negative forms of Cul-3 in the clock neurons alters locomotor behavior and dampens Per and Tim oscillations in light-dark cycles. In constant conditions, Cul-3 deregulation induces behavioral arrhythmicity and rapidly abolishes Tim cycling, with slower effects on Per. Cul-3 affects Tim accumulation more strongly in the absence of Per and forms protein complexes with hypo-phosphorylated Tim. In contrast, Slmb affects Tim more strongly in the presence of Per and preferentially associates with phosphorylated Tim. Cul-3 and Slmb show additive effects on Tim and Per, suggesting different roles for the two ubiquitination complexes on Per and Tim cycling. This work thus shows that Cul-3 is a new component of the Drosophila clock, which plays an important role in the control of Tim oscillations (Grima, 2012).

Hipk proteins dually regulate Wnt/Wingless signal transduction

The Wnt/Wingless (Wg) pathway is an evolutionarily conserved signaling system that is used reiteratively, both spatially and temporally, to control the development of multicellular animals. The stability of cytoplasmic β-catenin/Armadillo, the transcriptional effector of the pathway, is controlled by sequential N-terminal phosphorylation and ubiquitination that targets it for proteasome-mediated degradation. Orthologous members of the Homeodomain-interacting protein kinase family from Drosophila to vertebrates have been implicated in the regulation of Wnt/Wingless signaling. In Drosophila, as a consequence of Hipk activity, cells accumulate stabilized Armadillo that directs the expression of Wg-specific target genes. Hipk promotes the stabilization of Armadillo by inhibiting its ubiquitination (and hence subsequent degradation) by the SCF(Slimb) E3 ubiquitin ligase complex. Vertebrate Hipk2 impedes β-catenin ubiquitination to promote its stability and the Wnt signal in a mechanism that is functionally conserved. Moreover, this study describes that Hipk proteins have a role independent of their effect on β-catenin/Armadillo stability to enhance Wnt/Wingless signaling (Verheyen, 2012).

In summary, Hipk proteins play multiple important roles during developmental signaling events, and have the capacity to simultaneously impact the outcomes of both Wg and Hh signal transduction. The finding that Hipk proteins act by blocking Slimb/β-TrCP-mediated ubiquitination of substrates suggests that additional targets may also be affected through the action of these kinases. Additionally a nuclear role for Hipk was observed that is independent of its role in stabilizing the Wg pathway effector Arm. The data suggest that Hipk can enhance the transcriptional activation by the Arm/TCF complex. This role thought to be independent of its role in blocking substrate ubiquitination (Verheyen, 2012).

Multiple roles have been described for Hipk family members in regulation of Wnt/Wg pathway activity. Such findings are reminiscent of the work performed to decipher the roles of several other kinases in the Wnt pathway. Both GSK3 and CK1 have distinct roles at different steps in the transduction of Wnt signaling and act to either promote or inhibit pathway activity and as a result gene expression. The finding that Hipk-stabilized Arm is phosphorylated by GSK3 and CK1 reveals a specific point of action in the targeting and destruction of Arm/β- catenin. Future work should reveal whether the stabilized N-terminally phosphorylated Arm is associated with the destruction complex, in effect blocked from being transferred to the proteasome as a result of Hipk acting on Slimb. The regulation of β-catenin stability and activity has important implications for normal growth and patterning, tissue homeostasis and the development of cancer (Verheyen, 2012).

Regulation of Autophosphorylation Controls PLK4 Self-Destruction and Centriole Number

Polo-like kinase 4 (PLK4) is a major player in centriole biogenesis: in its absence centrioles fail to form, while in excess leads to centriole amplification. The SCF-Slimb/betaTrCP-E3 ubiquitin ligase controls PLK4 levels through recognition of a conserved phosphodegron. SCF-Slimb/betaTrCP substrate binding and targeting for degradation is normally regulated by phosphorylation cascades, controlling complex processes, such as circadian clocks and morphogenesis. This study shows that PLK4 is a suicide kinase, autophosphorylating in residues that are critical for SCF-Slimb/betaTrCP binding. A multisite trans-autophosphorylation mechanism, likely to ensure that both a threshold of PLK4 concentration is attained, is demonstrated, and a sequence of events is observed before PLK4 can autodestruct. It was shown that PLK4 trans-autophosphorylates other PLK4 molecules on both Ser293 and Thr297 within the degron and that these residues contribute differently for PLK4 degradation, the first being critical and the second maximizing auto-destruction. Second, PLK4 trans-autophosphorylates a phospho-cluster outside the degron, which regulates Thr297 phosphorylation, PLK4 degradation, and centriole number. Finally, the importance was shown of PLK4-Slimb/betaTrCP regulation as it operates in both soma and germline. As betaTrCP, PLK4, and centriole number are deregulated in several cancers, this work provides novel links between centriole number control and tumorigenesis (Cunha-Ferreira, 2013).

Polo-like Kinase 4 autodestructs by generating its Slimb-binding phosphodegron

Polo-like kinase 4 (Plk4) is a conserved master regulator of centriole assembly. Previous study has found that Drosophila Plk4 protein levels are actively suppressed during interphase. Degradation of interphase Plk4 prevents centriole overduplication and is mediated by the ubiquitin-ligase complex SCFSlimb/betaTrCP. Since Plk4 stability depends on its activity, the consequences were examined of inactivating Plk4 or perturbing its phosphorylation state within its Slimb-recognition motif (SRM). Plk3 was shown to be directly responsible for extensively autophosphorylating and for generating its Slimb-binding phosphodegron (the residues that direct the starting place of degradation). Phosphorylatable residues within this regulatory region were systematically mutated to determine their impact on Plk4 protein levels and centriole duplication when expressed in S2 cells. Notably, autophosphorylation of a single residue (Ser293) within the SRM is critical for Slimb binding and ubiquitination. These data also demonstrate that autophosphorylation of numerous residues flanking S293 collectively contribute to establishing a high-affinity binding site for SCFSlimb. Taken together, these findings suggest that Plk4 directly generates its own phosphodegron and can do so without the assistance of an additional kinase(s) (Klebba 2013).

The SCFSlimb E3 ligase complex regulates asymmetric division to inhibit neuroblast overgrowth

Drosophila larval brain neuroblasts divide asymmetrically to balance between self-renewal and differentiation. This study demonstrates that the SCFSlimb E3 ubiquitin ligase complex, which is composed of Cul1, SkpA, Roc1a and the F-box protein Supernumerary limbs (Slimb), inhibits ectopic neuroblast formation and regulates asymmetric division of neuroblasts. Hyperactivation of Akt leads to similar neuroblast overgrowth and defects in asymmetric division. Slimb associates with Akt in a protein complex, and SCFSlimb acts through SAK and Akt to inhibit neuroblast overgrowth. Moreover, Beta-transducin repeat containing, the human ortholog of Slimb, is frequently deleted in highly aggressive gliomas, suggesting a conserved tumor suppressor-like function (Li, 2014).

It has been shown that Akt associates with Slimb and can be ubiquitinated by Slimb. It was determined which domain of Akt is important for its association with Slimb. The central region of Akt, which contains its protein kinase domain (T2), but not the N-terminus (T1, containing a pleckstrin homology domain) or the C-terminus (T3, containing an AGC kinase domain), interacted with Slimb in S2 cells by co-immunoprecipitation. Taken together, these biochemical and genetic data suggest that the SCF^Slimb complex inhibits ectopic NB formation in part through an association with Akt (Li, 2014).

Beta-transducin repeat containing (BTRC/β-TrCP; human homologue of Slimb) showed a significant loss in 72.5% of glioma patients. Its copy number was an independent predictor of prognosis in a multivariate analysis. BTRC copy loss was observed in patients with glioblastoma (82%) and oligodendroglioma (68%). It was also observed in patients with mesenchymal and proliferative (85 and 88%, respectively), which are frequently associated with activated AKT signaling, a central oncogenic pathway regulating glioblastoma (GBM) growth and survival. The BTRC functional module was derived by mining mRNA expression databases and mapped to 544 mRNA transcripts. This module was able to stratify patients into two subgroups and was significantly associated with survival. A multivariate Cox Regression model confirmed that the BTRC functional subgroup was independently associated with survival. Furthermore, BTRC copy changes have a significant inverse correlation with the gene expression of AKT2 and PIK3CD (Li, 2014).

β-TRCP, the mammalian Slimb homolog, is speculated to play a greater role as an oncogene than as a tumor suppressor. This study has demonstrated that the SCF^Slimb complex plays an important role in NB self-renewal and asymmetric division. It acts at least in part through the oncogenic protein Akt, a key player that regulates NB self-renewal and asymmetric division. The activation of Akt in NBs appears to be independent of Phosphatase and tensin homologue (PTEN), a negative regulator of PI3K/Akt signaling, because no NB overgrowth were observed in two loss-of-function alleles, PTEN^C494 and PTEN¹. A recent report suggested a role for Target of rapamycin (TOR) signaling in restraining tumorigenesis in larval brains. PI3K/TOR also interacts genetically with Pins in the suppression of tumor growth in larval brains, suggesting the complex role of PI3K/Akt and the interconnected TOR pathway in NB homeostasis (Li, 2014).

Crumbs promotes expanded recognition and degradation by the SCFSlimb/beta-TrCP ubiquitin ligase

In epithelial tissues, growth control depends on the maintenance of proper architecture through apicobasal polarity and cell-cell contacts. The Hippo signaling pathway has been proposed to sense tissue architecture and cell density via an intimate coupling with the polarity and cell contact machineries. The apical polarity protein Crumbs (Crb) controls the activity of Yorkie (Yki)/Yes-activated protein, the progrowth target of the Hippo pathway core kinase cassette, both in flies and mammals. The apically localized Four-point-one, Ezrin, Radixin, Moesin domain protein Expanded (Ex) regulates Yki by promoting activation of the kinase cascade and by directly tethering Yki to the plasma membrane. Crb interacts with Ex and promotes its apical localization, thereby linking cell polarity with Hippo signaling. This study shows that, as well as repressing Yki by recruiting Ex to the apical membrane, Crb promotes phosphorylation-dependent ubiquitin-mediated degradation of Ex. Skp/Cullin/F-boxSlimb/beta-transducin repeats-containing protein (SCFSlimb/beta-TrCP) was identifed as the E3 ubiquitin ligase complex responsible for Ex degradation. Thus, Crb is part of a homeostatic mechanism that promotes Ex inhibition of Yki, but also limits Ex activity by inducing its degradation, allowing precise tuning of Yki function (Ribeiro, 2014).

Recent work in flies and mammals has implicated the apical polarity determinant Crb as a transmembrane receptor for Hpo signaling. Accordingly, clonal loss of crb function leads to Yki derepression and increased growth. However, the observation that Crb is required for Ex membrane localization apparently conflicts with the finding that Crb^intra overexpression reduces Ex levels and, like crb loss of function, leads to increased Yki activity. This study reconciles these findings by showing that Crb is not only required for Ex tethering at the apical membrane but also for promoting its degradation via the SCF^{Slimb/β-TrCP} E3 ubiquitin ligase. Indeed, immediately downstream of its FERM domain, Ex contains a sequence that conforms to the D/S/TSGφXS consensus sequence for canonical Slmb targets, which is conserved in Ex orthologs from arthropod species but absent from related FERM domain proteins such as Moe and Mer. In addition, loss of Slmb increases Ex levels in vivo, whereas Slmb depletion prevents Crb^intra-induced Ex degradation in cell culture. Thus, in ^crb mutants, Ex no longer reaches the apical membrane and is protected from degradation in the cytoplasm, where it accumulates but is presumably unable to repress Yki. When Crb^intra is overexpressed, Ex turnover at the membrane (or in an endocytic compartment if Ex degradation occurs after Crb internalization) is accelerated, leading to its depletion and consequent Yki activation. Therefore, in both cases, the outcome is Yki derepression, albeit for different reasons (Ribeiro, 2014).

How is the Slmb:Ex association regulated by Crb? Previous observations point to the involvement of a phosphorylation-dependent degradation mechanism, because Crb induces Ex phosphorylation. Accordingly, mutation of the phosphodegron (Ser⁴⁵³) or the putative priming site (Ser⁴⁶²) leads to loss of Slmb:Ex association and Ex stabilization. The most obvious candidate Ex kinase in this context is aPKC, which is known to interact with the Crb polarity complex and to phosphorylate Crb at its FBM. However, aPKC is thought to be recruited to the Crb complex via Par-6, which interacts with Crb through the PBM at the C terminus of the Crb intracellular domain. This is inconsistent with the fact that the PBM is dispensable for Crb to promote both Ex phosphorylation and degradation. Moreover, a dominant-negative version of aPKC is unable to rescue the overgrowth phenotype of Crb^intra overexpression in the wing. The Hpo downstream kinase Wts is another attractive candidate, because mammalian LATS1/2 promotes YAP phosphorylation on Ser³⁸¹, providing priming for phosphorylation by CK1δ/ε and triggering degradation by SCF^β-TrCP. However, neither Ser⁴⁶² nor Ser⁴⁵³ conform to the Wts/LATS consensus. The identity of the kinases therefore remains open (Ribeiro, 2014).

Regulation of tissue growth during development and adult life depends on the maintenance of tissue architecture, which, in turn, relies on cell-cell and cell-matrix interactions. Due to its intimate coupling to polarity and cytoskeletal regulators, the Hpo signaling pathway is thought to sense epithelial integrity and couple tissue architecture to growth control. In particular, YAP has been shown to sense contact inhibition in cell culture, such that its progrowth activity is silenced as cultured epithelial cells reach confluence. This is thought to depend on the assembly of tight junctions, leading to repression of YAP by the mammalian Crb complex. Recent work in flies and zebrafish has suggested that Crb can form homodimers in trans. This suggests that Crb mediates local cell-cell communication in epithelial tissues. Indeed, clonal loss of Crb leads to Crb depletion in the junctions of wild-type cells abutting the crb mutant tissue. This loss of Crb at the clone boundary is mirrored by loss of Ex, which is dependent on Crb for its apical localization. Thus, loss of Crb caused by loss of polarity or cell death can be transmitted to neighboring cells. This has been proposed as a means of cell- cell communication to induce regenerative proliferation through Yki activation. Indeed, genetic induction of epithelial wounds in imaginal discs has been shown to up-regulate Yki activity in cells neighboring the wound. In addition, Yki/ YAP is activated and promotes tissue regeneration upon injury in the vertebrate and fly intestine, as well as in the mouse liver (Ribeiro, 2014).

These findings suggest that Crb (and perhaps other polarity proteins) functions as a sensor of cell density and tissue integrity during development. In this model, disruption of Crb function would lead to Yki/YAP derepression, which, upon tissue injury, would allow regenerative growth to ensue. Another interesting question is whether liganded Crb behaves differently to unliganded Crb with respect to regulation of Ex stability. For example, it is possible that unliganded Crb promotes Ex turnover faster than its liganded counterpart, which might provide a sensitive means of responding to the status of neighboring cells. Further work will be needed to resolve this issue. The present work indicates that Crb fulfills a dual function in Hpo signaling, both recruiting Ex apically to repress Yki activity and promoting Ex turnover through phosphorylation and Slmb-dependent degradation. This mechanism could ensure constant turnover of Ex at the apical membrane, allowing Yki activity to rapidly respond to changing environmental conditions. This dynamic equilibrium could be particularly important to promote fast tissue regeneration upon injury (Ribeiro, 2014).

The F-box protein Slmb restricts the activity of aPKC to polarize epithelial cells

The Par-3/Par-6/aPKC complex is the primary determinant of apical polarity in epithelia across animal species, but how the activity of this complex is restricted to allow polarization of the basolateral domain is less well understood. In Drosophila, several multiprotein modules antagonize the Par complex through a variety of means. This study identified a new mechanism involving regulated protein degradation. Strong mutations in supernumerary limbs (slmb), which encodes the substrate adaptor of an SCF-class E3 ubiquitin ligase, cause dramatic loss of polarity in imaginal discs accompanied by tumorous proliferation defects. Slmb function is required to restrain apical aPKC activity in a manner that is independent of endolysosomal trafficking and parallel to the Scribble module of junctional scaffolding proteins. The involvement of the Slmb E3 ligase in epithelial polarity, specifically limiting Par complex activity to distinguish the basolateral domain, points to parallels with polarization of the C. elegans zygote (Skwarek, 2014).

This study extends the mechanisms involved in epithelial polarity to include a new function: targeted protein degradation. Targeted degradation can create spatial asymmetries in protein distributions, and there is precedent for roles of E3 ubiquitin ligases, including SCFSlmb, in polarizing different aspects of cells. The involvement of Slmb in Drosophila apicobasal polarity has gone unnoticed due to the previous use of hypomorphic alleles. The strong alleles described in this study display potent expansion of the apical pole of imaginal epithelia, demonstrating that Slmb is a new polarity regulator that functions to restrict the apical domain (Skwarek, 2014).

Loss of Slmb phenocopies the polarity defects associated with mutations in two classes of 'apical antagonists': the Scrib module of core polarity regulators, and endocytic regulators that control trafficking through the early endosome. Despite the similar polarity defects, slmb mutations do not alter endolysosomal cargo traffic, nor do they display protein recruitment defects characteristic of Scrib module mutants; furthermore, no genetic interactions are seen with either pathway. Nevertheless, the downstream consequences of polarity misregulation - including tumor-like transformation and the upregulation of specific target genes - are again shared between slmb and the other apical antagonists, and, moreover, slmb and Scrib module mutant cells share a distinctive trafficking defect associated with elevated aPKC activity. It is therefore suggestrf that Slmb acts in parallel to the Scrib module to antagonize the Par complex and other apical regulators (Skwarek, 2014).

The role for Slmb defined in this study points to the existence of an apical polarity-regulating protein substrate, the levels of which must be controlled. A number of validated Slmb substrates have been ruled out as the relevant target. Bioinformatic scans of Drosophila proteins for Slmb degron sequences suggest other candidates, including Expanded (Ex), but overexpression of Ex is not sufficient to induce polarity defects resembling those of slmb. Although a contribution from the elevation of multiple substrates cannot be ruled out, slmb-like polarity phenotypes can be induced by the elevated activity of individual proteins, including Crb or aPKC. Despite evidence that aPKC undergoes ubiquitin-mediated degradation in embryos, neither aPKC nor Crb levels appear to be controlled by Slmb-mediated degradation in imaginal discs. Nevertheless, the data together suggest that the substrate of Slmb in polarity regulation will function as a positive regulator of aPKC-driven outcomes (Skwarek, 2014).

