cAMP-dependent protein kinase 1

Activation of protein kinase A (PKA) at discrete intracellular sites facilitates oogenesis and development in Drosophila. Thus, PKA-anchor protein complexes may be involved in controlling these crucial biological processes. Evaluation of this proposition requires knowledge of PKA binding/targeting proteins in the fly. A novel, Drosophila A kinase anchor protein, DAKAP550, has been isolated and characterized. DAKAP550 is a large (>2300 amino acids) acidic protein that is maximally expressed in anterior tissues. It binds regulatory subunits (RII) of both mammalian and Drosophila PKAII isoforms. The tethering region of DAKAP550 includes two proximal, but non-contiguous RII-binding sites (B1 and B2). The B1 domain (residues 1406-1425) binds RII approximately 20-fold more avidly than B2 (amino acids 1350-1369). Affinity-purified anti-DAKAP550 IgGs were exploited to demonstrate that the anchor protein is expressed in many cells in nearly all tissues throughout the lifespan of the fly. The protein seems to be ubiquitous but exhibits differing levels in different tissues. For example, at gastrulation, anchor protein content is elevated in the ventral furrow and adjacent mesectodermal cells. Protein remains high in mesoderm for several hours and is elevated in neuroblasts delaminating from the ectoderm. Peripheral neurons, subsets of central neurons, hindgut, the tracheal system and salivary glands show substantial levels of anchor protein; the protein is especially enriched in neural cells, such as the photoreceptor neurons of the developing eye. DAKAP550 is asymmetrically positioned in subpopulations of neurons and in apical portions of cells in gut and trachea. The combination of RII (PKAII) binding activity with differential expression and polarized localization is consistent with a role for DAKAP550 in creating target loci for the reception of signals carried by cAMP. The DAKAP550 gene maps to the 4F1.2 region of the X chromosome; flies that carry a deletion for this portion of the X chromosome lack DAKAP550 protein (Han, 1997).

In the developing Drosophila eye, cell fate determination and pattern formation are directed by cell-cell interactions mediated by signal transduction cascades. Mutations at the rugose locus (rg) result in a rough eye phenotype due to a disorganized retina and aberrant cone cell differentiation, which leads to reduction or complete loss of cone cells. The cone cell phenotype is sensitive to the level of rugose gene function. Molecular analyses show that rugose encodes a Drosophila A kinase anchor protein (DAKAP 550). Genetic interaction studies show that rugose interacts with the components of the Egfr- and Notch-mediated signaling pathways. These results suggest that rg is required for correct retinal pattern formation and may function in cell fate determination through its interactions with the Egfr and Notch signaling pathways. rugose has also been identified in a genetic screen for modifiers of Hairless (H), a Notch pathway antagonist (Schreiber, 2002) and rugose interacts with Egfr and N signaling pathways (Shamloula, 2002).

Differentiation of the R7 photoreceptor cell is dependent on the Sevenless receptor tyrosine kinase, which activates the Ras1/mitogen-activated protein kinase signaling cascade. Kinase suppressor of ras (Ksr) functions genetically downstream of Ras1 in this signal transduction cascade. Expression of dominant-negative Ksr (KDN) in the developing eye blocks Ras pathway signaling, prevents R7 cell differentiation, and causes a rough eye phenotype. To identify genes that modulate Ras signaling, a screened was carried out for genes that alter Ras1/Ksr signaling efficiency when misexpressed. In this screen, three known genes, Lk6, misshapen, and Akap200, were recovered. Seven previously undescribed genes were recovered; one encodes a novel rel domain member of the NFAT family, and six encode novel proteins. These genes may represent new components of the RAS pathway or components of other signaling pathways that can modulate signaling by RAS (Huang, 2000).

One of the misexpression interactors, MESR2, was an insertion upstream of the Akap200 locus. DAKAP200 refers to Drosophila A kinase anchor protein of molecular weight 200 kd, and binds the regulatory II (rII) subunit of cyclic AMP-dependent protein kinase (PKA). The Akap200 gene produces two different transcripts, one that contains the binding site for RII and one where the exon encoding for the RII binding site is spliced out to generate a protein that does not interact directly with PKA. Both isoforms of AKAP200 are expressed at relatively similar levels throughout development as well as in adult heads (Huang, 2000).