The demonstration that Slmb limits aPKC activity to distinguish the epithelial basolateral domain reveals intriguing parallels to polarization of the worm zygote. In this context, Par-2 is the primary antagonist that restricts aPKC/Par activity, while Lgl homologs function in a parallel, redundant role. Par-2 contains a RING finger domain that is characteristic of single-subunit E3 ligases, but Par-2 homologs have not been identified outside of nematodes, Par-2 does not affect aPKC/Par levels, and a degraded substrate in polarity regulation has yet to be identified. The discovery of a Drosophila E3 ligase with a similar function to Par-2 raises the possibility of a conserved molecular logic to polarity in these two paradigmatic systems; determination of the relevant substrate will shed further light on this question (Skwarek, 2014).

Slmb antagonises the aPKC/Par-6 complex to control oocyte and epithelial polarity

The Drosophila anterior-posterior axis is specified when the posterior follicle cells signal to polarise the oocyte, leading to the anterior/lateral localisation of the Par-6/aPKC complex and the posterior recruitment of Par-1, which induces a microtubule reorganisation that localises bicoid and oskar mRNAs. This study shows that oocyte polarity requires Slmb, the substrate specificity subunit of the SCF E3 ubiquitin ligase that targets proteins for degradation. The Par-6/aPKC complex is ectopically localised to the posterior of slmb mutant oocytes, and Par-1 and oskar mRNA are mislocalised. Slmb appears to play a related role in epithelial follicle cells, as large slmb mutant clones disrupt epithelial organisation, whereas small clones show an expansion of the apical domain, with increased accumulation of apical polarity factors at the apical cortex. The levels of aPKC and Par-6 are significantly increased in slmb mutants, whereas Baz is slightly reduced. Thus, Slmb may induce the polarisation of the anterior-posterior axis of the oocyte by targeting the Par-6/aPKC complex for degradation at the oocyte posterior. Consistent with this, overexpression of the aPKC antagonist Lgl strongly rescues the polarity defects of slmb mutant germline clones. The role of Slmb in oocyte polarity raises an intriguing parallel with C. elegans axis formation, in which PAR-2 excludes the anterior PAR complex from the posterior cortex to induce polarity, but its function can be substituted by overexpressing Lgl (Morais-de-Sa, 2014).

Very little is known about how the posterior follicle cells signal to polarise the AP axis of the oocyte, except that signalling is disrupted when the germline is mutant for components of the exon junction complex, such as Mago nashi. The current results reveal that Slmb also plays an essential role in this pathway, where it acts to establish the complementary cortical domains of Baz/Par-6/aPKC and Par-1. Although Slmb might act in a variety of ways to establish this asymmetry, the observation that it regulates the levels of the Par-6/aPKC complex suggests a simple model in which Slmb directly or indirectly targets a component of the complex for degradation at the posterior of the oocyte. Since aPKC phosphorylates Par-1 to exclude the latter from the cortex, the degradation of aPKC would allow the posterior recruitment of Par-1, which would then maintain polarity by phosphorylating and antagonising Baz. Indeed, this might explain the observation that Par-6 is excluded from the posterior cortex before Baz. The polarisation of the oocyte therefore appears to occur in two phases. During the initiation phase, Slmb removes the Par-6/aPKC complex from the posterior cortex to allow the recruitment of Par-1. Par-1 then maintains and reinforces this asymmetry by phosphorylating Baz to exclude it from the posterior cortex, thereby removing the cortical anchor for the Par-6/aPKC complex (Morais-de-Sa, 2014).

Slmb is usually recruited to its targets by binding to phosphorylated residues that lie 9-14 amino acids downstream from the ubiquitylated lysine. Although both aPKC and Par-6 contain several sequences that could serve as atypical Slmb binding sites, neither contains a classic Slmb-dependent degron sequence. It is therefore unclear whether the SCFSlmb complex directly ubiquitylates either protein to target it for degradation or whether it targets another, unknown component of the complex that is required for the stability of Par-6 and aPKC. Nevertheless, this model leads to the prediction that the polarising signal from the follicle cells will induce the activation of a kinase that phosphorylates a Slmb substrate at the posterior of the oocyte, thereby triggering the local degradation of the Par-6/aPKC complex (Morais-de-Sa, 2014).

The demonstration that Slmb is required for the exclusion of the Par-6/aPKC complex from the posterior of the Drosophila oocyte raises interesting parallels with AP axis formation in C. elegans. Although Drosophila does not have an equivalent of the main symmetry-breaking step in the worm, in which a contraction of the actomyosin cortex removes the anterior PAR proteins from the posterior, the function of Slmb is analogous to that of PAR-2 in the alternative polarity induction pathway. Both proteins act to remove the Par-6/aPKC complex from the posterior cortex to allow the posterior recruitment of Par-1, which then reinforces polarity by excluding Baz/PAR-3 by phosphorylation. Furthermore, the polarity phenotypes of both slmb and par-2 mutants can be rescued by the overexpression of Lgl. Slmb and PAR-2 act by different mechanisms, since the former is a subunit of the SCF ubiquitin ligase complex and promotes the degradation of the Par-6/aPKC complex, whereas the latter functions by recruiting PAR-1. Nevertheless, it is intriguing that PAR-2 contains a RING finger domain that is typically found in ubiquitin ligases, suggesting that it might have lost this activity during evolution (Morais-de-Sa, 2014).

The Drosophila Bcl-2 family protein Debcl is targeted to the proteasome by the beta-TrCP homologue Slimb

The ubiquitin-proteasome system is one of the main proteolytic pathways. It inhibits apoptosis by degrading pro-apoptotic regulators, such as caspases or the tumor suppressor p53. However, it also stimulates cell death by degrading pro-survival regulators, including IAPs. In Drosophila, the control of apoptosis by Bcl-2 family members is poorly documented. Using a genetic modifier screen designed to identify regulators of mammalian bax-induced apoptosis in Drosophila, this study identified the ubiquitin activating enzyme Uba1 as a suppressor of bax-induced cell death. Uba1 was demonstrated to regulate apoptosis induced by Debcl, the only counterpart of Bax in Drosophila. Furthermore, these apoptotic processes were shown to involve the same multimeric E3 ligase-an SCF complex consisting of three common subunits and a substrate-recognition variable subunit identified in these processes as the Slimb F-box protein. Thus, Drosophila Slimb, the homologue of beta-TrCP targets Bax and Debcl to the proteasome. These new results shed light on a new aspect of the regulation of apoptosis in fruitfly that identifies the first regulation of a Drosophila member of the Bcl-2 family (Colin, 2014).

This paper reports the regulation of bax- and debcl-induced apoptosis by the ubiquitin-proteasome pathway. The stimulation of this pathway by overexpressing Uba1, which encodes the ubiquitin activating enzyme, leads to an almost complete loss of bax-induced cell death. This regulation seems conserved through evolution, as debcl-induced apoptosis is also regulated in this way. However, since Bax seems to necessitate Debcl in order to kill Drosophila eye cells, one could wonder whether suppression of Bax-induced cell death depends on the direct effect of Uba1 on Debcl. Nevertheless, since both Bax and Debcl proteins are degraded when Uba1 is overexpressed, this seems unlikely unless Debcl stabilizes Bax (Colin, 2014).

Since Buffy inhibits autophagy in response to starvation, it is hypothesized that Debcl induces an autophagic cell death. Autophagy was monitored by using a UAS-Atg8-GFP transgene. It was found that actually no autophagy could be detected upon Debcl expression. Uba1 has been shown in the literature to be required for autophagy and reduction of cell size in the intestine. This study shows that Debcl-induced cell death in the wing disc is not only suppressed by Uba1 but also by proteasome mutants. These data suggest that the UPS pathway is the main proteolytic pathway involved in the suppression of Bax and Debcl-induced apoptosis by Uba1. However, given that slmb has been shown to regulate Wg and Dpp pathways, it cannot be excluded that these pathways are partially involved in phenotypic suppression (Colin, 2014).

Studies of the proteasome-dependent regulation of members of the Bcl-2 family in mammals have only rarely led to the identification of the specific E3 ligases. This study identified an SCFSlmb complex as the E3 ubiquitin ligase that regulates the Debcl pathway and may target it to proteasomal degradation. It would be interesting to determine whether the mammalian homologue of Slmb, β-TrCP, targets Bax to the proteasome in mammalian cells (Colin, 2014).

This study has show Debcl is a target of the UPS, thus finding a new regulation of apoptosis that differs from the control of Dronc, Drice and RHG protein levels by Diap1. Indeed, Diap1 is a key enzyme that decides of cell fate by degrading either pro-apoptotic regulators or itself, leading to either cell survival or apoptosis. Since Diap1 levels are downregulated by the F-box protein Morgue in presence of Rpr or Grim, it could be hypothesized that the Morgue/Diap1 pathway is involved in bax- and debcl-induced apoptosis regulation. This does not seem to be the case because a hypomorphic allele of morgue did not increase bax- and debcl-induced apoptosis. Furthermore, overexpression of diap1 does not inhibit bax- and debcl-induced apoptosis. Thus, the proteasome-dependent regulation that this study has identified is independent of Diap1 and differs from the Morgue/Diap1 regulation of cell death. The existence of different UPS-modulated cell death pathways is also supported by the reported absence of genetic interaction between RHG pathway components and Debcl (Colin, 2014).

The results indicate an anti-apoptotic role of Uba1 in the wing tissue. However, two other studies revealed a pro-apoptotic role of Uba1 in the eye; strong Uba1 loss-of-function alleles lead to apoptosis and compensatory proliferation in the developing eye. As previously shown in other systems, these processes seem to involve the RHG/Diap1/Dronc pathway. Hypomorphic alleles of Uba1 have shown opposite effects as they suppressed hid- or grim-induced apoptosis in the eye. These results are consistent with previous data indicating that Uba1 overexpression, using the Uba1 EP2375 allele, increases RHG-induced apoptosis. In principle, this apparent contradiction may result from either the cell death signal or the studied tissue. The use of a GMR-Gal4 driver shows that Uba1 overexpression also inhibits debcl-induced apoptosis in the eye tissue, which suggests that the Uba1 effect is specific of debcl-induced apoptosis. In contrast, rpr-induced cell death is enhanced by Uba1 overexpression in the eye, whereas it is suppressed by Uba1 in the wing. These results suggest that Debcl could be involved in rpr-induced cell death in the wing but not in the eye. RHG proteins are known to mediate their pro-apoptotic function by stimulating Diap1 degradation by the UPS while Debcl is a direct target of ubiquitination. Therefore, RHG-induced degradation of Diap1 through its ubiquitination by Morgue could explain the pro-apoptotic role of Uba1 in the eye whereas the anti-apoptotic Uba1 function mediated by Slmb in Debcl-induced cell death would rely on Debcl degradation. By showing that different pro-apoptotic pathways are regulated by the UPS in Drosophila, this work suggests that the tissue-dependent effect of the pleiotropic enzyme Uba1 must result from a change in the balance between UPS pro- and anti-apoptotic effects. It is proposed that this change relies on the availability in E3 enzymes, the incoming signals and relative amounts of pro- and anti-apoptotic regulators of cell fate (Colin, 2014).

Protection of Armadillo/beta-Catenin by Armless, a novel positive regulator of Wingless signaling

The Wingless (Wg/Wnt) signaling pathway is essential for metazoan development, where it is central to tissue growth and cellular differentiation. Deregulated Wg pathway activation underlies severe developmental abnormalities, as well as carcinogenesis. Armadillo/β-Catenin plays a key role in the Wg transduction cascade; its cytoplasmic and nuclear levels directly determine the output activity of Wg signaling and are thus tightly controlled. In all current models, once Arm is targeted for degradation by the Arm/β-Catenin destruction complex, its fate is viewed as set. This study identified a novel Wg/Wnt pathway component, Armless (Als; CG5469) that is required for Wg target gene expression in a cell-autonomous manner. Genetic and biochemical analyses showed that Als functions downstream of the destruction complex, at the level of the SCF/Slimb/βTRCP E3 Ub ligase. In the absence of Als, Arm levels are severely reduced. Biochemical and in vivo studies showed that Als interacts directly with Ter94, an AAA ATPase known to associate with E3 ligases and to drive protein turnover. It is suggested that Als antagonizes Ter94's positive effect on E3 ligase function, and it is proposed that Als promotes Wg signaling by rescuing Arm from proteolytic degradation, spotlighting an unexpected step where the Wg pathway signal is modulated (Reim, 2014).

The wingless (wg) gene was found nearly forty years ago with the characterization of a Drosophila mutant without wings. The gene encodes a secreted glycoprotein, the founding member of the Wnt family of signaling proteins. In the decades following its discovery, Wg/Wnt signaling has been shown to be essential during embryogenesis. Indeed, it is important throughout an organism's life, controlling also the homeostasis of different organs, for example, regeneration of epithelial cells in the intestine - the aberrant behavior of these cells in cancer is caused by constitutive Wg/Wnt signaling, which is consequently a key focus of medical and translational research (Reim, 2014).

The relay of the Wg signal is controlled at different levels. However, the pivotal step is the regulation of the levels of Armadillo (Arm)/β-Catenin, the key transducer of the Wg/Wnt pathway. A multiprotein complex consisting of the scaffold proteins Axin and APC and the kinases Shaggy/GSK3β and Casein kinase I (CKI) recruits and phosphorylates Arm/β-Catenin. This marks Arm/β-Catenin for ubiquitination by the SCF/Slimb/βTRCP E3 ubiquitin ligase and subsequent degradation by the ubiquitin-proteasome system (UPS). When Wg/Wnt binds its receptors at the cell membrane, degradation of Arm/β-Catenin is prevented, presumably by protein interactions that lead to the dissociation of the E3 ubiquitin ligase from the Arm/β-Catenin destruction complex. As a consequence, Arm/β-Catenin translocates into the nucleus, where it adopts its role as a transcriptional effector of Wg/Wnt signaling. Although this step is crucial, and is a potential point of regulation, little is known about the players involved in the processing of Arm/β-Catenin and its ultimate degradation (Reim, 2014).

In a genome-wide RNA interference (RNAi) screen Armless (Als) was isolated as a regulator of proximodistal growth of Drosophila limbs, and has been shown in subsequent analyses to exert its function in the Wg pathway. Detailed genetic studies demonstrate that Als acts downstream of the destruction complex, at the level of the SCF/Slimb/βTRCP E3 Ub ligase. Cells depleted for Als exhibit strongly reduced Arm protein levels. Importantly, the activity of a constitutively active form of Arm, which cannot be phosphorylated and hence escapes ubiquitination and proteasomal degradation, is insensitive to depletion of Als. Using immunopurification and mass spectrometry analysis this study found that Ter94 interacts with Als. Ter94 is an AAA ATPase associated with protein turnover and proteasomal degradation. In sum, these data suggest that Als acts downstream of the Arm/β-Catenin destruction complex to positively regulate Arm protein levels, possibly by rescuing Arm from ubiquitination via Slimb. The human ortholog of Als, UBXN6, can substitute for Als in Drosophila, and Wnt target gene expression was impaired upon knock-down of UBXN6 in HEK-293 cells. It is thus infered that Als and UBXN6 represent regulators of a conserved mechanism that ensures appropriate levels of Armadillo/β-Catenin by antagonizing its entry into the UPS (Reim, 2014).

A prevalent mechanism for controlling information flow in signaling pathways is the alteration of the protein levels of key components. In the Wg/Wnt pathway, the Arm/β-Catenin destruction complex targets Arm/β-Catenin for ubiquitination by the SCF/Slimb/βTRCP E3 Ub ligase, resulting in proteasomal degradation and low cytoplasmic levels of Arm/β-Catenin in the Wnt pathway off state. If the pathway is turned on, Slimb-mediated ubiquitination is prevented, thus rescuing Arm from its proteasomal fate and causing a concomitant increase in Arm protein levels. This study describes Als as a new component of this control system; Als was found to be required to prevent the degradation of Arm/β-Catenin (Reim, 2014).

This study has identified als in a genome-wide in vivo RNAi screen in Drosophila. Because no EMS- or P-element-induced null allele was isolated, and because another gene overlaps with als, particularly thorough evidence validating als gene function was obtained. (1) The als phenotypes could be reproduced by nine different UAS-RNAi transgenes encoding independent RNA target sites. Together with an extended off target analysis, unintentional RNAi was ruled out as a cause for the als phenotypes. (2) RNAi-mediated inhibition of als expression was ascertained by monitoring als mRNA expression via real-time PCR and antisense mRNA in situ hybridization. (3) Expression of Als with different RNAi-insensitive rescue transgenes, as well as with its human ortholog UBXN6, rescued als phenotypes (Reim, 2014).

These analyses show that als encodes an essential positive Wg signaling component. This conclusion is based on the following evidence. als depletion caused wings with notched wing margins and loss of sensory bristles, which is characteristic of impaired Wg signaling. The distal wing region is most sensitive to als levels, as is the case for other positive components of Wg signaling. In agreement with this, increased als expression was found in the central wing pouch, at least in earlier L3 larval stages. Stimulation of the Wg pathway in wing imaginal discs or Kc-167 cells caused higher als expression, suggesting that als can be positively controlled by Wg signaling. However, Als levels must be precisely controlled since already mild overexpression of UAS-als elicits a dominant-negative effect on Wg signaling. The function of als for Wg signaling is not restricted to the wing: also in other tissues, such as the thorax, eyes, legs, and the embryo, als phenotypes are identical to those seen when Wg signaling is disturbed. Also in human HEK-293 cells UBXN6/UBXD1, the ortholog of Als, was found to be required for Wnt signaling, and human UBXN6 largely rescues the als phenotypes in Drosophila, which suggests their functional conservation. Depletion of als also enhanced Wg-sensitized phenotypes, further supporting the notion that its product is a Wg pathway component. Moreover, the expression of positively regulated Wg target genes is reduced or abolished upon loss of als function, while Wg-repressed target gene expression is ectopically activated. Importantly, while interfering with als function suppressed Wg signaling, it did not affect other pathways, such as Notch and Hh, Jak/Stat, or EGFR signaling. However, it cannot be ruled out that als is not required in another pathway in a different biological context. In humans, UBXN6 is reported to play a role in diverse scenarios: for example, it was shown to play a role in Caveolin turnover in human osteosarcoma U2OS cells. This might indicate a broader role of UBXN6 in mammalians (Reim, 2014).

The data show that Als regulates Armadillo protein levels. Based on epistasis experiments, Als acts downstream of Shaggy/GSK3β and upstream of the SCF/Slimb/βTRCP E3 Ub ligase, which is known to ubiquitinate Arm, a prerequisite for proteasomal degradation. Consistent with this, the degradation-resistant form of Arm could completely bypass the requirement for als, in contrast to the wild-type form of Arm. This suggests that proteasomal degradation acts downstream of als; however, this cannot be taken as an unambiguous proof. Importantly, depletion of ubiquitin and overexpression of CSN6, a negative regulator of SCF/Slimb/βTRCP E3 Ub ligase, could ameliorate the als phenotype (as well as phenotypes based on the overexpression of Axin or Shaggy, which overactivate the destruction complex, thus resulting in enhanced Arm degradation). In contrast, altering these factors did not ameliorate the Lgs phenotype, which is caused by interfering more downstream in the Wg pathway. These findings suggest that als works upstream of proteasomal degradation. A further informative experiment was monitoring Wg pathway components with respect to protein levels: Arr, Fz, Axin, APC, Sgg, and Arm. The only change in the absence of Als function was Arm: its levels were strongly reduced upon als depletion. The effects on Arm levels could be due either to a direct effect on Arm or to an indirect effect on a negative component. Importantly, the rate-limiting factor Axin as well as other key negative components of the Arm/β-Catenin destruction complex were unaltered uponals depletion (Reim, 2014).

Some further mechanistic insight was obtained with the finding that Ter94 interacts in vitro and in vivo with Als. Interestingly, Als-Ter94 was found to localize at the cell cortex, as was similarly observed for the Arm/β-Catenin destruction complex. The studies are consistent with earlier work that showed that the human ortholog of Ter94, p97, interacts with UBXN6. Ter94/p97/Cdc48 is a conserved and highly abundant AAA ATPase that was found to associate with SCF/Slimb/βTRCP E3 Ub ligases or proteasomal shuttle factors to mediate UPS-mediated protein degradation. Specifications of the diverse activities of Ter94/p97 and the fate of its substrates are mainly exerted by UBX domain protein co-factors, which eventually either promote or hinder p97's function in protein turnover; an example of the latter involves the dissociation of the SCF/Slimb/βTRCP E3 Ub ligase complex, eventually leading to its inactivation. Interestingly, it was recently reported that inactivation of the E3 ligase complex upon Wnt signaling is achieved by its dissociation from the destruction complex. Based on the current experiments and what is known about Ter94/p97, a possible mechanism is suggested that Als antagonizes Ter94's positive effect on E3 ligase function, thereby rescuing Arm levels. No increased protein levels were observed of Slimb, Axin, Shaggy, or APC in this analyses; thus, the results favor a model in which Als antagonizes Ter94 to hinder the transfer of Arm to the proteasome by interfering with the SCF/Slimb/βTRCP E3 Ub ligase function or its assembly. Importantly, no interaction was found between Arm and Als. This is consistent with the finding that UBX domain family members lacking an UBA domain, such as UBXN6/Als, do not directly interact with substrate proteins, but are necessary for the activity or fate of the Ter94/p97 (Reim, 2014).

Interestingly, another study found that ter94 depletion affected the partial proteolysis of Ci. However, that study observed neither any typical consequence of disturbed Hh signaling per se (i.e., no alteration of Hh target gene expression in genes such as ptc) nor any phenotypical consequence upon overexpression of a dominant negative form of Ter94 (i.e., aberrant wing patterning and growth typical for Hh signaling). This is consistent with the current data that neither Ci target expression nor Hh signaling was affected upon als or ter94 depletion (Reim, 2014).

p97/Ter94 is known as a highly pleiotropic AAA ATPase associated with many cellular functions. Further, p97/Ter94 acts in multifaceted and large protein–protein complexes, and it is its regulatory co-factors, including UBX domain proteins, that render p97/Ter94 specific for a certain task in a particular cellular context. For example, p47/Shp1 is a co-factor of p97/Ter94 that blocks other co-factors from Ter94 binding. Interestingly, in Kc-167 cell mass spectroscopy experiments, this study found p47 in Ter94/Als protein complexes, but only in the absence of Wg stimulation. On the other hand, als transcript and Als protein levels were elevated upon Wg signaling. These findings suggest a dynamic regulation of the Ter94 complex upon signaling inputs. The identification and functional analysis of all key components of the Als-Ter94 complex will be needed to obtain a refined insight into Als-Ter94's molecular mechanism (Reim, 2014).

Critically, this work spotlights an underappreciated facet in the control of the output of the entire canonical Wg/Wnt pathway - how Arm/β-Catenin is handed over to the proteasome— and the potential for regulating this step; this works also indicates that this step, in contrast to the conventional wisdom, is tunable. The identification and characterization of the UBX protein Als as a positive regulator of Wg/Wnt signaling contributes to this layer of pathway control (Reim, 2014).

Ter94 ATPase complex targets k11-linked ubiquitinated Ci to proteasomes for partial degradation

The Cubitus interruptus (Ci)/Gli family of transcription factors can be degraded either completely or partially from a full-length form [Ci155/Gli^FL] to a truncated repressor (Ci75/Gli(R)) by proteasomes to mediate Hedgehog (Hh) signaling. The mechanism by which proteasomes distinguish ubiquitinated Ci/Gli to carry out complete versus partial degradation is not known. This study shows that Ter94 ATPase and its mammalian counterpart, p97, are involved in processing Ci and Gli3 into Ci75 and Gli3^R, respectively. Ter94 regulates the partial degradation of ubiquitinated Ci by Cul1-Slimb-based E3 ligase through its adaptors Ufd1-like and dNpl4. Cul1-Slimb-based E3 ligase, but not Cul3-Rdx-based E3 ligase, modifies Ci by efficient addition of K11-linked ubiquitin chains. Ter94^{Ufd1-like/dNpl4} complex interacts directly with Cul1-Slimb, and, intriguingly, it prefers K11-linked ubiquitinated Ci. Thus, Ter94 ATPase and K11-linked ubiquitination in Ci contribute to the selectivity by proteasomes for partial degradation (Zhang, 2013).

Hh signaling plays important roles in metazoan development, and its malfunction is implicated in numerous human congenital disorders and cancers. Secreted Hh proteins bind Patched (Ptc)-iHog coreceptors to relieve an inhibitory effect of Ptc on Smoothened (Smo), which leads to activation of the Ci/Gli family of zinc finger transcription factors. Biochemical and genetic studies in Drosophila have revealed several important steps in the regulation of Ci/Gli activity. In the absence of Hh, full-length Ci, Ci155, is sequentially phosphorylated by protein kinase A (PKA), glycogen synthase kinase 3 (GSK3), and casein kinase I (CKI) and then ubiquitinated by Cullin 1 (Cul1)-Supernumerary limbs (Slimb, known also as β-TrCP)-based E3 ligase. This results in partial degradation by proteasomes, leaving the N terminus of Ci intact (Ci75) as a transcriptional repressor. In the presence of Hh, unphosphorylated Ci155 accumulates and enters nucleus to activate Hh target genes. As a feedback control of the pathway, active Ci155 induces the expression of roadkill (rdx)/Hib to form Cul3-Rdx-based E3 ligase and promotes complete proteasomal degradation of Ci155 (Zhang, 2013).

Although it is well established that Ci is ubiquitinated by Cul1-Slimb and Cul3-Rdx-based E3 ligases under different conditions, it remains unknown how proteasomes distinguish ubiquitinated Ci for partial versus complete degradation. As ubiquitinated proteins are transferred to proteasomes by different pathways, it is hypothesized that some specific components are involved in the recruitment of ubiquitinated Ci for partial degradation. Transitional elements of the endoplasmic reticulum 94 kDa (Ter94) was identified as the Drosophila homolog of yeast CDC48, which is a member of the ATPase associated with various cellular activities (AAA) family. In mammals, the CDC48/Ter94 homolog p97 (also known as VCP) mainly functions in endoplasmic reticulum-associated degradation (ERAD). Proteomic analysis revealed that p97 might play a broad role in regulating the turnover of ubiquitin proteasome system (UPS) substrates. This study has shown that Ter94 is a component of the Ci processing machinery and is critical for Ci75 formation (Zhang, 2013).

The control of partial versus complete proteasomal degradation of Ci and Gli3 is a major regulatory step in Hh signal transduction. How proteasomes distinguish ubiquitinated Ci to carry out either partial or complete degradation is not known. Based on the current findings, the following model is proposed. In the absence of Hh, Ci155 is phosphorylated and ubiquitinated by Cul1-Slimb-based E3 ligase to generate Ci75. In this process, K11-linked ubiquitin chains are added onto Ci155. Ter94Ufd1-like/dNpl4 forms a complex with Cul1-Slimb-based E3 ligase through Ufd1-like and Roc1a, and another component dNpl4 is bound to the K11-linked ubiquitin chains on Ci155. Through ATP hydrolysis, Ter94Ufd1-like/dNpl4 facilitates the delivery of ubiquitinated Ci155 to the proteasomes for processing (Zhang, 2013).

Besides Ci and Gli3, the best example of partial degradation is the processing of human nuclear factor-κB (NF-κB) and its yeast homologs, SPT23 and MGA2. Previous studies have suggested that some common features of processing determinant domain (PDD) are involved in the partial proteasomal degradation of Ci and NF-κB. However, Ci also undergoes complete proteasomal degradation by Cul3-Rdx-based E3 ligase. Why such 'degradation stop signals' fail to work in such instances and how proteasomes make the decision for partial or complete degradation are unknown. Based on the current results and previous studies, it is proposed that Ter94/p97 complex may specifically target 'partial-degradation-proteins' to proteasomes through K11-linked ubiquitin chains. Further investigation is needed to provide direct evidence to support this hypothesis. As many similarities are shared between Ci and NF-κB precursors in partial degradation, it will be interesting to test whether p97 and K11-linked ubiquitination are also involved in the partial degradation and/or maturation of p100 in NF-κB signaling (Zhang, 2013).

This study found that K11-linked chains are added onto Ci by Cul1-Slimb-based E3 ligase in the absence of Hh pathway activity, whereas Cul3-Rdx-based E3 ligase mainly adds K48-linked chains on Ci when the pathway is active. This illustrates a phenomenon that the same protein can be modified with different types of ubiquitin chains by distinct E3 ligases. Although K11-linked chains added on APC substrates lead to complete degradation, the data demonstrate that K11-linked chains are involved in the partial degradation of Ci. These findings also raise the interesting possibility that the topology of ubiquitin chains may be recognized as a selective signal for proteasomal degradation. As mixed or heterologous ubiquitin chains may exist, further investigation is essential to determine whether mixed ubiquitin chains are formed by Cul1-Slimb-based E3 ligase on Ci (Zhang, 2013).

DEVELOPMENTAL BIOLOGY / EFFECTS OF MUTATION

In the Drosophila leg disc, wingless and decapentaplegic are expressed in a ventral-anterior and a dorsal-anterior stripe of cells, respectively. This pattern of expression is essential for proper limb development. While the hedgehog (Hh) pathway regulates dpp and wg expression in the anterior-posterior (A/P) axis, mechanisms specifying their expression in the dorsal-ventral (D/V) axis are not well understood. Evidence is presented that slimb mutant clones in the disc deregulate wg and dpp expression in the D/V axis. This suggests for the first time that their expression in the D/V axis is actively regulated during imaginal disc development. Furthermore, slimb is unique in that it also deregulates wg and dpp in the A/P axis. The misexpression phenotypes of slimb- clones indicate that the regulation of wg and dpp expression is coordinated in both axes, and that slimb plays an essential role in integrating A/P and D/V signals for proper patterning during development. Genetic analysis further reveals that slimb intersects the A/P pathway upstream of smoothened (smo) (Theodosiou, 1998).

slimb was identified in a mutant screen. To identify recessive overproliferation mutations in genes that are lethal in homozygous mutant animals, genetic screens were performed in mosaic flies containing homozygous mutant patches in otherwise wild-type backgrounds. Two classes of recessive overproliferation mutations have been identified. Mutations of the first group cause mutant cells to undergo extensive proliferation and form unpatterned, tumorous outgrowths in mosaic adults. Mutations of the second group induce both patterned and irregular outgrowths. slimb affects developmental signals that regulate cell proliferation and pattern organization. The slimb transcript encodes a Cdc4-related protein containing F-box and WD-40 motifs. Jiang (1998) has independently reported the identification of this gene. Using a Drosophila slimb cDNA, a human homolog (H-slimb) has been isolated. The fly and human proteins share 78% amino acid identity throughout, suggesting that slimb is functionally conserved (Theodosiou, 1998).

slimb-induced outgrowths are reminiscent of the phenotypes caused by misexpression of dpp and wg. dpp and wg expression were examined in slimb mosaic leg discs using wg-lacZ and dpp-lacZ reporter genes. slimb clones ectopically express both wg and dpp in a cell-autonomous fashion. slimb mutant clones deregulate wg and dpp in both D/V and A/P axes. Ectopic wg expression is observed in both ventral and dorsal regions. Similar results are also observed for dpp. In slimb mutant clones situated within or near the endogenous dpp expression zone, dpp is expressed in the mutant cells but down-regulated in adjacent wild-type cells. Previously it had been shown that Wg and Dpp signaling mutually antagonize each other's expression, which prevents expression of the two molecules in the same cells. Ectopic expression of both wg and dpp in slimb- clones in the dorsal-anterior of the leg disc indicates a disruption of this mutual antagonism. To test whether ectopic wg and dpp expression are responsible for the outgrowth phenotype in slimb mosaic animals, flies were generated carrying clones of cells mutant for both slimb and wg, or slimb and dpp. In comparison to slimb mutant clones, double mutant clones do not cause any significant outgrowths. Therefore, Wg and Dpp are two primary effector molecules responsible for the induction of outgrowths in slimb mosaic animals. These results are consistent with previous observations that wg and dpp are both required for defining the proximodistal outgrowth center (Theodosiou, 1998).

The slimb phenotype differs from those of all previously known genes, in that it is the first gene found to deregulate both wg and dpp expression in the D/V axis. Disrupting components of the Hh signaling pathway deregulate wg and dpp only along the A/P axis. Thus, the control of wg and dpp expression in the D/V axis is not disrupted by disruption of the Hh pathway. The mechanism restricting wg and dpp in the D/V axis is not known. The mutant phenotype of slimb- clones in discs provides the first evidence that wg and dpp expression in the D/V axis is actively regulated during imaginal disc development, and is not solely defined during embryonic development. Since the Hh pathway regulates wg and dpp expression in the A/P axis, these results suggest that a pathway different from Hh may operate in imaginal discs to restrict their expression in the D/V axis. This pathway cannot be either the Wg or Dpp signaling pathway since inactivation of Wg or Dpp signaling is known to affect either dpp or wg expression, but not both. The slimb phenotypes described here were not observed in the previous study which used weak slimb alleles and revealed only A/P defects (Jiang, 1998). Jiang proposed that Slimb protein normally targets Ci and Arm for processing or degradation by the ubiquitin/proteasome pathway, and that Hh and Wg regulate gene expression, at least in part, by inducing changes in Ci and Arm, which protect both Ci and Arm from Slimb-mediated proteolysis. The phenotypic differences probably reflect the fact that a null allele was used in the current study instead of hypermorphic alleles. In addition to D/V defects, slimb mutant clones also deregulate wg and dpp expression in the A/P axis. slimb is the first identified gene that regulates both wg and dpp expression in the A/P as well as D/V axes (Theodosiou, 1998).

To further explore how slimb regulation and function correlates with A/P signaling, double mutant analysis was carried out with slimb mutants and with mutants of hh and smo. No reduction of outgrowths was observed in slimb-, hh- double mutant clones. Furthermore, slimb mutant clones have no effect on hh expression. This indicates that slimb acts downstream or independent of Hh signaling. In contrast, slimb-, smo- double mutant clones almost completely suppress slimb induced outgrowths. Consistent with the adult phenotype, discs carrying slimb- , smo- clones fail to ectopically express either dpp or wg. These data suggest that slimb intersects the A/P signal upstream of smo. Jiang (1998) suggested that slimb acts downstream of smo. This difference may be explained by the use of different alleles for smo and slimb. The Slimb product contains WD-40 repeats believed to act as a scaffold for the binding of multiple proteins. It is possible that this structure may allow for proteins such as Smo and components of a D/V pathway to converge. The Slimb-related protein Cdc4 from Saccharomyces cerevisiae along with Cdc53, and Cdc34 are part of the ubiquitin proteolysis machinery. The current data that Slimb acts upstream of Smo, together with its sequence homology with Cdc4, suggests that Slimb could be involved in the regulation of Smo protein degradation (Theodosiou, 1998).

Slimb (Slmb) is an F-box/WD40 protein that potentially participates in the ubiquitin proteolysis machinery. During development, Slmb is required in limb discs to repress Hedgehog (Hh) target genes, i.e. wingless and decapentaplegic, as well as the Wg signal transduction pathway. These repression functions have been proposed from studies using weak slmb alleles. Interestingly, experiments with strong slmb alleles have revealed additional functions in which slmb is required, such as leg dorsal-ventral restriction. New alleles of the slmb gene have been isolated in a screen for new negative regulators of dpp: several amorphs (characterized by genetic and molecular criteria) and a cold-sensitive hypomorph. By performing somatic clone experiments with these new amorphic slmb alleles, it has been determined that regulation of Dpp and Wg by Slmb could be different from what has already been published. In leg discs, lack of slmb function derepresses the transcription of wg, independent of Hh signaling. Ectopic legs resulting from slmb^- clone induction come only from wg misexpression in the normal dpp domain, since ectopic proximo-distal axes are induced dorsally, and adult ectopic legs are often perfect with respect to antero-posterior polarity. In wing discs, transcription of wg, which is normally independent of Hh signaling, is also derepressed in the absence of slmb function. In discs bearing amorphic slmb clones and in discs of two other slmb^- contexts, a novel pattern of dpp expression is described consisting of an enlargement of the normal dpp domain. Strong evidence indicates that this dpp modification can be linked to imaginal disc regeneration following slmb^- cell elimination. The fate of slmb^- clones, which disappear before adulthood, has been investigated. It was found that two mechanisms of cell elimination can account for imaginal cell regeneration: an early apoptosis and a mechanism of sorting-out that excludes all slmb^- clones from all imaginal discs. This result suggests that Slmb is likely to be involved, in addition to its repression role on Dpp and Wg, in some other essential cellular mechanism, since, in the absence of Slmb, cell affinities are dramatically modified regardless of the deregulated morphogen and of the type of imaginal disc (Miletich, 2000).