PKA is the principal mediator of signals that activate adenylate cyclase. cAMP signals are often targeted to effectors that accumulate to discrete intracellular locations. This targeting is due to a nonuniform distribution of PKA molecules within cells. In Drosophila, PKA has been implicated in normal developmental events in all imaginal tissues through the Hedgehog signaling pathway and is involved in signaling pathways that generate cell polarity: this requires that Hh be localized to distinct intracellular locations. Subcellular localization of PKA occurs through association with AKAPs. AKAPs are a functionally related family of proteins, defined by their ability to associate with PKA. Each AKAP contains a unique targeting domain that directs the complex to a defined intracellular location where PKA is placed proximal to both a signal generator (adenylate cyclase) as well as to potential PKA effector molecules. Coordinate binding of specific combinations of enzymes can allow such complexes to respond to distinct second messenger-mediated signals (Huang, 2000).

Studies in mammalian cells have suggested that PKA signaling via Rap1, another small molecular weight GTP-binding protein, antagonizes RAS1 signaling by competing for RAS pathway components such as B-Raf and MAPK. However, more recent studies suggest no genetic interaction between Drosophila Rap1 and RAS1. In Drosophila, overexpression of Rap1 in a heterozygous RAS1 mutant background has no effect on photoreceptor determination, suggesting no interaction between the two gene functions. A heterozygous Rap1 mutation does not reduce the number of R7 cells in a sev-RAS1^V12 rough eye, also suggesting that the two pathways do not interact. Although there is no direct evidence linking PKA activation to MAPK activation via Rap1, there may be a still unknown pathway by which these molecules can signal (Huang, 2000).

The screen isolated Akap200 as a misexpression enhancer of KDN and suppressor of RAS1^V12. This suggests that overexpression of this AKAP decreases signaling through RAS1. Overexpression of an AKAP might cause mislocalization of PKA molecules to the plasma membrane. This could activate a signaling pathway that normally is not utilized in this cell or at this time in development. If PKA and Rap1 are involved in RAS signaling, why were they not uncovered in previous loss-of-function screens? One possibility is that mutations in either gene may not be dose sensitive and therefore be unable to dominantly modify a rough eye phenotype. Another is the possibility that overexpression of an AKAP causes abnormal targets of PKA to become activated. Whether PKA signals through Rap1 is still unclear; however, the reported effects of attenuating RAS1/MAPK signaling are supported by this study. The enhancement of the KDN rough eye phenotype could be due to the additive effects of inefficient signaling due to KDN as well as the attenuation of MAPK by mislocalized PKA. In the activated RAS1^V12 background, the attenuating effects of activated PKA due to mislocalization to the plasma membrane might reduce the amount of signaling through the pathway to suppress the RAS1-dependent rough eye phenotype (Huang, 2000).

The Drosophila homeodomain-containing protein Fushi tarazu (Ftz) is expressed sequentially in the embryo, first in alternate segments, then in specific neuroblasts and neurons in the central nervous system, and finally in parts of the gut. During these different developmental stages, the protein is heavily phosphorylated on different subsets of Ser and Thr residues. This stage-specific phosphorylation suggests possible roles for signal transduction pathways in directing tissue-specific Ftz activities. One of the Ftz phosphorylation sites, T263 in the N-terminus of the Ftz homeodomain, is phosphorylated in vitro by Drosophila embryo extracts and protein kinase A. In the embryo, mutagenesis of this site to the non-phosphorylatable residue Ala results in loss of ftz-dependent segments. Conversely, substitution of T263 with Asp, which is also non-phosphorylatable, but which successfully mimics phosphorylated residues in a number of proteins, rescues the mutant phenotype. This suggests that T263 is in the phosphorylated state when functioning normally in vivo. The T263 substitutions of Ala and Asp do not affect Ftz DNA-binding activity in vitro, nor do they affect stability or transcriptional activity in transfected S2 cells. This suggests that T263 phosphorylation is most likely required for a homeodomain-mediated interaction with an embryonically expressed protein. Preliminary experiments with Ftz T263 mutants suggest that the phosphorylation state of T263 does not affect either the Ftz-Prd or the Ftz-Ftz-F1 interaction. Hence, a yet to be identified protein expressed in embryos is likely to be the relevant target (Dong, 1998a).

In approximately half of all HOX proteins, residue 7 of the homeodomain (analogous to Ftz T263) is either a Thr or Ser residue, while position 5 is an Arg residue. This conserves the PKA recognition site, and suggests that each of these proteins probably shares the ability to be phosphorylated at this position. This includes the more divergent POU class homeodomain proteins Pit-1 and Oct-1, which like Ftz are phosphorylated efficiently by PKA, or a PKA-like kinase in vitro. It is also worth noting that some homeodomain proteins, such as En and Even-skipped (Eve), normally possess alanines at position 7. Hence, the substitution of Thr263 with Ala is a conservative one that is unlikely to exert its effect at the level of general homeodomain structure. This is consistent with the findings that this substitution has no apparent effect on DNA-binding activity, transactivating activity, protein stability or subcellular localization (Dong, 1998a).