These results indicate that slmb is involved in the repression of wg transcription independent of Hh signaling. In the wing pouch, wg transcription depends on communications between dorsal and ventral cells involving the Notch receptor. This finding suggests that slmb is involved in the proteasome-dependent degradation or proteolytic cleavage of a putative regulatory protein of the Notch signaling pathway. This is in good agreement with a proposal that the proteasome is involved in the degradation of an active form of Notch, thus limiting the activation of the Notch targets. In the leg disc, the mechanism responsible for wg transcription in the posterior cells is unknown. In the anterior compartment of imaginal discs, the transcription factor Ci is necessary to activate (or repress) Hh target genes. In the posterior compartment cells, engrailed represses transcription of ci. Thus, a deregulation of wg transcription depending on Hh signaling must be linked to ectopic ci transcription. Ectopic transcription of ci is not observed in the posterior compartment of leg discs bearing slmb^- clones; neither is a switching-off of en transcription observed. It is concluded that a repression of wg transcription occurs in leg discs irrespective of the Hh/Ci signaling, and that slmb is involved in this process (Miletich, 2000).

In conclusion, two mechanisms appear to eliminate slmb^- cells in imaginal discs: an early apoptosis that only concerns some cells, and a mechanism for sorting-out that excludes all slmb^- clones from all imaginal tissues. The early apoptosis is possibly induced by differential cell adhesion: in this case, it would be classified as an early sorting-out. Another possibility is that apoptosis is an alternative path to sorting-out when mutant cells form a 2-4 cell group (the theoretical size of a clone aged 24 h) rather than an organized population of cells (as found 48 h after clone induction). This could be answered by investigating whether early apoptosis is causally linked to a modification of cell affinity. It is also important to investigate whether slmb plays a direct role in the appearance of apoptotic cell death. If so, slmb would act as an anti-apoptotic gene; preliminary results favoring this result have been obtained by generating slmb^- clones in a context of apoptosis inhibition (Miletich, 2000).

An important feature of the disappearance of null slmb^- clones is that sorting-out occurs regardless of the deregulated signaling pathway and of the type of imaginal disc. Therefore, it seems that this exclusion is not a result of the deregulation of these pathways but rather is the result of the deregulation of some other essential cellular mechanism shared by all imaginal cells. Since slmb encodes an F-box protein that would be involved in targeting degradation of proteins by the proteasome, it is proposed that slmb is necessary in a general process required for the proper functioning of many cellular mechanisms. Alteration of this process would then lead to such dramatic changes in cell affinities that all slmb^- cells would be excluded from all types of imaginal discs (Miletich, 2000).

The duplication of the centrosome is a key event in the cell-division cycle. Although defects in centrosome duplication are thought to contribute to genomic instability and are a hallmark of certain transformed cells and human cancer, the mechanism responsible for centrosome duplication is not understood. Recent experiments have established that centrosome duplication requires the activity of cyclin-dependent kinase 2 (Cdk2) and cyclins E and A. The stability of cyclin E is regulated by the ubiquitin ligase SCF, which is a protein complex composed of Skp1, Cdc53 (Cullin) and F-box proteins. The Skp1 and Cullin components have been detected on mammalian centrosomes, and shown to be essential for centrosome duplication and separation in Xenopus. Slimb, an F-box protein that targets proteins to the SCF complex, plays a role in limiting centrosome replication. In Drosophila the hypomorphic mutation slimb^crd causes the appearance of additional centrosomes and mitotic defects in mutant larval neuroblasts (Wojcik, 2000).

A mutant Drosophila line, initially named centrosome replication defective (crd), was detected in a screen of late larval and pupal lethal mutants from a collection of third chromosome P-element insertion mutants. Examination of larval neuroblast chromosome spreads revealed that homozygous crd mutants display two types of abnormal mitotic figures: metaphase figures comprising overcondensed chromosomes, and polyploid figures suggestive of defects in progressing through the mitotic cycle. The single P element in this line maps to position 93B10-13 by in situ hybridization to polytene chromosomes. The chromosomal deficiency Df(3R)e^R1 uncovered the crd mutation and the resulting hemizygous animals also have a mitotic phenotype similar to the homozygous crd mutants. Whereas crd homozygotes die at the larval-pupal boundary, the mitotic defects and lethality of crd were reverted by excision of the P element (Wojcik, 2000).

Isolation of genomic DNA flanking the crd P-element insertion revealed that it is inserted 296 bp into the 5' untranslated region (UTR) of a previously identified locus, supernumerary limbs (slimb). Genetic analysis confirmed that crd and the slimb mutations are allelic and responsible for the observed centrosome replication defect. Accordingly, the allele was renamed slimb^crd (Wojcik, 2000).

To characterize further the slimb^crd mitotic phenotype, the morphology of hemizygous slimb^crd neuroblasts was examined by confocal microscopy. Most striking of the abnormalities in mutant mitotic cells was the excessive number of centrosomes revealed by the distribution of the centrosomal antigen CP190. Diploid cells were observed that contained more than two and as many as seventeen centrosomes. Polyploid giant neuroblasts were also observed in mutant brain discs, containing far greater numbers of centrosomes than predicted by a failure in cleavage alone (Wojcik, 2000).

To confirm that the CP190-positive structures identify centrosomes, tests were performed for the presence of the majority of the known intrinsic components of Drosophila microtubule-organizing centers, including gamma-tubulin, Centrosomin (CNN) and Abnormal spindle (ASP). Each component was present at the putative centrosomes. Double-label experiments using antibodies against gamma-tubulin, CP190, CNN or ASP showed that signals from all these antigens always coincide with the putative centrosome foci in both wild-type and slimb^crd mutant mitotic neuroblasts. In addition, the size and shape of the multiple foci in slimb^crd neuroblasts are uniform and comparable to centrosomes in wild-type cells. Taken together, these observations are most consistent with the presence of excess numbers of centrosomes in diploid slimb^crd mutant cells, and are not readily explained by the aberrant aggregation of centrosomal antigens or the fragmentation of a single pair of centrosomes (Wojcik, 2000).

Analysis of centrosome number in mutant diploid neuroblasts revealed that a majority of cells (66%) contained excess centrosomes. The aberrant number of centrosomes often exceeds the four foci that would be expected if the defect arose from the precocious separation of the centriole pair associated with each centrosome. Instead, the aberrant centrosome numbers suggest that repeated rounds of centrosome duplication occur during individual cell cycles in slimb^crd cells. Significantly, the increase in the number of centrosomes is not random, with 74% of cells, excluding normal cells with two centrosomes, containing even numbers of centrosomes. This result suggests that not all of the extant centrosomes are licensed to replicate, but is more consistent with the continuous replication of the starting pair of centrosomes. This phenotype is distinct from an assembly/fragmentation defect in centrosome morphogenesis recently reported for a Drosophila Hsp90 mutation (Wojcik, 2000).

It is striking that, despite the presence of excess centrosomes in slimb^crd cells, the spindles are bipolar and show no indication of branching or multipolarity as seen in other mitotic mutants. For centrosomes considerably displaced from the poles, no substantial or stable microtubule-organizing activity was apparent, further suggesting that the additional centrosomes are 'immature' or functionally distinct. Still, the low incidence of hyperploid cells associated with the slimb mutations suggests that, despite the lack of severe spindle defects, excess centrosomes may disrupt the downstream events associated with cytokinesis or cleavage (Wojcik, 2000).

The failure of homozygous slimb- clones to proliferate in mosaic animals is consistent with the cell-cycle defects observed. In Drosophila embryos, Xenopus and sea-urchin extracts, as well as mammalian cells, centrosome duplication is closely tied to known mitotic regulators. Therefore, it is possible that slimb mutations affect centrosome replication indirectly by generally modulating progression through mitosis. Because it has previously been shown that centrosome replication is coupled to S phase in vitro, and that an abnormally prolonged S phase can result in centrosome over-replication, an examination was made to determine whether the duration of S phase was increased in slimb^crd cells. If S phase is lengthened in the slimb mutant cells, then an increase in the total number of S-phase cells seen at any given moment in mutant, compared with wild-type, tissues, would be expected. Instead, a decrease was found in the number of S-phase cells in slimb mutant brains. Therefore, it is proposed that Slimb is not likely to lengthen the progression of mitosis and, instead, acts more directly to stop centrosome replication (Wojcik, 2000).

The slimb gene was first identified as a negative regulator of the Hedgehog (Hh) and Wingless (Wnt/Wg) signaling pathways in Drosophila. It was recognized as a member of the F box/WD40 class of proteins that can act as targeting factors for the SCF complex, an E3 ubiquitin ligase first identified from cell-cycle studies in yeast. SCF activity is also required to degrade cell-cycle regulatory proteins in metazoans, as is evident from the accumulation of cyclin E in mouse knockouts deficient for its Cullin 1 component. In Drosophila neuroblasts, Slimb is required to restrict centrosome duplication during the cell cycle. The target whose presumed degradation is regulated by Slimb is not known. One possibility is that the target is cyclin E, since this Cdk2 subunit is known to be degraded by the SCF complex and is also required for centrosome duplication. This may in part explain why antibodies to either the Skp1 or Cullin1 components of SCF can block the initial separation of replicating centrioles in vitro. Nevertheless, because individual F-box proteins, such as Slimb, can interact with more than one target protein, and multiple F-box proteins localize at centrosomes during mitosis, other centrosomal targets and distinct steps in centrosome replication are likely to be involved (Wojcik, 2000).

The requirement for an E3 ubiquitin ligase targeting component to regulate both signaling pathways and centrosome duplication offers one means of coordinating the regulation of developmental processes, and signals for cell proliferation, with the mechanics of cell-cycle progression. It is not difficult to imagine how competition for rate-limiting levels of Slimb protein might regulate the division of cells within a particular developmental program. Similar regulatory networks may also be relevant in the significant number of human cancers in which the degradation of ß-catenin is dysregulated and in human tumor cells with the known occurrence of excess centrosomes (Wojcik, 2000).

Photoreceptor differentiation in the Drosophila eye disc progresses from posterior to anterior in a wave driven by the Hedgehog and Decapentaplegic signals. Cells mutant for the hyperplastic discs gene misexpress both of these signaling molecules in anterior regions of the disc, leading to premature photoreceptor differentiation and overgrowth of surrounding tissue. hyperplastic discs encodes a HECT domain E3 ubiquitin ligase that is likely to act by targeting Cubitus interruptus and an unknown activator of hedgehog expression for proteolysis (Lee, 2002).

If hyd regulates dpp expression by altering Ci activity, loss of hyd should lead to upregulation of full-length, active Ci. Increased levels of full-length Ci are indeed observed in hyd mutant clones in the anterior of the eye disc. However, this could be due to misexpression of hh in the same clones. To determine whether hyd has a direct effect on Ci, hyd;hh double mutant clones anterior to the morphogenetic furrow were examined. High levels of full-length Ci accumulated in these clones, confirming that Hyd normally reduces Ci levels independently of Hh activity (Lee, 2002).

The F-box protein Slmb has been shown to promote processing of Ci to Ci₇₅ as a component of an SCF ubiquitin ligase complex. Therefore the effects were compared of slmb and hyd mutations on Ci levels in the wing disc. Ci₁₅₅ is much more dramatically increased in slmb clones than in hyd clones. An interesting difference was also observed between hyd and slmb in the regulation of dpp. dpp expression is increased in hyd mutant clones close to the AP border, but is very rarely activated outside this domain. In contrast, slmb mutant clones activated dpp expression only when they lay outside the wing pouch, perhaps because of activation of Wg signaling, which represses dpp expression, within the wing pouch. Consistent with these third instar phenotypes, anterior duplications like those resulting from loss of slmb are not observed in adult wings containing hyd mutant clones, although outgrowths did arise from internal regions of the wing. Such duplications would require dpp to be misexpressed at a distance from its normal domain of expression. Ptc expression, which requires activation of the full-length form of Ci, was not altered in either hyd or slmb mutant clones. Slmb and Hyd thus appear to have distinct effects on Ci protein accumulation and activity, suggesting that they have either different substrates or different effects on the same substrate (Lee, 2002).

Cullins are the major components of a series of multimeric ubiquitin ligases that control the degradation of a broad range of proteins. The ubiquitin-like protein, Nedd8, covalently modifies members of the Cullin family. Nedd8 modifies Cullin 1 (Cul1, also known as Lin-19-like or simply Lin-19) in Drosophila. In mutants of Drosophila Nedd8 and Cul1, levels of the signal transduction effectors, Cubitus interruptus (Ci) and Armadillo, and the cell cycle regulator, Cyclin E (CycE), are unusually high, suggesting that the Cul1-based multimeric SCF ubiquitin ligase complex requires Nedd8 modification for the degradation processes of Ci, Arm, and CycE in vivo. Two distinct degradation mechanisms modulating Ci stability in the developing eye disc are separated by the morphogenetic furrow (MF) in which retinal differentiation is initiated. In cells anterior to the MF, Ci proteolytic processing promoted by PKA requires the activity of the Nedd8-modified Cul1-based SCF^Slimb complex. In posterior cells, Ci degradation is controlled by a mechanism that requires the activity of Cul3, another member of the Cullin family. This posterior Ci degradation mechanism, which partially requires Nedd8 modification, is activated by Hedgehog (Hh) signaling and is PKA-independent (Ou, 2002).

The Cul1-based SCF^Slimb complex controls Ci^FL processing in the anterior cells of the eye disc. In contrast, Cul3-mediated Ci degradation mechanism is specific to the posterior cells. These specific activities in controlling Ci protein stability are not caused by differential gene expression of Cul1 or Cul3 in the eye disc. Ubiquitous mRNA expression patterns of both Cul1 and Cul3, and ubiquitous Cul1 protein expression are found all along the eye disc, suggesting that control of specificity is mediated by mechanisms other than regulation of Cul1 and Cul3 expression (Ou, 2002).

The genetic evidence suggests that Nedd8 is directly required for Ci^FL proteolytic processing, consistent with the hypothesis that neddylation affects Ci^FL proteolysis through regulating SCF^Slimb activity. Cullin proteins are the identified targets for Nedd8 modification. In the Drosophila genome, six Cullin proteins are identified, each corresponding to a mammalian homolog. Among them, Cul1 is involved in the formation of SCF complexes that function as E3 ligase. The null Cul1 allele, Cul1^EX was generated. In Cul1^EX homozygous larvae in the first instar stage, the Cul1 signal detected by anti-Cul1 antibodies is almost completely absent. The residual Cul1 protein in Cul1^EX larvae is probably maternally contributed (Ou, 2002).

In addition to the Nedd8-Cul1 core component, the SCF complex also includes a substrate-specific F-box protein. To investigate whether SCF activity in Ci^FL processing is limited to the anterior cells of the eye disc, the mutant phenotype of slimb was examined. slimb is required for Ci^FL proteolytic processing in tissues such as wing and leg discs. When slimb¹ mutant clones were generated in eye discs, high levels of Ci^FL accumulation were detected exclusively in clones located anterior to the MF. No accumulation of Ci^FL could be detected in posterior slimb¹ clones. Suppression of Ci^FL accumulation in the posterior cells was not due to possible residual activity present in hypomorphic slimb¹, because identical results of Ci^FL accumulation were observed in the strong hypomorphic allele slimb² and the null allele slimb^P (Ou, 2002).

In summary, the results strongly suggested that in vivo, the Nedd8-modified, Cul1-based SCF^Slimb complex controls Ci^FL proteolysis in anterior cells. Following the sweep of the MF, Ci^FL stability in the posterior cells is controlled by an SCF^Slimb-independent mechanism (Ou, 2002).

A ubiquitin-proteasome pathway represses the Drosophila immune deficiency signaling cascade

The inducible production of antimicrobial peptides is a major immune response in Drosophila. The genes encoding these peptides are activated by NF-κB transcription factors that are controlled by two independent signaling cascades: the Toll pathway that regulates the NF-κB homologs, Dorsal and DIF; and the IMD pathway that regulates the compound NF-κB-like protein, Relish. Although numerous components of each pathway that are required to induce antimicrobial gene expression have been identified, less is known about the mechanisms that either repress antimicrobial genes in the absence of infection or that downregulate these genes after infection. In a screen for factors that negatively regulate the IMD pathway, two partial loss-of-function mutations were isolated in the SkpA gene that constitutively induce the antibacterial peptide gene, Diptericin, a target of the IMD pathway. These mutations do not affect the systemic expression of the antifungal peptide gene, Drosomycin, a target of the Toll pathway. SkpA encodes a homolog of the yeast and human Skp1 proteins. Skp1 proteins function as subunits of SCF-E3 ubiquitin ligases that target substrates to the 26S proteasome, and mutations affecting either the Drosophila SCF components, Slimb and Cullin1, or the proteasome also induce Diptericin expression. In cultured cells, inhibition of SkpA and Slimb via RNAi increases levels of both the full-length Relish protein and the processed Rel-homology domain. It is concluded that in contrast to other NF-κB activation pathways, the Drosophila IMD pathway is repressed by the ubiquitin-proteasome system. A possible target of this proteolytic activity is the Relish transcription factor, suggesting a mechanism for NF-κB downregulation in Drosophila (Khush, 2002).

In wild-type flies Diptericin is tightly controlled by the IMD pathway. Therefore, to identify genes that normally function to repress the IMD pathway, 2,000 yellow, white (y,w) F1 male progeny from male flies mutagenized with ethyl methanesulfonate were screened for constitutive expression of a Green Fluorescent Protein (GFP) reporter gene under the control of the Diptericin promoter. Two males, J6 and G49, expressed Diptericin-GFP, and this gene was constitutively expressed in larvae and adults in homozygous lines derived from these males. Although flies carrying the J6 and G49 mutations are viable and fertile at 25°C, G49 is pupal lethal at 29°C, indicating temperature-sensitive phenotypes associated with this mutation (Khush, 2002).

Using recombination mapping, the J6 and G49 mutations were shown to be tightly linked to the y locus on the proximal tip of the X chromosome. To further localize the two mutations, deletions were used to determine that J6 falls in the area defined by the overlap of Df(1)74k24.1, Df(1)svr, and Df(1)su(s)83, placing it in cytological region 1B10 near the Dredd gene. Two lethal P-element insertions in the Bloomington stock center collection, l(1)G0389 and l(1)G0109, which map near this region, were shown to not complement the constitutive Diptericin expression in the J6 and G49 lines. By sequencing DNA flanking the P elements in the two insertion lines, both elements were ascertained to lie within 200 bp of each other in the 5' untranslated region of the SkpA gene. To confirm that J6 and G49 are mutations in SkpA, a wild-type SkpA transgene on the second chromosome was shown to suppress the constitutive Diptericin expression phenotype in G49 flies. The J6 and G49 lines were shown to each contain a point mutation in the SkpA gene that generates a single amino acid change in the SkpA protein: J6, renamed SkpA^J6, converts threonine 98 to an isoleucine, and G49, renamed SkpA^G49, replaces glutamic acid 101 with a lysine. These alleles are hypomorphic mutations of SkpA since the P-element insertions are pupal lethal at 25°C. SkpA^G49 is pupal lethal at 29°C, and homozygous SkpA^G49 adults transferred to 29°C express Diptericin at similar levels as flies heterozygous for SkpA^G49 and either the P-element insertions or deletions that remove SkpA. At 29°C, therefore, SkpA^G49 behaves like a null mutation, which probably reflects the significant change from the negatively charged glutamic acid to the positively charged lysine in this allele (Khush, 2002).

The SkpA gene encodes a protein that is highly similar to Skp1 proteins in humans and yeast. Skp1 proteins are components of SCF ubiquitin ligases that target substrates to the proteasome, and crystal structures of human Skp1 complexed with the F-box protein Skp2 and the cullin protin Cul1 have been solved. SkpA^J6 and SkpA^G49 both affect a conserved region of SkpA that corresponds to helix 5 of Skp1; helix 5 forms part of the core interface between Skp1, the F-box region of Skp2, and the amino-terminal domain of Cul1, with some amino acids in this helix making direct contact with residues in Skp2 and Cul1. This suggests that the SkpA^J6 and SkpA^G49 mutations disrupt interactions between SkpA and the F-box protein and cullin components of an SCF complex. Protein interaction studies indicate that SkpA functions with the F-box protein Slimb and the Cullin-like protein Cullin1 (Cul1) in a Drosophila SCF complex. In support of this model, slimb¹ and dcul1^l(2)02074 mutant larvae, as well as larvae carrying the DTS5 mutation, a dominant-negative mutation that affects the β6 subunit of the 26S proteasome, were shown to express Diptericin at levels comparable to those in the SkpA mutants. To further test the DTS5 phenotype, the UAS-Gal4 system was used to overexpress a UAS-DTS5 transgene in larval fat bodies: DTS5 overexpression induces Diptericin to levels that are comparable to those generated by bacterial infection with Erwinia carotovora carotovora 15 (Ecc15). Flies heterozygous for mutations at both the SkpA and slimb loci were generated: these flies constitutively express Diptericin, indicating a synergistic interaction between SkpA and slimb. The constitutive Diptericin expression in the slimb¹, dcul1^l(2)02074, and DTS5 mutants and the interaction between SkpA and slimb together suggest that an SCF^{SkpA/Cul1/Slimb} ubiquitin ligase represses Diptericin expression by targeting a regulatory factor for degradation by the 26S proteasome (Khush, 2002).

To determine if the constitutive Diptericin expression in the SCF complex mutants is mediated through the IMD pathway, Diptericin levels were examined in larvae homozygous for mutations in either SkpA, or slimb and various genes of the IMD pathway: SkpA^J6;imd¹ and SkpA^G49;dtak1¹ double mutants display constitutive Diptericin expression, although Diptericin levels are slightly reduced in the SkpA^G49;dtak1¹ larvae. Mutations in DmIkkγ, DmIkkβ, and Relish, however, completely block Diptericin expression in the SkpA^J6 background, and a Dredd mutation completely blocks Diptericin expression in the slimb¹ background. The constitutive Diptericin expression observed in SkpA and slimb mutants, therefore, does not require IMD and dTak1, but it is dependent on the DmIKK complex, Dredd, and Relish. These results imply that, in wild-type flies, the SCF^{SkpA/Cul1/Slimb} negatively regulates the IMD pathway by targeting one of these factors, or an additional unidentified component of the IMD pathway, for degradation by the proteasome. In contrast to fat body cells, the IMD pathway is the primary regulator of all antimicrobial genes, including Drosomycin, in surface epithelial tissues. A Drosomycin-GFP transgene is constitutively expressed in tracheal cells but not in fat body cells of slimb¹ mutant larvae; this expression pattern further demonstrates that the IMD pathway, but not the Toll pathway, is constitutively activated when the SCF^{SkpA/Cul1/Slimb} complex is compromised (Khush, 2002).

Although the genetic results do not allow differentiating between the DmIKK complex, Dredd, Relish, or other unidentified downstream components of the IMD pathway as targets of the ubiquitin-proteasome pathway, the mammalian Relish homolog, P105, is regulated by an SCF complex that contains the Slimb homolog β-TrCP/E3RS^IκB. Consequently, RNA-mediated interference (RNAi), an effective technique for specifically inhibiting targeted proteins, was used in cultured Drosophila S2 cells to test for interactions between the SCF^{SkpA/Cul1/Slimb} complex and Relish. Initially, SkpA and Slimb activity were blocked in S2 cells via RNAi; then, transient expression of a full-length Relish protein, modified by an N-terminal FLAG tag, was induced in the same S2 cells and the effects of the SkpA and slimb RNAi treatments on FLAG-Relish protein stability was monitored using Western blots and anti-FLAG antibodies (Khush, 2002).

Reducing Slimb activity, in the absence of LPS stimulation, visibly increases steady-state levels of both full-length Relish and the active N-terminal Rel-homology domain; levels of both polypeptides are further increased by inhibiting Slimb and SkpA simultaneously. This effect is specific since RNAi of the SkpA homologs, SkpB and SkpD, does not increase Relish levels. Dredd RNAi does increase Relish levels at day 1, but this is probably because Dredd inhibition blocks Relish processing. Previous studies show that Relish processing in S2 cells is induced by lipopolysaccharide (LPS) and requires Dredd activity. As expected, therefore, RNAi of Dredd blocks LPS-induced Relish processing. Simultaneous RNAi of SkpA and Slimb in the presence of LPS, however, results in higher steady-state levels of the Rel-homology domain up to 4 days after Relish induction. Higher levels of the Rel-homology domain after SkpA and Slimb RNAi could be caused by increased processing of full-length Relish. However, because full-length Relish levels also mount, the explanation is favored that Rel-homology domain turnover is reduced. Although the Slimb and SkpA RNAi treatments appear to inhibit Relish turnover, Relish levels do eventually diminish. This suggests that RNAi efficiency decreases with time, possibly due to degradation of the transfected double-stranded RNA. These RNAi experiments indicate that the constitutive antimicrobial gene expression in SkpA and slimb mutant flies is caused by higher Relish levels, and they suggest that the SCF^{SkpA/Cul1/Slimb} complex represses the IMD pathway by promoting the degradation of both full-length and processed Relish proteins (Khush, 2002).

If the constitutive antimicrobial gene expression in flies carrying mutations that affect the SCF^{SkpA/Cul1/Slimb} complex or proteasome is due to higher Relish levels, this would imply some level of steady-state Relish activation. Low levels of the Rel-homology domain have been reported in nuclear extracts from unstimulated S2 cells, and these low levels indicate that Relish is constitutively processed. Increasing Relish levels in larvae and adults via the Gal4-UAS system is sufficient to induce low levels of Diptericin expression. These results indicate that Relish is constitutively processed and activated to some level, supporting the hypothesis that Relish activity, in the absence of infection, is countered by ubiquitination and degradation (Khush, 2002).

Multiple roles of the F-box protein Slimb in Drosophila egg chamber development

Substrate-specific degradation of proteins by the ubiquitin-proteasome pathway is a precise mechanism that controls the abundance of key cell regulators. SCF complexes are a family of E3 ubiquitin ligases that target specific proteins for destruction at the 26S-proteasome. These complexes are composed of three constant polypeptides -- Skp1, Cullin1/3 and Roc1/Rbx1 -- and a fourth variable adapter, the F-box protein. Slimb (Slmb) is a Drosophila F-Box protein that fulfills several roles in development and cell physiology. Slmb participation in egg chamber development was analyzed and slmb was found to be required in both the follicle cells and the germline at different stages of oogenesis. In slmb somatic clones, morphogenesis of the germarium and encapsulation of the cyst are altered, giving rise to egg chambers with extra germline cells and two oocytes. Furthermore, in slmb somatic clones, ectopic Fasciclin 3 expression was observed, suggesting a delay in follicle cell differentiation, that correlates with the occurrence of ectopic polar cells, lack of interfollicular stalks and mislocalization of the oocyte. Later in oogenesis, Slmb is required in somatic cells to specify the position, size and morphology of dorsal appendages. Mild overactivation of the Dpp pathway causes similar phenotypes that are antagonized by simultaneous overexpression of Slmb, suggesting that Slmb normally downregulates the Dpp pathway in follicle cells. Indeed, ectopic expression of a dad-LacZ enhancer trap reveals that the Dpp pathway is upregulated in slmb somatic clones and, consistent with this, ectopic accumulation of the co-Smad protein, Medea, occurs. By analyzing slmb germline clones, it was found that loss of Slmb provokes a reduction in E2f2 and Dp levels, which correlate with misregulation of mitotic cycles during cyst formation, abnormal nurse cell endoreplication and impairment of dumping of the nurse cell content into the oocyte (Muzzopappa, 2005).

Thus Slmb is required for oogenesis in both the germline and FC. In the germline, Slmb plays a role in the control of mitotic cycles during cyst formation, in regulation of nurse cell endoreplication and in nurse cell dumping. Recent reports have demonstrated that Slmb can control cell cycle progression in different experimental settings. Following DNA replication, Slmb is required in larval wing discs for proteolysis of the cell cycle modulator E2f1. Remarkably, the E2f complex is implicated in cell cycle control of ovarian germ cells, in nurse cell transition from polyteny to polyploidy and in dumping of the nurse cell content into the oocyte. This study shows that two subunits of the E2f complex, Dp and E2f2, are downregulated in ovaries bearing slmb germline clones, while E2f1 does not change. Differences in Cyclin E levels, another cell cycle regulator involved in cyst formation, could not be detected in these clones. A good correlation exists between the phenotypes observed in slmb germline clones and in Dp germline clones; in both cases an additional round of cystocyte mitotic divisions occurs. In order to understand the molecular mechanism causing Dp and E2f2 reduction in slmb germline clones, a detailed analysis of the alterations of the network regulating the cell cycle is required (Muzzopappa, 2005).

Although expression levels in somatic cells in the germarium are too low to be detected through an enhancer trap or by in situ hybridization, loss-of-function experiments suggest that slmb is needed in these cells for normal morphogenesis of the egg chamber and for encapsulation of the cyst. In addition, the results suggest that Slmb is required for timely differentiation of FC that is reflected by the refinement of Fas3 expression; this is accompanied by the occurrence of ectopic polar cells, lack of interfollicular stalks and disruption of normal egg chamber polarity. Later in oogenesis, Slmb is expressed at high levels in FC surrounding the oocyte and participates in chorion patterning, contributing to define the shape and position of DA (Muzzopappa, 2005).

It has been reported that slmb mutant clones induce ectopic activation of the Hedgehog (Hh) pathway in limb discs. Notably, some of the phenotypes observed upon slmb somatic clone induction are similar to those originated by overactivation of the Hh pathway in FC. These include a delay in FC differentiation, development of ectopic polar cells and mislocalization of the oocyte. Nevertheless, excessive activation of the Hh pathway also causes FC over-proliferation that results in excess of undifferentiated somatic cells that form very long interfollicular stalks between egg chambers. By contrast, slmb loss of function in FC caused a lack rather than an excess of interfollicular cells. Finally, dominant genetic interactions were not detected between slmb and negative regulators of Hh pathway and the ptc-LacZ enhancer trap, which has been reported to be activated in FC by the Hh pathway, is not induced ectopically in slmb mutant clones. These results indicate that, despite some similarities between slmb loss-of-function and hh gain-of-function phenotypes, Slmb is unlikely to be a negative regulator of Hh pathway during oogenesis (Muzzopappa, 2005).

In limb discs, Slmb is a negative regulator of the Dpp pathway, although the molecular mechanism involved is still unclear. Mild overexpression of Dpp causes a wide spectrum of phenotypes that are largely coincident with those caused by slmb loss of function in FC. Supporting the idea that loss of slmb might cause hyperactivation of the Dpp pathway, the strongest chorion phenotypes originated by overexpression of Dpp are completely antagonized by simultaneous overexpression of Slmb in FC. Moreover, expansion of dad-lacZ expression occurs in slmb mutant follicles, further suggesting that ectopic induction of the Dpp pathway indeed occurs as a consequence of slmb loss of function. Consistent with this, a downstream component of the Dpp pathway, the co-Smad protein Medea, is upregulated in slmb mutant egg chambers. Because in mammalian cell culture it was demonstrated that Smad4 is a direct target of the mammalian Slmb ortholog, ßTrcp1, it is believed that Medea could be a direct target of Slmb. Further molecular work is required to assess whether this is indeed the case or if alternatively, the effect of Slmb on Medea is indirect (Muzzopappa, 2005).

Wnt signaling cross-talks with JH signaling by suppressing Met and gce expression

Juvenile hormone (JH) plays key roles in controlling insect growth and metamorphosis. However, relatively little is known about the JH signaling pathways. Until recent years, increasing evidence has suggested that JH modulates the action of 20-hydroxyecdysone (20E) by regulating expression of broad (br), a 20E early response gene, through Met/Gce and Kr-h1. To identify other genes involved in JH signaling, a novel Drosophila genetic screen was designed to isolate mutations that derepress JH-mediated br suppression at early larval stages. It was found that mutations in three Wnt signaling negative regulators in Drosophila, Axin (Axn), supernumerary limbs (slmb), and naked cuticle (nkd), caused precocious br expression, which could not be blocked by exogenous juvenile hormone analogs (JHA). A similar phenotype was observed when armadillo (arm), the mediator of Wnt signaling, was overexpressed. qRT-PCR revealed that Met, gce and Kr-h1expression are suppressed in the Axn, slmb and nkd mutants as well as in arm gain-of-function larvae. Furthermore, ectopic expression of gce restored Kr-h1 expression but not Met expression in the arm gain-of-function larvae. Taken together, it is concluded that Wnt signaling cross-talks with JH signaling by suppressing transcription of Met and gce, genes that encode for putative JH receptors. The reduced JH activity further induces down-regulation of Kr-h1expression and eventually derepresses br expression in the Drosophila early larval stages (Abdou, 2011).

JH transduces its signal through Methoprene-tolerant (Met), Germ cell-expressed (Gce) and Krüppel-homolog 1 (Kr-h1) and the p160/SRC/NCoA-like molecule (Taiman in Drosophila and FISC in Aedes). The Drosophila Met and gce genes encode two functionally redundant bHLH-PAS protein family members, which have been proposed to be components of the elusive JH receptor. Both Met and gce mutants are viable and resistant to JH analogs (JHA) as well as to natural JH III. However, Met-gce double mutants are prepupal lethal and phenocopies CA-ablation flies. The Met protein binds JH III with high affinity. In Tribolium, suppression of Met activity by injecting double-stranded (ds) Met RNA causes precocious metamorphosis. Kr-h1 is considered as a JH signaling component working downstream of Met. In both Drosophila and Tribolium, Kruppel-homolog1 (Kr-h1) mRNA exhibits high levels during the embryonic stage and is continuously expressed in the larvae; then, it disappears during pupal and adult development. Kr-h1 expression can be induced in the abdominal integument by exogenous JH analog (JHA) at pupariation. Suppression of Kr-h1 by dsRNA in the early larval instars of Tribolium causes precocious br expression and premature metamorphosis after one succeeding instar. Thus, Kr-h1 is necessary for JH to maintain the larval state during a molt by suppressing br expression. Studies in Aedes, Drosophila and Tribolium have demonstrated that the p160/SRC/NCoA-like molecule is also required for JH to induce expression of Kr-h1 and other JH response genes. For example, Aedes FISC forms a functional complex with Met on the JH response element in the presence of JH and directly activates transcription of JH target genes (Abdou, 2011 and references therein).

In an attempt to isolate other genes involving JH signaling, a novel genetic screen was conducted, and mutations in were identified in three Wnt signaling component genes, Axin (Axn), supernumerary limbs (slmb), and naked cuticle (nkd), induced precocious br expression, which was similar to a loss of JH activity. The evolutionarily conserved Wnt signaling pathway controls numerous developmental processes. The key mediator of the Drosophila Wnt pathway is Armadillo (Arm, the homolog of vertebrate β-catenin). When the Wnt signaling ligand, Wingless (Wg), is absent, the destruction complex is active and phosphorylates Arm, earmarking it for degradation. Upon Wg stimulation, the destruction complex is inactivated; as a result, unphosphorylated Arm accumulates in the cytosol and is targeted to the nucleus to stimulate transcription of Wnt target genes. Many players in the Wnt signaling pathway negatively regulate its activity. For example, Axin (Axn) is one of the main components of the destruction complex. Supernumerary limbs (Slmb) recognizes phosphorylated Arm and targets it for polyubiqitination and proteasomal destruction. Naked cuticle (Nkd) antagonizes Wnt signaling by inhibiting nuclear import of Arm. The current investigations reveal that the high activity of Wnt signaling in the Axn, slmb, and nkd mutants suppresses the transcription of Met and gce, genes encoding for putative JH receptors, thus linking Wnt signaling to JH signaling and insect metamorphosis for the first time (Abdou, 2011).

The 'status quo' action of JH in controlling insect metamorphosis is conserved in hemimetabous and most holometabous insects. However, the larval-pupal transition in higher Diptera, such as Drosophila, has largely lost its dependence on JH. For instance, in most insects, the addition of JH in larvae at the last instar causes the formation of supernumerary larvae. However, exogenous JH does not prevent pupariation and pupation in Drosophila, and instead disrupts the development of only the adult abdominal cuticle and some internal tissues. The molecular mechanisms underlying these differential responses to JH are not clear (Abdou, 2011).

Broad is a JH-dependent regulator that specifies pupal development and mediates the 'status quo' action of JH. In the relatively basal holometabolous insects, such as beetles and moths, JH is both necessary and sufficient to repress br expression during all of the larval stages. These studies revealed that JH is also required during the early larval stages in the more derived groups of the holometabolous insects, such as Drosophila, but it is not sufficient to repress br expression at the late 3^rd instar. During the early larval stages, overexpression of the JH-degradative enzyme JHE, reduction of JH biosynthesis or disruption of the JH signaling always causes precocious br expression in the fat body. However, exogenous JHA treatment can not repress br expression in the fat body of late 3^rd instar larvae. The molecular mechanism underlying the developmental stage-specific responses of the br gene to JH signaling remains to be clarified (Abdou, 2011).