Control over the nuclear import of transcription factors represents a level of gene regulation integral to cellular processes such as differentiation and transformation. The Drosophila transcription factor Dorsal shares with other family members of rel oncogene a phosphorylation site for the cAMP-dependent protein kinase (PKA) located 22 amino acids N-terminal to the nuclear localization signal (NLS) at amino acids 335-340. This study examines the nuclear import kinetics of Dorsal fusion proteins in rat hepatoma cells in vivo and in vitro. Nuclear uptake is not only NLS-dependent, but also strongly dependent on the PKA site, where by using site-directed mutagenesis, the substitution of Ser312 by either Ala or Glu severely reduces nuclear accumulation. Either exogenous cAMP or PKA catalytic subunit significantly enhance the nuclear import of wild-type proteins both in vivo and in vitro. Using a direct binding assay, the molecular basis of PKA site enhancement of Dorsal fusion protein nuclear import was determined to be PKA site-mediated modulation of NLS recognition by the importin 58/97 complex. The physiological relevance of these results is supported by the observation that Drosophila embryos expressing PKA site Dorsal mutant variants are impaired in development. It is concluded that the Dorsal NLS and PKA site constitute a phosphorylation-regulated NLS essential to Dorsal function and able to function in heterologous mammalian cell systems, where phosphorylation modulates the affinity of NLS recognition by importin (Briggs, 1998).

In Drosophila, dorsal-ventral polarity is determined by a maternally encoded signal transduction pathway that culminates in the graded nuclear localization of the Rel protein, Dorsal. Dorsal is retained in the cytoplasm by the IkappaB protein, Cactus. Signal-dependent phosphorylation of Cactus results in the degradation of Cactus and the nuclear targeting of Dorsal. An in-depth study of the functional importance of Dorsal phosphorylation is presented. Dorsal is phosphorylated by the ventral signal while associated with Cactus, and Dorsal phosphorylation is essential for its nuclear import. In vivo phospholabeling of Dorsal is limited to serine residues in both ovaries and early embryos. A protein bearing mutations in six conserved serines abolishes Dorsal activity; it is constitutively cytoplasmic, and appears to eliminate Dorsal phosphorylation, but still interacts with Cactus. Two individual serine-to-alanine mutations have produced unexpected results. In a wild-type signaling background, a mutation in the highly conserved PKA site (S312) produces only a weak loss-of-function; however, it completely destabilizes the protein in a cactus mutant background. Significantly, the phosphorylation of another completely conserved serine (S317) regulates the high level of nuclear import found in ventral cells. It is concluded that the formation of a wild-type Dorsal nuclear gradient requires the phosphorylation of both Cactus and Dorsal. The strong conservation of the serines suggests that phosphorylation of other Rel proteins is essential for their proper nuclear targeting (Drier, 1999).

Two of the serines that were changed to alanines are predicted to be phosphorylated by specific kinases. S79 is part of a predicted casein kinase II (CK II) site, and interestingly, this is the only mutant that retains wild-type function, suggesting that if the serine is phosphorylated, the modification is not essential. S312 is part of a PKA recognition site. This site may or may not be phosphorylated, but it is not the target of the signal-dependent phosphorylation that occurs on Dorsal. Rather, if S312 is phosphorylated, the modification may occur already in the ovary, and stabilize the protein. A serine (S317) that is not part of any known kinase recognition site is the likely target of the signal-dependent phosphorylation. This raises the question as to what kinase is responsible for the modification. Tube and Pelle are components of the dorsal-ventral signal transduction pathway that function downstream from the Toll receptor. They interact directly with Dorsal, albeit in the amino-terminal domain 1 of the Rel homology region (RHR). This interaction represents an essential step in the transduction of the ventral signal. Pelle is a serine-threonine kinase and may be directly responsible for the phosphorylation of Dorsal. However, as S317 is in domain 2 of the RHR, it is possible that additional kinases, such as the IB kinases, phosphorylate Dorsal and that Pelle controls their activity (Drier, 1999).