As knowledge of signal transduction increases, the next step is to understand how individual signaling pathways integrate into the broader signaling networks that regulate fundamental biological processes. In vertebrates, Wnt signaling has been found to interact with different hormone signaling pathways to mediate various developmental events. For example, the Wnt/beta-catenin signaling pathway interacts with thyroid hormones in the terminal differentiation of growth plate chondrocytes and interacts with estrogen to regulate early gene expression in response to mechanical strain in osteoblastic cells. In insects, both Wnt and JH signaling are important regulatory pathways, each controlling a wide range of biological processes. This study reports that the Wnt signaling pathway interacts with JH in regulating insect development. During the Drosophila early larval stages, elevated Wnt signaling activity in the Axn, slmb, nkd mutants and arm-GAL4/UAS-arm^S10 flies represses Met and gce expression, which down-regulates Kr-h1 and causes precocious br expression in the fat body. Ectopic expression of UAS-gce in the arm-GAL4/UAS-arm^S10 larvae is sufficient for restoring Kr-h1 expression and then repressing br expression (Abdou, 2011).

Arm is a co-activator that interacts with Drosophila TCF homolog Pangolin (Pan), a Wnt-response element-binding protein, to stimulate expression of Wnt signaling target genes. In the absence of nuclear Arm, Pan interacts with Groucho, a co-repressor, to repress transcription of Wingless-responsive genes. Upon the presence of nuclear Arm, it binds to Pan, converting it into a transcriptional activator to promote the transcription of Wingless-responsive genes. It is proposed that Wnt signaling indirectly suppresses Met and gce expression by activating an unknown transcriptional repressor (Abdou, 2011).

JH signaling is well known to be a systemic factor that decides juvenile versus adult commitment. Wg is a morphogen that tissue-autonomously promotes proliferation and patterning during organogenesis. The current studies show that ectopically activating Wg signaling, either by mutations of negative regulators or by the ectopic expression of Arm, results in br derepression via loss of Met and Gce. How and why does the localized Wg signaling regulate the global JH signaling during insect development? It is hypothesized that though JH signaling activity is globally controlled by JH titer in the hemolymph, distinct tissues may response to JH with different sensitivity, which could be regulated by Wnt signaling-mediated Met and gce expression. Actually, it was found that precocious br expression is detectible in the fat body but not midgut of the Axn mutant 2^nd instar larvae. This is one line of evidence to support that Wnt signaling regulates Met and gce expression in a tissue-specific manner (Abdou, 2011).

EVOLUTIONARY HOMOLOGS

Slimb homologs and protein degradation in yeast

In budding yeast, ubiquitination of the cyclin-dependent kinase (Cdk) inhibitor Sic1 is catalyzed by the E2 ubiquitin conjugating enzyme Cdc34 in conjunction with an E3 ubiquitin ligase complex composed of Skp1, Cdc53 and the F-box protein, Cdc4 (the SCFCdc4 complex). Skp1 binds a motif called the F-box; in turn, F-box proteins appear to recruit specific substrates for ubiquitination. Skp1 has been shown to interact with Cdc53 in vivo, and Skp1 bridges Cdc53 to three different F-box proteins: Cdc4, Met30, and Grr1. Cdc53 contains independent binding sites for Cdc34 and Skp1, suggesting it functions as a scaffold protein within an E2/E3 core complex. F-box proteins show remarkable functional specificity in vivo: Cdc4 is specific for degradation of Sic1; Grr1 is specific for degradation of the G1 cyclin Cln2, and Met30 is specific for repression of methionine biosynthesis genes. In contrast, the Cdc34-Cdc53-Skp1 E2/E3 core complex is required for all three functions. Combinatorial control of SCF complexes may provide a basis for the regulation of diverse cellular processes (Patton, 1998).

Ubiquitin-dependent degradation of regulatory proteins controls many cellular processes, including cell cycle progression, morphogenesis, and signal transduction. Skp1p-cullin-F-box protein (SCF) complexes are ubiquitin ligases composed of a core complex, including Skp1p, Cdc53p, one of multiple F-box proteins that are thought to provide substrate specificity to the complex, and the ubiquitin-conjugating enzyme, Cdc34p. It is not understood how SCF complexes are regulated and how physiological conditions alter their levels. Three F-box proteins, Grr1p, Cdc4p, and Met30p, are unstable components of the SCF, and are themselves degraded in a ubiquitin- and proteasome-dependent manner in vivo. Ubiquitination requires all the core components of the SCF and an intact F-box, suggesting that ubiquitination occurs within the SCF complex by an autocatalytic mechanism. Cdc4p and Grr1p are intrinsically unstable, and their steady-state levels do not fluctuate through the cell cycle. Taken together, these results suggest that ubiquitin-dependent degradation of F-box proteins allows rapid switching among multiple SCF complexes, thereby enabling cells to adapt quickly to changing physiological conditions and progression through different phases of the cell cycle (Galan, 1999).

SCFCdc4 (Skp1, Cdc53/cullin, F-box protein) defines a family of modular ubiquitin ligases (E3s) that regulate diverse processes including cell cycle, immune response, and development. Mass spectrometric analysis of proteins copurifying with Cdc53 has identified the RING-H2 finger protein Hrt1 as a subunit of SCF. Hrt1 shows striking similarity to the Apc11 subunit of anaphase-promoting complex. Conditional inactivation of hrt1(ts) results in stabilization of the SCFCdc4 substrates Sic1 and Cln2 and cell cycle arrest at G1/S. Hrt1 assembles into recombinant SCF complexes and individually binds Cdc4, Cdc53 and Cdc34, but not Skp1. Hrt1 stimulates the E3 activity of recombinant SCF potently and enables the reconstitution of Cln2 ubiquitination by recombinant SCFGrr1. Surprisingly, SCF and the Cdc53/Hrt1 subcomplex activate autoubiquitination of Cdc34 E2 enzyme by a mechanism that does not appear to require a reactive thiol. The highly conserved human HRT1 complements the lethality of hrt1Delta, and human HRT2 binds CUL-1. It has been concluded that Cdc53/Hrt1 comprise a highly conserved module that serves as the functional core of a broad variety of heteromeric ubiquitin ligases (Seol, 1999).

Centrosomes organize the mitotic spindle to ensure accurate segregation of the chromosomes in mitosis. The mechanism that ensures accurate duplication and separation of the centrosomes underlies the fidelity of chromosome segregation, but remains unknown. In Saccharomyces cerevisiae, entry into S phase and separation of spindle pole bodies each requires CDC4 and CDC34, which encode components of an SCF (Skp1-cullin-F-box) ubiquitin ligase, but a direct (SCF) connection to the spindle pole body is unknown. Using immunofluorescence microscopy, it has been shown that in mammalian cells the Skp1 protein and the cullin Cul1 are localized to interphase and mitotic centrosomes and to the cytoplasm and nucleus. Deconvolution and immunoelectron microscopy suggest that Skp1 forms an extended pericentriolar structure that may function to organize the centrosome. Purified centrosomes also contain Skp1, and Cul1 modified by the ubiquitin-like molecule NEDD8: this suggests a role for NEDD8 in targeting. Using an in vitro assay for centriole separation in Xenopus extracts, antibodies to Skp1 or Cul1 block separation. Proteasome inhibitors block both centriole separation in vitro and centrosome duplication in Xenopus embryos. Candidate centrosomal F-box proteins have been identified, suggesting that distinct SCF complexes may direct proteolysis of factors mediating multiple steps in the centrosome cycle (Freed, 1999).

Among the domains of Cdc4p that are crucial for function are the F-box, which links Cdc4p to Cdc53p through Skp1p, and the WD-40 repeats, which are required for binding the substrate for Cdc34p. In addition to Cdc4p, other F-box proteins, including Grr1p and Met30p, may similarly act together with Cdc53p and Skp1p to function as ubiquitin ligase complexes. Because the relative abundance of these complexes, known collectively as SCFs, is important for cell viability, evidence of mechanisms that modulate F-box protein regulation have been sought. The abundance of Cdc4p is subject to control by a peptide segment that is termed the R-motif (for 'reduced abundance'). Binding of Skp1p to the F-box of Cdc4p inhibits R-motif-dependent degradation of Cdc4p. These results suggest a general model for control of SCF activities (Mathias, 1999).

C. elegans Slimb homologs

In multicellular eukaryotes, a complex program of developmental signals regulates cell growth and division by controlling the synthesis, activation and degradation of G1 cell cycle regulators. The lin-23 gene of C. elegans is required to restrain cell proliferation in response to developmental cues. In lin-23 null mutants, all postembryonic blast cells undergo extra divisions, creating supernumerary cells that can differentiate and function normally. In contrast to the inability to regulate the extent of blast cell division in lin-23 mutants, the timing of initial cell cycle entry of blast cells is not affected. lin-23 encodes an F-box/WD-repeat protein that is orthologous to the Saccharomyces cerevisiae gene MET30, the Drosophila gene slmb, and the human gene ßTRCP, all of which function as components of SCF ubiquitin-ligase complexes. Loss of function of the Drosophila slmb gene causes the growth of ectopic appendages in a non-cell autonomous manner. In contrast, lin-23 functions cell autonomously to negatively regulate cell cycle progression, thereby allowing cell cycle exit in response to developmental signals (Kipreos, 2000).

In lin-23 mutants, cells appear to require normal signals to start cell division. Only blast cells and their descendants divide in lin-23 larval stages, while postmitotic non-blast cells do not divide. Blast cells do not divide precociously, but rather wait until the appropriate developmental stage to divide. Further, among the VPCs, those closest to the AC, which secretes an inductive/proliferative signal, divide more than those further from the AC, as is true for wild type. Final differentiation states of lin-23 cells appear normal. Distinct neuronal, hypodermal, somatic gonad, muscle, and intestine phenotypes are readily observed by DIC microscopy (Kipreos, 2000).

Morphogenesis is disorganized in several larval tissues in lin-23 mutants relative to wild type, e.g., spermathecae and uterus, and in embryos lacking lin-23 maternal product. lin-23 may be required for morphogenic processes: alternatively, the disorganization may be the byproduct of excess cell proliferation. In both cyclin-dependent kinase inhibitor cki-1 mutants and cul-1 loss-of-function animals, which also exhibit hyperplasia, there is a similar disorganization of larval tissues and lack of clear morphogenesis in embryos. In contrast to mitotic cell cycles, lin-23 is not required for restraining purely endoreplicative cycles. The ploidy of intestine cells, which endoreplicate in wild type to achieve 32n, does not increase beyond 32n in lin-23 mutants. The first endoreplication for most wild-type intestine cells is preceded by a nuclear division (segregation of chromosomes but no cytokinesis). Interestingly, the first endoreplication after the nuclear division is converted to a second nuclear division in lin-23 mutants, suggesting that lin-23 is required to allow these cells to bypass mitosis. Later endoreplications are not converted to nuclear divisions in lin-23 mutants, suggesting that mitotic bypass is maintained by a lin-23-independent mechanism, potentially via the downregulation of mitotic cyclin, as is observed in Drosophila endoreplicative cycles (Kipreos, 2000).

lin-23 is expressed at its highest levels in embryos and the adult germline, but has some expression in all developmental stages. The complete S. cerevisiae genome has just two F-box/WD-repeat proteins, Cdc4p and Met30p. Both Cdc4p and Met30p function as components of an SCF E3 complex to facilitate the recognition of substrates by a ubiquitin-conjugating enzyme (E2) for ubiquitin-mediated proteolysis. Met30p and Cdc4p function as the substrate recognition subunits of different SCF complexes, SCF Met30 and SCF Cdc4 . Each complex also includes Skp1p, the cullin Cdc53p, and Rbx1p/Roc1p/Hrt1p, and interacts with the ubiquitin-conjugating enzyme Cdc34p. Both Cdc4p and Met30p are involved in the degradation of cell cycle regulators. Cdc4p is required for the degradation of G1 cyclins (Cln1p and Cln2p), cyclin-dependent kinase inhibitors (Sic1p and Far1p), and the DNA replication protein Cdc6p. Met30p is required for the degradation of the CDK inhibitory kinase Swe1p, and for the repression of the sulfur network genes by inactivating the transcription factor Met4p. Increased activity of Met4p in met30 mutants produces a G1 arrest with increased turnover of the mRNA for the G1 cyclins CLN1, CLN2 and PCL2 (Kipreos, 2000).

Parsimony analysis suggests that lin-23 is more closely related to Met30p, while another C. elegans protein SEL-10 is more closely related to Cdc4p. SEL-10 functions to negatively regulate LIN-12 activity and has been found to physically interact with the intracellular domain of LIN-12, suggesting that SEL-10 functions in the turnover of LIN-12. Surprisingly, a null allele of sel-10 has only minor, impenetrant phenotypic consequences in a wild-type lin-12 genetic background. sel-10 is the apparent ortholog of the yeast cell cycle regulator CDC4. While the sel-10 mutant phenotype does not indicate a role in cell cycle regulation, it is possible that another gene functions redundantly with sel-10 to effect cell cycle regulation. However, a double mutant of lin-23 and sel-10 fail to uncover synthetic cell cycle phenotypes, suggesting that lin-23 does not share critical functions with sel-10. Finally, the dissimilar mutant phenotypes of lin-23 (defective cell cycle exit) and met30 (cell cycle arrest) further suggests that the cellular functions of SCF complexes have not been conserved between yeast and metazoa. lin-23 has a mutant hyperplasia phenotype similar to that of the cul-1 gene, which also encodes an SCF component. Orthologs of lin-23 and cul-1 function together in SCF complexes in both yeast and humans, making it likely that lin-23 and CUL-1 also form an SCF complex in C. elegans. The phenotypes of cul-1 and lin-23, while similar, do show differences. Whereas lin-23 maternal products perdure only through embryogenesis, cul-1 maternal products can suffice through the L1 stage. Despite the later onset of the cul-1 mutant phenotype, cul-1 larvae arrest development earlier with a more severe hyperplasia, e.g., cul-1 mutants exhibit on average twice as much vulval hyperplasia as lin-23 mutants (Kipreos, 2000).

Hyperplasia in lin-23 mutants does not appear to be due to morphogen secretion, as the hyperplasia occurs throughout development in all tissue types, and mosaic analysis indicates that lin-23 mutant hyperplasia occurs through a cell autonomous mechanism. It is currently unclear why the two orthologs have such different modes of action in Drosophila and C. elegans. It is tempting to speculate that both genes evolved to negatively regulate cell proliferation, however, differences in the way cell proliferation is regulated in Drosophila, via the action of morphogens to pattern surrounding cells, compared to C. elegans, where cell intrinsic decisions are much more important, have shaped the mechanism of action of the two gene products (Kipreos, 2000).

Cdc25 phosphatases are key positive cell cycle regulators that coordinate cell divisions with growth and morphogenesis in many organisms. Intriguingly in C. elegans, two cdc-25.1(gf) mutations induce tissue-specific and temporally restricted hyperplasia in the embryonic intestinal lineage, despite stabilization of the mutant CDC-25.1 protein in every blastomere. This study investigated the molecular basis underlying the CDC-25.1(gf) stabilization and its associated tissue-specific phenotype. Both mutations were found affect a canonical β-TrCP phosphodegron motif, while the F-box protein LIN-23, the β-TrCP orthologue, is required for the timely degradation of CDC-25.1. Accordingly, depletion of lin-23 in wild-type embryos stabilizes CDC-25.1 and triggers intestinal hyperplasia, which is, at least in part, cdc-25.1 dependent. lin-23(RNAi) causes embryonic lethality owing to cell fate transformations that convert blastomeres to an intestinal fate, sensitizing them to increased levels of CDC-25.1. This characterization of a novel destabilizing cdc-25.1(lf) intragenic suppressor that acts independently of lin-23 indicates that additional cues impinge on different motifs of the CDC-25.1 phosphatase during early embryogenesis to control its stability and turnover, in order to ensure the timely divisions of intestinal cells and coordinate them with the formation of the developing gut (Hebeisen, 2008).

The early cell divisions of the C. elegans embryo are precisely controlled by gene products that are provided from the maternal germ line. The isolation of two gain-of-function alleles of CDC-25.1 that demonstrate a strict maternal effect indicates that this regulation can be perturbed, resulting in supernumerary cell divisions specifically within the E lineage. How these mutations give rise to the extra divisions is unclear, although CDC-25.1(rr31) is more stable than its wild-type counterpart. By developing a GFP-based transgenic assay to assess the dynamics of protein degradation during early embryogenesis, this study shows that the stabilization of CDC-25.1 is mediated by a point mutation within a conserved DSGX₄S β-TrCP-like phosphodegron in both cdc-25.1(gf) mutants. This stabilizes the protein, resulting in the abnormal presence of CDC-25.1 during a short, yet crucial, window during early development, which is presumably the cause of the observed cell cycle defect (Hebeisen, 2008).

Mammalian Slimb homologs

Ubiquitin-mediated destruction of regulatory proteins is a frequent means of controlling progression through signaling pathways. F-box proteins are components of modular E3 ubiquitin protein ligases called SCFs, which function in phosphorylation-dependent ubiquitination. F-box proteins contain a carboxy-terminal domain that interacts with substrates and a 42-48 amino-acid F-box motif, which binds to the protein Skp1. Skp1 binding links the F-box protein with a core ubiquitin ligase composed of the proteins Cdc53/Cul1, Rbx1 (also called Hrt1 and Roc1) and the E2 ubiquitin-conjugating enzyme Cdc34. The genomes of the budding yeast Saccharomyces cerevisiae and the nematode worm C. elegans contain, respectively, 16 and more than 60 F-box proteins; in S. cerevisiae, the F-box proteins Cdc4, Grr1 and Met30 target cyclin-dependent kinase inhibitors, G1 cyclins and transcriptional regulators for ubiquitination. Only four mammalian F-box proteins (Cyclin F, Skp1, beta-TRCP and NFB42) have been identified so far. Here, the identification of a family of 33 novel mammalian F-box proteins is reported. The large number of these proteins in mammals suggests that the SCF system controls a correspondingly large number of regulatory pathways in vertebrates. Four of these proteins contain a novel conserved motif: the F-box-associated (FBA) domain, which may represent a new protein-protein interaction motif. The identification of these genes will help uncover pathways controlled by ubiquitin-mediated proteolysis in mammals (Winston, 1999b).

In normal and transformed cells, the F-box protein p45SKP2 is required for S phase and forms stable complexes with p19SKP1 and cyclin A-cyclin-dependent kinase (CDK)2. Human CUL-1, a member of the cullin family, and the ubiquitin-conjugating enzyme CDC34 are identified as additional partners of p45SKP2 in vivo. CUL-1 also associates with cyclin A and p19SKP1 in vivo and, with p45SKP2, they assemble into a large multiprotein complex. In Saccharomyces cerevisiae, a complex of similar molecular composition (an F-box protein, a member of the cullin family and a homolog of p19SKP1) forms a functional E3 ubiquitin protein ligase complex, designated SCFCDC4, that facilitates ubiquitination of a CDK inhibitor by CDC34. The data presented here imply that the p45SKP2-CUL-1-p19SKP1 complex may be a human representative of an SCF-type E3 ubiquitin protein ligase. It is proposed that all eukaryotic cells may use a common ubiquitin conjugation apparatus to promote S phase. Multiprotein complex formation involving p45SKP2-CUL-1 and p19SKP1 is governed, in part, by periodic, S phase-specific accumulation of the p45SKP2 subunit and by the p45SKP2-bound cyclin A-CDK2. The dependency of p45SKP2-p19SKP1 complex formation on cyclin A-CDK2 may ensure tight coordination of the activities of the cell cycle clock with those of a potential ubiquitin conjugation pathway (Lisztwan, 1999).

HOS, a human homolog of Slimb, forms an SCF complex with Skp1 and Cullin1 and targets the phosphorylation-dependent degradation of IkappaB and beta-catenin. SCF E3 ubiquitin ligases mediate ubiquitination and proteasome-dependent degradation of phosphorylated substrates. A human F-box/WD40 repeats protein (HOS), has been identified that is homologous to Slimb/h betaTrCP. Being a part of SCF complex with Skp1 and Cullin1, HOS specifically interacts with the phosphorylated IkappaB and beta-catenin, targeting these proteins for proteasome-dependent degradation in vivo. This targeting requires Cullin1 because expression of a mutant Cullin1 abrogates the degradation of IkappaB and of beta-catenin. Mutant HOS, which lacks the F-box, blocks TNF alpha-induced degradation of IkappaB as well as GSK3beta-mediated degradation of beta-catenin. This mutant also inhibits NF-kappaB transactivation and increases the beta-catenin-dependent transcription activity of Tcf. These results demonstrate that SCF(HOS) E3 ubiquitin ligase regulate both NF-kappaB and beta-catenin signaling pathways (Fuchs, 1999).

The SCF ubiquitin ligase complex of budding yeast triggers DNA replication by catalyzing ubiquitination of the S phase cyclin-dependent kinase inhibitor, SIC1. SCF is composed of three proteins (ySKP1, CDC53 [Cullin], and the F-box protein CDC4) that are conserved from yeast to humans. As part of an effort to identify components and substrates of a putative human SCF complex, hSKP1 has been isolated in a two-hybrid screen with hCUL1, the closest human homolog of CDC53. hCUL1 associates with hSKP1 in vivo and directly interacts with both hSKP1 and the human F-box protein SKP2 in vitro, forming an SCF-like particle. Moreover, hCUL1 complements the growth defect of yeast cdc53(ts) mutants; it associates with ubiquitination-promoting activity in human cell extracts, and can assemble into functional, chimeric ubiquitin ligase complexes with yeast SCF components. Taken together, these data suggest that hCUL1 functions as part of an SCF ubiquitin ligase complex in human cells. Further application of biochemical assays similar to those described here can now be used to identify regulators/components of hCUL1-based SCF complexes, to determine whether the hCUL2-hCUL5 proteins also are components of ubiquitin ligase complexes in human cells, and to screen for chemical compounds that modulate the activities of the hSKP1 and hCUL1 proteins (Lyapina, 1998).

HIV-1 Vpu interacts with CD4 in the endoplasmic reticulum and triggers CD4 degradation, presumably by proteasomes. Human beta TrCP identified by interaction with Vpu connects CD4 to this proteolytic machinery; CD4-Vpu-beta TrCP ternary complexes have been detected by coimmunoprecipitation. beta TrCP binding to Vpu and its recruitment to membranes require two phosphoserine residues in Vpu essential for CD4 degradation. In beta TrCP, WD repeats at the C terminus mediate binding to Vpu, and an F box near the N terminus is involved in interaction with Skp1p, a targeting factor for ubiquitin-mediated proteolysis. An F-box deletion mutant of beta TrCP has a dominant-negative effect on Vpu-mediated CD4 degradation. These data suggest that beta TrCP and Skp1p represent components of a novel ER-associated protein degradation pathway that mediates CD4 proteolysis (Margottin, 1998).

Mammalian Slimb homologs: Roles in Wnt signaling

Defects in beta-catenin regulation contribute to the neoplastic transformation of mammalian cells. Dysregulation of beta-catenin (Drosophila homolog: Armadillo) can result from missense mutations that affect critical sites of phosphorylation by glycogen synthase kinase 3beta (GSK3beta). Given that phosphorylation can regulate targeted degradation of beta-catenin by the proteasome, beta-catenin might interact with an E3 ubiquitin ligase complex containing an F-box protein, as is the case for certain cell cycle regulators. Accordingly, disruption of the Drosophila F-box protein Slimb upregulates the beta-catenin homolog Armadillo. It was reasoned that the human homologs of Slimb (beta-TrCP and its isoform beta-TrCP2 [KIAA0696]) might interact with beta-catenin. The binding of beta-TrCP to beta-catenin is direct and dependent upon the WD40 repeat sequences in beta-TrCP and on phosphorylation of the GSK3beta sites in beta-catenin. Endogenous beta-catenin and beta-TrCP can be coimmunoprecipitated from mammalian cells. Overexpression of wild-type beta-TrCP in mammalian cells promotes the downregulation of beta-catenin, whereas overexpression of a dominant-negative deletion mutant upregulates beta-catenin protein levels and activates signaling dependent on the transcription factor Tcf. In contrast, beta-TrCP2 does not associate with beta-catenin. It is concluded that beta-TrCP is a component of an E3 ubiquitin ligase that is responsible for the targeted degradation of phosphorylated beta-catenin (Hart, 1999).

beta-catenin plays an essential role in the Wingless/Wnt signaling cascade and is a component of the cadherin cell adhesion complex. Deregulation of beta-catenin accumulation as a result of mutations in adenomatous polyposis coli (APC) tumor suppressor protein is believed to initiate colorectal neoplasia. beta-catenin levels are regulated by the ubiquitin-dependent proteolysis system and beta-catenin ubiquitination is preceded by phosphorylation of its N-terminal region by the glycogen synthase kinase-3beta (GSK-3beta)/Axin kinase complex. FWD1 (the mouse homolog of Slimb/betaTrCP), an F-box/WD40-repeat protein, specifically forms a multi-molecular complex with beta-catenin, Axin, GSK-3beta and APC. Mutations at the signal-induced phosphorylation site of beta-catenin inhibit beta-catenin association with FWD1. FWD1 facilitates ubiquitination and promotes degradation of beta-catenin, resulting in reduced cytoplasmic beta-catenin levels. In contrast, a dominant-negative mutant form of FWD1 inhibits the ubiquitination process and stabilizes beta-catenin. These results suggest that the Skp1/Cullin/F-box protein FWD1 (SCFFWD1)-ubiquitin ligase complex is involved in beta-catenin ubiquitination and that FWD1 serves as an intracellular receptor for phosphorylated beta-catenin. FWD1 also links the phosphorylation machinery to the ubiquitin-proteasome pathway to ensure prompt and efficient proteolysis of beta-catenin in response to external signals. SCFFWD1 may be critical for tumor development and suppression through regulation of beta-catenin protein stability (Kitagawa, 1999).

Regulation of beta-catenin stability is essential for Wnt signal transduction during development and tumorigenesis. It is well known that serine-phosphorylation of beta-catenin by the Axin-glycogen synthase kinase (GSK)-3beta complex targets beta-catenin for ubiquitination-degradation, and mutations at critical phosphoserine residues stabilize beta-catenin and cause human cancers. Phosphorylated beta-catenin is specifically recognized by beta-Trcp, an F-box/WD40-repeat protein that also associates with Skp1, an essential component of the ubiquitination apparatus. beta-Trcp is a homolog of Drosophila Slimb. beta-catenin harboring mutations at the critical phosphoserine residues escapes recognition by beta-Trcp, thus providing a molecular explanation for why these mutations cause beta-catenin accumulation that leads to cancer. Inhibition of endogenous beta-Trcp function by a dominant negative mutant stabilizes beta-catenin, activates Wnt/beta-catenin signaling, and induces axis formation in Xenopus embryos. Therefore, beta-Trcp plays a central role in recruiting phosphorylated beta-catenin for degradation and in dorsoventral patterning of the Xenopus embryo (Liu, 1999).

Mammalian Slimb homologs: Roles in NF-kappaB signaling

NF-kappaB, a ubiquitous, inducible transcription factor involved in immune, inflammatory, stress and developmental processes, is retained in a latent form in the cytoplasm of non-stimulated cells by inhibitory molecules: IkappaBs (Homologs of Drosophila Cactus). Its activation is a paradigm for a signal-transduction cascade that integrates an inducible kinase and the ubiquitin-proteasome system to eliminate inhibitory regulators. The pIkappaBalpha-ubiquitin ligase (pIkappaBalpha-E3) has been isolated. This ligase attaches ubiquitin, a small protein that marks other proteins for degradation by the proteasome system, to the phosphorylated NF-kappaB inhibitor pIkappaBalpha. Taking advantage of its high affinity to pIkappaBalpha, this ligase has been isolated from HeLa cells by single-step immunoaffinity purification. Using nanoelectrospray mass spectrometry, the specific component of the ligase that recognizes the pIkappaBalpha degradation motif has been identified as an F-box/WD-domain protein belonging to a recently distinguished family of beta-TrCP/Slimb proteins. This component, which is denoted E3RSIkappaB (pIkappaBalpha-E3 receptor subunit), binds specifically to pIkappaBalpha and promotes its in vitro ubiquitination in the presence of two other ubiquitin-system enzymes, E1 and UBC5C, one of many known E2 enzymes. An F-box-deletion mutant of E3RS(IkappaB), which tightly binds pIkappaBalpha but does not support its ubiquitination, acts in vivo as a dominant-negative molecule, inhibiting the degradation of pIkappaBalpha and consequently NF-kappaB activation. E3RS(IkappaB) represents a family of receptor proteins that are core components of a class of ubiquitin ligases. When these receptor components recognize their specific ligand, which is a conserved, phosphorylation-based sequence motif, they target regulatory proteins containing this motif for proteasomal degradation (Yaron, 1998).

FWD1 (the mouse homolog of Drosophila Slimb and Xenopus betaTrCP, a member of the F-box- and WD40 repeat-containing family of proteins, and a component of the SCF ubiquitin ligase complex) interacts with IkappaBalpha and thereby promotes IkappaBalpha ubiquitination and degradation. FWD1 also binds to IkappaBbeta and IkappaBepsilon and induces their ubiquitination and proteolysis. FWD1 recognizes the conserved DSGPsiXS motif (where Psi represents the hydrophobic residue) present in the NH(2)-terminal regions of these three IkappaB proteins only when the component serine residues are phosphorylated. However, in contrast to IkappaBalpha and IkappaBbeta, the recognition site in IkappaBepsilon for FWD1 is not restricted to the DSGPsiXS motif; FWD1 also interacts with other sites in the NH(2)-terminal region of IkappaBepsilon. Substitution of the critical serine residues in the NH(2)-terminal regions of IkappaBalpha, IkappaBbeta, and IkappaBepsilon with alanines also markedly reduces the extent of FWD1-mediated ubiquitination of these proteins and increases their stability. These data indicate that the three IkappaB proteins, despite their substantial structural and functional differences, all undergo ubiquitination mediated by the SCF(FWD1) complex. FWD1 may thus play an important role in NF-kappaB signal transduction through regulation of the stability of multiple IkappaB proteins (Shirane, 1999).

Ubiquitin-mediated proteolysis has a central role in controlling the intracellular levels of several important regulatory molecules, such as cyclins, CKIs, p53, and IkappaBalpha. Many diverse proinflammatory signals lead to the specific phosphorylation and subsequent ubiquitin-mediated destruction of the NF-kappaB inhibitor protein IkappaBalpha. Substrate specificity in ubiquitination reactions is, in large part, mediated by the specific association of the E3-ubiquitin ligases with their substrates. One class of E3 ligases is defined by the recently described SCF complexes, the archetype of which was first described in budding yeast and contains Skp1, Cdc53, and the F-box protein Cdc4. These complexes recognize their substrates through modular F-box proteins in a phosphorylation-dependent manner. A biochemical dissection is described of a novel mammalian SCF complex, SCFbeta-TRCP, that specifically recognizes a 19-amino-acid destruction motif in IkappaBalpha (residues 21-41) in a phosphorylation-dependent manner. This SCF complex also recognizes a conserved destruction motif in beta-catenin, a protein with levels also regulated by phosphorylation-dependent ubiquitination. Endogenous IkappaBalpha-ubiquitin ligase activity cofractionates with SCFbeta-TRCP. Furthermore, recombinant SCFbeta-TRCP assembled in mammalian cells contains phospho-IkappaBalpha-specific ubiquitin ligase activity. These results suggest that an SCFbeta-TRCP complex functions in multiple transcriptional programs by activating the NF-kappaB pathway and inhibiting the beta-catenin pathway (Winston, 1999a).

Activation of the transcription factor nuclear factor kappa B (NF-kappaB) is controlled by the proteolysis of its inhibitory subunit (IkappaB) via the ubiquitin-proteasome pathway. Signal-induced phosphorylation of IkappaBalpha by a large multisubunit complex containing IkappaB kinases is a prerequisite for ubiquitination. Here, FWD1 (a mouse homolog of Slimb/betaTrCP), a member of the F-box/WD40-repeat proteins, is associated specifically with IkappaBalpha only when IkappaBalpha is phosphorylated. The introduction of FWD1 into cells significantly promotes ubiquitination and degradation of IkappaBalpha in concert with IkappaB kinases, resulting in nuclear translocation of NF-kappaB. In addition, FWD1 strikingly evokes the ubiquitination of IkappaBalpha in the in vitro system. In contrast, a dominant-negative form of FWD1 inhibits the ubiquitination, leading to stabilization of IkappaBalpha. These results suggest that (1) the substrate-specific degradation of IkappaBalpha is mediated by a Skp1/Cull 1/F-box protein (SCF) FWD1 ubiquitin-ligase complex, and (2) that FWD1 serves as an intracellular receptor for phosphorylated IkappaBalpha. Skp1/Cullin/F-box protein FWD1 might play a critical role in transcriptional regulation of NF-kappaB through control of IkappaB protein stability (Hatakeyama, 1999).

The SCF complex containing Skp1, Cul1, and the F-box protein FWD1 (the mouse homolog of Drosophila Slimb and Xenopus beta-TrCP) functions as the ubiquitin ligase for IkappaBalpha. FWD1 associates with Skp1 through the F-box domain and also recognizes the conserved DSGXXS motif of IkappaBalpha. The structural requirements for the interactions of FWD1 with IkappaBalpha and with Skp1 have now been investigated further. The D31A mutation (but not the G33A mutation) in the DSGXXS motif of IkappaBalpha abolishes the binding of IkappaBalpha to FWD1 and its subsequent ubiquitination without affecting the phosphorylation of IkappaBalpha. The IkappaBalpha mutant D31E still exhibits binding to FWD1 and undergoes ubiquitination. These results suggest that, in addition to site-specific phosphorylation at Ser(32) and Ser(36), an acidic amino acid at position 31 is required for FWD1-mediated ubiquitination of IkappaBalpha. Deletion analysis of Skp1 reveals that residues 61-143 of this protein are required for binding to FWD1. In contrast, the highly conserved residues Pro(149), Ile(160), and Leu(164) in the F-box domain of FWD1 are dispensable for binding to Skp1. Together, these data delineate the structural requirements for the interactions among IkappaBalpha, FWD1, and Skp1 that underlie substrate recognition by the SCF ubiquitin ligase complex (Hattori, 1999).

Homologue of Slimb (HOS)/beta-transducin repeats-containing proteins up-regulate nuclear factor kappaB activity by targeting its inhibitor (IkappaB) for ubiquitination and subsequent degradation. Whether inhibition of HOS function may modulate apoptosis in human melanoma cells has been investigated. Forced expression of the dominant negative HOSdeltaF construct inhibits IkappaB degradation and leads to sensitization of melanoma cells to apoptosis induced by tumor necrosis factor alpha with cycloheximide, as well as by cisplatin and ionizing and UV irradiation. These data indicate that HOS plays an important role in controlling the IkappaB-dependent apoptotic pathways in human melanoma (Soldatenkov, 1999).

Mammalian Slimb homologs: Roles in Hedgehog signaling

Drosophila Suppressor of fused [Su(fu)] encodes a novel 468-amino-acid cytoplasmic protein which, by genetic analysis, functions as a negative regulator of the Hedgehog segment polarity pathway. Described here is the primary structure, tissue distribution, biochemical and functional analyses of a human Su(fu): [hSu(fu)]. Two alternatively spliced isoforms of hSu(fu) were identified, predicting proteins of 433 and 484 amino acids, with a calculated molecular mass of 48 and 54 kDa, respectively. The two proteins differ only by the inclusion or exclusion of a 52-amino-acid extension at the carboxy terminus. Both isoforms are expressed in multiple embryonic and adult tissues, and exhibit a developmental profile consistent with a role in Hedgehog signaling. The hSu(fu) contains a high-scoring PEST-domain, and exhibits an overall 37% sequence identity (63% similarity) with the Drosophila protein and 97% sequence identity with the mouse Su(fu). The hSu(fu) locus maps to chromosome 10q24-q25, a region that is deleted in glioblastomas, prostate cancer, malignant melanoma and endometrial cancer. HSu(fu) represses activity of the zinc-finger transcription factor Gli, which mediates Hedgehog signaling in vertebrates, and physically interacts with Gli, Gli2 and Gli3 as well as with Slimb, an F-box containing protein which, in the fly, suppresses the Hedgehog response, in part by stimulating the degradation of the fly Gli homolog. Coexpression of Slimb with Su(fu) potentiates the Su(fu)-mediated repression of Gli. Taken together, these data provide biochemical and functional evidence for the hypothesis that Su(fu) is a key negative regulator in the vertebrate Hedgehog signaling pathway. The data further suggest that Su(fu) can act by binding to Gli and inhibiting Gli-mediated transactivation as well as by serving as an adaptor protein, which links Gli to the Slimb-dependent proteasomal degradation pathway (Stone, 1999).

Hedgehog-regulated processing of the transcription factor Cubitus interruptus (Ci) in Drosophila depends on phosphorylation of the C-terminal region of Ci by cAMP-dependent protein kinase and subsequently by Casein kinase 1 and Glycogen synthase kinase 3. Ci processing also requires Slimb, an F-box protein of SCF (Skp1/Cullin/F-box proteins) complex, and the proteasome, but the interplay between phosphorylation and the activity of Slimb and the proteasome remains unclear. This study shows that processing of the Gli3 protein, a homolog of Ci, also depends on phosphorylation of a set of four cAMP-dependent protein kinase sites that primes subsequent phosphorylation of adjacent casein kinase 1 and glycogen synthase kinase 3. Gain- and loss-of-function analyses in cultured cells further reveal that ßTrCP, the vertebrate homolog of Slimb, is required for Gli3 processing, and ßTrCP can bind phosphorylated Gli3 both in vitro and in vivo. Gli3 protein is polyubiquitinated in the cell, and its processing depends on proteasome activity. These findings provide evidence for a direct link between phosphorylation of Gli3/Ci proteins and ßTrCP/Slimb action, thus supporting the hypothesis that the processing of Gli3/Ci is affected by the proteasome (Wang, 2006).