Calcium-dependent potassium (KCa) channels carry ionic currents that regulate important cellular functions. Like some other ion channels, KCa channels are modulated by protein phosphorylation. The recent cloning of complementary DNAs encoding Slowpoke KCa channels has enabled KCa channel modulation to be investigated. Protein phosphorylation modulates the activity of Drosophila Slo KCa channels expressed in Xenopus oocytes. Application of ATP-gamma S to detached membrane patches increases Slo channel activity by shifting channel voltage sensitivity. This modulation is blocked by a specific inhibitor of cyclic AMP-dependent protein kinase (PKA). Mutation of a single serine residue in the channel protein also blocks modulation by ATP-gamma S, demonstrating that phosphorylation of the Slo channel protein itself modulates channel activity. The results also indicate that KCa channels in oocyte membrane patches can be modulated by an endogenous PKA-like protein kinase which remains functionally associated with the channels in the detached patch (Esguerra, 1994).

Tyrosine hydroxylase (TH) catalyzes the first step in dopamine biosynthesis in Drosophila, just as it does in vertebrates. Tissue-specific alternative splicing of the TH primary transcript generates two distinct TH isoforms in Drosophila, DTH I and DTH II. Expression of DTH I is restricted to the central nervous system, whereas DTH II is expressed in non-nerve tissue, like the epidermis. The two enzymes present a single structural difference; DTH II specifically contains a very acidic segment of 71 amino acids inserted in the regulatory domain. The enzymatic and regulatory properties of vertebrate TH are generally conserved in insect TH and the isoform DTH II presents unique characteristics. The two DTH isoforms were expressed as apoenzymes in Escherichia coli and purified by fast protein liquid chromatography. The recombinant DTH isoforms are enzymatically active in the presence of ferrous iron and a tetrahydropteridine co-substrate. However, the two enzymes differ in many of their properties. DTH II has a lower Km value for the co-substrate (6R)-tetrahydrobiopterin and for its activation requires a lower level of ferrous ion than DTH I. The two isoforms also have a different pH profile. As for mammalian TH, enzymatic activity of the Drosophila enzymes is decreased by dopamine binding, and this effect is dependent on ferrous iron levels. However, DTH II appears comparatively less sensitive than DTH I to dopamine inhibition. The central nervous system isoform DTH I is activated through phosphorylation by cAMP-dependent protein kinase (PKA) in the absence of dopamine. In contrast, activation of DTH II by PKA is only manifest in the presence of dopamine. Site-directed mutagenesis of Ser32, a serine residue occurring in a PKA site conserved in all known TH proteins, abolishes phosphorylation of both isoforms and activation by PKA. It is proposed that tissue-specific alternative splicing of TH has a functional role for differential regulation of dopamine biosynthesis in the nervous and non-nervous tissues of insects (Vié, 1999).

Dopamine and other catecholamines inhibit and stabilize vertebrate TH activity, and iron stimulates dopamine binding. This regulation is conserved in Drosophila TH; the level of inhibition by dopamine depends on ferrous iron concentration. For vertebrate TH, it has been demonstrated that catecholamine binds to the active, iron-oxidized site in a ferric redox state, thus stabilizing the enzyme in an inactivated form. Phosphorylation by PKA decreases the binding affinity of human TH for dopamine, and conformation studies suggest that the catalytic site lies in close proximity or interacts with the PKA phosphorylation site of the regulatory domain. Deletion of the NH2-terminal regulatory domain of human TH1 (up to the PKA phosphorylation site [Ser40]) abolishes the inhibitory effect of dopamine. All these data strongly suggest that: (1) there is a direct interaction between the region surrounding the PKA site in the regulatory domain, and the iron binding site occurring close to the active site in the catalytic domain; (2) this interaction is required for dopamine binding and inhibition of TH activity, and (3) this interaction is relieved by phosphorylation of Ser40. Interestingly, DTH II seems to be more resistant than DTH I to dopamine inhibition, suggesting again that this isoform is at least partially in an activated state comparable to that of phosphorylated TH (Vié, 1999 and references).

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 (Supernumerary limbs), 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 ptc and en appears to be exclusively controlled by the activator form of Ci whereas dpp 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 Fu 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).