Mammalian Slimb homologs and Presenilin degradation

Mutations in either of two human presenilin genes (PS1 and PS2) cause Alzheimer's disease (see Drosophila Presenilin). Genetic and physical interactions between Caenorhabditis elegans SEL-10, a member of the Cdc4p family of proteins, and SEL-12, a C. elegans presenilin are described. Loss of sel-10 activity can suppress the egg-laying defective phenotype associated with reducing sel-12 activity, and SEL-10 can physically complex with SEL-12. Proteins of the Cdc4p family have been shown to target proteins for ubiquitin-mediated turnover. The functional and physical interaction between sel-10 and sel-12 therefore offers an approach to understanding how presenilin levels are normally regulated (Wu, 1998).

Developmental roles for Slimb homologs

Mutations that influence lin-12 activity in C. elegans may identify conserved factors that regulate the activity of lin-12/Notch proteins (see Drosophila Notch). Genetic evidence indicates that sel-10 is a negative regulator of lin-12/Notch-mediated signaling in C. elegans. Sequence analysis shows that SEL-10 is a member of the CDC4 family of proteins and has a potential human ortholog. Coimmunoprecipitation data indicate that C. elegans SEL-10 complexes with LIN-12 and with murine Notch4. It is proposed that SEL-10 promotes the ubiquitin-mediated turnover of LIN-12/Notch proteins (Hubbard, 1997).

Proteolysis of LIN-12/Notch proteins might occur in response to ligand binding or occur constitutively. For a variety of cell surface receptors, ligand-induced polyubiquitination appears to be a mechanism for down-regulation. Although there is no direct evidence for ligand-induced ubiquitination of LIN-12/Notch receptors, it has been noted that LIN-12(intra), which genetically and physically interacts with SEL-10, behaves like an activated receptor. Alternatively, SEL-10 may target any form of LIN-12/Notch (activated or unactivated) for degradation. Although constitutive turnover is not strictly a mechanism for controlling receptor activity per se, it would, in effect, sensitize the system to other control mechanisms such as transcriptional regulation by generally reducing the amount of LIN-12 (Hubbard, 1997).

Constitutive turnover or ligand-induced down-regulation of LIN-12/Notch proteins may be important for cell fate decisions to occur normally. Potential roles for turnover or down-regulation can be illustrated with the AC/VU decision as an example. Initially, Z1.ppp and Z4.aaa have equal signaling and receiving potentials; ligand (LAG-2) and receptor (LIN-12) may interact, but signaling activity is below a critical threshold. SEL-10-mediated turnover or down-regulation of LIN-12 might prevent this initial signaling from causing both cells to achieve the threshold value of effector activity. Thus, one possible role for receptor turnover or down-regulation would be to limit the output from a single ligand-receptor interaction. Another potential role for receptor turnover or down-regulation is in enhancing differences in lin-12 activity between interacting cells. During the AC/VU decision, a small stochastic difference between the two cells is amplified by a feedback mechanism. The feedback mechanism appears to involve differential transcription of ligand and receptor genes: activation of LIN-12 appears to repress transcription of lag-2 and to stimulate transcription of lin-12. The feedback mechanism ensures that the cell with higher lin-12 activity becomes the VU whereas the cell with lower lin-12 activity becomes the AC. Down-regulation of LIN-12 would be necessary for differences in transcription to be manifest. In the absence of down-regulation, signaling from activated receptor would persist, masking the effects of differential transcription. Indeed, this situation is analogous to the role of ubiquitin-mediated degradation of G1 cyclins (Hubbard, 1997).

The Wnt/beta-catenin signaling pathway is responsible for the establishment of the dorsoventral axis of Xenopus embryos. The recent finding of the F-box/WD40-repeat protein Slimb in Drosophila, whose loss-of-function mutation causes ectopic activation of Wingless signaling, has led to an examination of the role of its vertebrate homolog betaTrCp in the Wnt/beta-catenin signaling and dorsal axis formation in Xenopus embryos. Co-injection of betaTrCp mRNA diminishes Xwnt8 mRNA-induced axis formation and expression of Siamois and Xnr3, suggesting that betaTrCP is a negative regulator of the Wnt/beta-catenin signaling pathway. An mRNA for a betaTrCp mutant construct (DeltaF), which lacks the F-box domain, induces an ectopic axis and expression of Siamois and Xnr3. Because this activity of DeltaF is suppressed by co-injection of DeltaF TrCP mRNA, DeltaF likely acts in a dominant negative fashion. The activity of DeltaF is diminished by C-cadherin, glycogen synthase kinase 3 and Axin, but not by a dominant negative dishevelled. These results suggest that betaTrCp can act as a negative regulator of dorsal axis formation in Xenopus embryos (Marikawa, 1998).

Developmental roles for Slimb homologs: Ebi, a Drosophila Slimb-like protein

ebi (the term for 'shrimp' in Japanese) regulates the epidermal growth factor receptor (Egfr) signaling pathway at multiple steps in Drosophila development. Mutations in ebi and Egfr lead to similar phenotypes and show genetic interactions. However, ebi does not show genetic interactions with other RTKs (e.g., torso) or with components of the canonical Ras/MAP kinase pathway. ebi encodes an evolutionarily conserved protein with a unique amino terminus, distantly related to F-box sequences, and six tandemly arranged carboxy-terminal WD40 repeats. The existence of closely related proteins in yeast, plants, and humans suggests that ebi functions in a highly conserved biochemical pathway. Proteins with related structures regulate protein degradation. Similarly, in the developing eye, ebi promotes Egfr-dependent down-regulation of Tramtrack88, an antagonist of neuronal development (Dong, 1999).

ebi mutations have been identified in a screen for enhancers of an eye mutant called roughex, which plays a key role in regulating cell cycle progression in the developing eye. As a consequence of cell cycle defects, photoreceptor differentiation and pattern formation in the eye are disrupted. Whereas cell cycle regulators enhance and suppress the primary cell cycle phenotype, mutations in other loci, such as Star and Epidermal growth factor receptor, only modify the differentiation phenotype, and not the earlier cell cycle defects. Like Star and Egfr, ebi enhances the differentiation phenotype. These observations led to a consideration of the relationship between the Egfr signaling pathway and ebi. Evidence shows that ebi participates in Egfr signaling pathways. ebi^E4, ebi^E90, and ebi^P7 are null, strong, and weak alleles, respectively (Dong, 1999).

That ebi functions in the Egfr pathway was initially suggested by phenotypes of a viable heteroallelic combination of ebi (i.e., ebi^P7/ebi^E90). These flies exhibit phenotypes similar to weak loss-of-function Egfr alleles (i.e., Egfr^top1/Egfr^f2) including partial female sterility resulting from partially ventralized eggs, wing vein defects, short bristles, and abnormal eyes (i.e., rough eyes). Further evidence that ebi participates in the Egfr pathway was provided by genetic interactions between ebi and Egfr components. For instance, flies carrying two different alleles of Egfr (Egfr^top1/Egfr^f2) have a weak rough-eye phenotype, which is enhanced in flies that are heterozygous for ebi. ebi and Egfr mutant embryos are also similar. Homozygous ebi null mutant embryos (ebi^E4) exhibit a tail-up or U-shaped embryo with head defects. Embryos lacking both the zygotic and maternal contributions of ebi were created using ovo^D and FRT/FLP-induced recombination. This results in a more severe phenotype, including the loss of ventral denticle belt structures and a tightly curled morphology indicating a marked failure in germ-band retraction. Severe head defects are also observed. In contrast to Egfr mutants, some residual ventral cuticular structures remain in embryos lacking both the zygotic and maternal contributions of ebi (Dong, 1999).

Loss of ebi also affects Egfr-dependent expression of genes in the embryo. The Egfr ligand Spitz is expressed along the ventral midline and induces expression of different target genes, including fasciclin III (fasIII) and orthodenticle (otd), in cells located in more lateral positions. In zygotic null Egfr mutants both otd and FasIII expression are lost. In wild-type stage 11/12 embryos, FasIII protein is broadly distributed in the visceral mesoderm and in a bilaterally symmetric cluster of cells flanking the midline of the ventral ectoderm. In ebi mutant embryos lacking both maternal and zygotic contribution, FasIII expression is largely abolished, although some residual patches of staining remain. Egfr-independent expression of FasIII in the anterior-most region of the embryo is unaffected in ebi mutants. In wild-type stage 10/11 embryos, otd mRNA is expressed in the preantennal head region and in the ventral-most ectoderm. In ebi mutant embryos, otd expression is markedly reduced. These data suggested that ebi may be a component in the Egfr signal transduction pathway. To assess whether ebi encoded a hitherto unidentified regulator in the Ras/MAP kinase pathway, its role in the Torso RTK pathway was assessed. Torso controls the development of the anterior and posterior termini of the embryo. Ras, Raf, MEK, and MAPK participate in both the Egfr and Torso RTK pathways. The expression of Torso target genes huckebein (hkb) and tailless (tll) in embryos entirely deficient in ebi (i.e., lacking both maternal and zygotic ebi) are indistinguishable from wild type. In summary, ebi mutant phenotypes assessed using both molecular and morphological criteria are similar to Egfr mutations. Furthermore, ebi does not function in all RTK pathways, since Torso-induced terminal development is ebi independent. These data indicate that ebi, either directly or indirectly, regulates Egfr signaling. As a step toward understanding the role of ebi in the context of a specific developmental process, the role of ebi in R7 development in the compound eye was assessed through both genetic and molecular studies (Dong, 1999).

The R7 equivalence group comprises five cells competent to become R7 neurons. They are the R7 precursor cell and the precursors to the four cone cells. Cone cell precursor cells can be induced to become R7 cells by ectopic activation of the R7 inductive pathway in these cells. Transformation of cone cells into R7 cells leads to a disorganized adult eye or a so-called rough-eye phenotype. The ability of loss-of-function ebi mutations to suppress this transformation was assessed in various genetic backgrounds. Whereas ebi dominantly suppresses R7 development induced by the activated Egfr expressed in the R7 equivalence group under the control of the sev enhancer (sev-Tor^DEgfr), it does not suppress R7 development induced by the activated Sev receptor (sev-Tor^DSev, SevS11, or activated forms of Ras, Raf, and MAPK. Hence, ebi is required for the transformation of cone cell precursors into R7 neurons by the activated Egfr (Dong, 1999).

To assess whether ebi participates in the induction of the R7 precursor cell into an R7 neuron, a genetically sensitized background in which only some 15%-20% of the R7 precursors become R7 neurons was used. The R7 inductive signal is attenuated by using a strong hypomorphic allele of sev (sev^E4) and a weak gain-of-function mutation in the Ras activator, encoded by the son-of-sevenless gene, Sos^JC2. Aside from the loss of the majority of the R7 cells, development of the eye in this genetic background is otherwise indistinguishable from wild type. ebi is a dominant enhancer of this phenotype, as are Egfr loss-of-function mutations. These data are consistent with studies demonstrating a requirement for both the Egfr and Sev receptor in R7 induction. Hence, ebi is required for induction of the R7 precursor cell into an R7 neuron and for transformation of cone cell precursors into R7 in response to ectopic activation of Egfr. Ttk88 down-regulation is required for R7 induction of the R7 precursor cell. This is supported by the finding that Ttk88 mutations are dominant suppressors of the Sev^E4;Sos^JC2/+ phenotype (Dong, 1999).

To assess the role of ebi on R7 development in an otherwise wild-type background, attempts were made to generate homozygous null mutant clones. Such clones could not be generated using X-ray and heat shock Flp-induced mitotic recombination. Hence, like Egfr, ebi is required for cell proliferation and/or survival during the proliferative phase of disc development. To increase the efficiency of Flp-induced mitotic recombination, a Flp source driven by the eyeless (ey) promoter was used. The ey promoter drives expression from the earliest cell divisions in the eye primordium until the last cell division of precursor cells in the third instar. This results in the production of multiple mutant clones throughout development. Mutant clones in the eye disc have been recognized by the loss of Ebi immunoreactivity. Rather small clones have been observed: clusters within these clones contain differentiating R cells. Each cluster contains a single R8 cell (i.e., stained with antibody to the Boss protein), and early clusters appear normal. Although clusters containing eight neurons form, disorganized clusters containing fewer differentiated neurons are also observed (Dong, 1999).

Adult ommatidia containing homozygous mutant cells are frequently highly disorganized and show a marked reduction in R cells. Mutant R cells, including R7 cells, are seen in adult mosaic ommatidia; some 80% of these cells show an altered cellular morphology. Hence, although ebi is required for R7 development in a genetically sensitized background, R7 neurons can develop in an ebi mutant. Although the formal possibility that these R7 neurons develop because of perdurance of Ebi protein in the R7 precursor cell cannot be ruled out, these data strongly suggest that R7 cells can form in an ebi-independent fashion, though less efficiently than in wild type. These data are consistent with ebi subserving a redundant function in R7 development. To gain clues to the molecular pathways regulated by ebi, the gene was cloned and sequenced (Dong, 1999). This transcription unit encodes a protein of 700 amino acids with a carboxy-terminal segment containing six WD40 repeats. The ebi^E4 and ebi^E90 alleles result in missense mutations. In ebi^E4 the methionine encoded by codon 1 is changed to an isoleucine, and in ebi^E90 a highly conserved cysteine, located at amino acid 510 between WD40 repeats 3 and 4, is changed to a tyrosine (Dong, 1999).

ebi-related human cDNA sequences and genomic sequences from S. cerevisiae and Arabidopsis thaliana, have been identified in the database. Because the initial human expressed sequence tag was not complete, additional cDNAs were isolated from adult human spleen cDNA library and sequenced. Both an amino-terminal 89-amino-acid segment and the carboxy-terminal WD40 repeats of fly ebi correspond remarkably well to these regions in the mammalian, plant, and yeast genes; the amino-terminal 89 amino acids and the WD40 repeat region share 81%, 34%, and 51% identity with the human, yeast, and plant sequences, respectively. In addition to these conserved regions the fly protein is predicted to contain an insertion of 160 amino acids between the amino terminus and the WD40 repeats (Dong, 1999).

The bipartite structure of Ebi is reminiscent of three proteins involved in protein degradation: Cdc4 from S. cerevisiae; Slimb from Drosophila melanogaster, and Sel-10 from C. elegans. All three proteins contain an amino-terminal F-box and carboxy-terminal WD40 repeats; these proteins have been shown (Cdc4) or proposed (Slimb and Sel-10) to target proteins for degradation by linking them to a ubiquitin-conjugase complex. Although the amino-terminal domain of Ebi is divergent from the Cdc4 F box (as is Slimb), it shares weak sequence and structural homology. The amino-terminal half of the F box is more highly related to ebi than the carboxy-terminal region. The periodic spacing of hydrophobic residues in both Ebi and F-box sequences is consistent with these regions being able to assume an alpha-helical amphipathic conformation. Three residues in the amino-terminal region of the Cdc4 F box have been shown to be required for binding to Skp1 (a component in the E3 complex). These amino acids are conserved in Ebi, and correspond to residues P45, I52, and L57 in the Ebi sequence (Dong, 1999).

Ebi is widely expressed in nuclei of the embryo and larvae. Immune staining is largely, if not exclusively, nuclear. Double staining of salivary gland nuclei with anti-Myc antibodies to detect Myc-tagged Ebi and the DNA stain DAPI demonstrates that Ebi was not associated with chromatin but, rather, is distributed in a reticular pattern throughout the nucleoplasm. The similarity of Ebi to F-box/WD40 repeat-containing proteins and its nuclear localization suggests that Ebi may regulate Egfr signaling through degradation of nuclear proteins. Recent studies have revealed an important role for both Egfr and degradation of a specific transcription factor Tramtrack88, for R7 development. The structural similarity between Ebi and F-box/WD40-repeat proteins involved in protein degradation prompted an exploration of the relationship between ebi and Ttk88 protein levels in the developing eye. Ttk88 is expressed at very low levels in undifferentiated cells in the developing eye disc and at high levels in developing cone cell nuclei; it is not expressed in developing photoreceptor cells. Transformation of cone cells into R7 by misexpression of phyl under the sev promoter leads to Ttk88 degradation. Ectopic R7 induction by Tor^DEgfr driven by the sev promoter also leads to marked degradation of Ttk88. sev-Tor^DEgfr-induced Ttk88 degradation is dominantly suppressed by ebi. Similarly, ebi dominantly suppresses the pGMR-phyl-induced decrease in Ttk88, as well as the pGMR-phyl-induced eye phenotype (Dong, 1999).

The role of ebi in regulating Ttk88 levels in an otherwise wild-type eye disc was examined. Analysis of Ttk88 levels on the small mutant clones generated with ey-Flp reveals no obvious differences. To explore this issue further, reduction in ebi was achieved by expressing the dominant-negative form of ebi in all cells posterior to the morphogenetic furrow in an ebi heterozygous background. Dominant-negative ebi contains the amino-terminal half of the protein from amino acids 1-334 expressed under the control of the pGMR promoter. In wild-type eye discs, Ttk88 staining is not observed in a focal plane in which photoreceptor cell nuclei are located. In contrast, in mutant discs, an average of 36 ± 6 Ttk88-positive nuclei are observed in this region. Most Ttk88-positive nuclei are found 8-10 rows posterior to the morphogenetic furrow. This increase in Ttk88-positive cells also parallels a concomitant decrease in the number of cells stained with the pan-neuronal stain, anti-Elav. In wild-type eye discs, all ommatidia 8-10 rows posterior to the morphogenetic furrow have at least seven Elav-positive cells (R1-R6 and R8). However, in mutant discs, many ommatidia in this region contained less than seven stained cells. Interestingly, a considerably smaller fraction of ommatidia in rows 11-13 contain less than eight Elav-positive R cells; in wild-type discs, all clusters contain eight Elav-positive cells in this region. Hence, a reduction in ebi activity delays neuronal development and this is correlated with persistent nuclear expression of the Ttk88 protein. In summary, both ebi and Egfr promote Ttk88 down-regulation, thereby promoting neuronal development. Further work is required to assess the relationship between ebi and ttk in Egfr signaling in other developmental contexts (Dong, 1999).

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