The Hedgehog (Hh) signal is transduced via Cubitus interruptus (Ci) to specify cell fates in the Drosophila wing. In the absence of Hh, the 155 kDa full-length form of Ci is cleaved into a 75 kDa repressor. Hh inhibits the proteolysis of full-length Ci and facilitates its conversion into an activator. Recently, it has been suggested that Hh promotes Ci nuclear import in tissue culture cells. The mechanism of Ci nuclear import in vivo and the relationship between nuclear import, stabilization and activation have been studied. Ci rapidly translocates to the nucleus in cells close to the anteroposterior (AP) boundary and this rapid nuclear import requires Hh signaling. The nuclear import of Ci is regulated by Hh even under conditions in which Ci is fully stabilized. Furthermore, cells that exhibit Ci stabilization and rapid nuclear import do not necessarily exhibit maximal Ci activity. It has been previously shown that stabilization does not suffice for activation. Consistent with this finding, the results suggest that the mechanisms regulating nuclear import, stabilization and activation are distinct from one another. cos2 and pka, two molecules that have been characterized primarily as negative regulators of Ci activity, also have positive roles in the activation of Ci in response to Hh (Wang, 2000).

In order to analyze the nuclear import of Ci in vivo, imaginal discs were subjected to whole-mount organ culture experiments, which allowed studies to be performed in different genetic backgrounds and under conditions that preserve the spatial context of Hh signaling. Because imaginal discs are sacs with only two layers of epidermal cells, drugs that inhibit cellular processes work as well in this context as on tissue culture cells. When protein export is blocked with LMB, a potent inhibitor of protein nuclear export, the amount of Ci peptides that enter the nucleus can be monitored, and a remarkable difference is observed in cells close to and those away from the AP boundary. Ci accumulates in the nucleus only in cells close to the boundary, and this has been further shown to be Hh dependent, since mutations in smo prevent nuclear accumulation of Ci. The rate of Ci nuclear import in the presence of Hh is very rapid in vivo, and the subcellular distribution of Ci shifts from predominantly cytoplasmic to predominantly nuclear within as short a time as half an hour of treatment. The rate difference of Ci nuclear import in cells receiving and not receiving Hh is also significant. The cytoplasmic localization of Ci is unchanged in cells away from the boundary after three hours of treatment. From these results, it can be concluded that the rate of Ci nuclear import is dramatically increased by Hh signaling in vivo (Wang, 2000).

pka mutation has a negative effect on the expression of hh/ci target genes. Mutations in pka are associated with disc overgrowth and duplication, signs of up-regulated Ci activity. The level of full-length Ci is elevated in pka loss-of-function clones and these clones ectopically express ptc and dpp. PKA activity is required for the cleavage of Ci. Nevertheless, PKA is not involved in nuclear import of Ci (Wang, 2000).

Another molecule that negatively regulates Ci stability and hh target gene expression is Cos2. To directly test the role of cos2 in Ci nuclear import, discs carrying cos2 clones were treated with LMB. All clones exhibit cell-autonomous nuclear accumulation of Ci regardless of the clone’s distance from the AP boundary. cos2 encodes a kinesin-like molecule that is part of a multi-protein complex including Ci. Over-expression of cos2 blocks nuclear entry of Ci in tissue culture cells, and it has been proposed that Cos2 regulates the microtubule-association of the complex and consequently the ability of Ci to translocate to the nucleus (Wang, 2000).

Discs mutant for fu show signs of compromised Ci activity, including fusion between wing veins 3 and 4, lack of late en expression in the anterior compartment, and diffuse ptc expression at lower levels. Examination of these discs after LMB treatment reveals a lack of Ci nuclear accumulation, suggesting that fu function is required for rapid Ci nuclear import. It has been shown previously that overexpression of ci can rescue the wing vein phenotype in fu mutants. Consistent with this finding, the Ci nuclear import phenotype is also partially rescued in fu discs in which a UAS-ci transgene is over-expressed along the AP boundary via ptcGAL4 (Wang, 2000).

The role of cos2 in Hh signal transduction has been studied by examining the expression of ptc in cos2 clones. High level ptc expression is dependent on Hh signaling and is normally found only in the Boundary Zone, namely the 2-3 rows of cells immediately anterior to the compartment boundary. Although cos2 clones away from the boundary can ectopically induce ptc expression, a small number of such clones do not express ptc. A striking variability in the expression of ptc is observed for cos2 clones abutting the boundary: approximately 50% of cos2 clones overlapping the Boundary Zone disrupt the wild-type high-level Ptc stripe. It is not known why cos2 activity is more critical along the boundary than away from the boundary. Nonetheless, this result suggests that cos2 is required for Ci to become fully active in the Boundary Zone in response to a high level of Hh signaling. A requirement for cos2 is more evident when an in vivo reporter of Ci activity, 4bslacZ, whose enhancer element contains only four Ci binding sites and four Scalloped binding sites, was examined and whose response to Ci exhibits a more stringent requirement for Hh signaling than that of ptc. Not only do cos2 clones away from the boundary fail to ectopically induce 4bslacZ, but clones abutting the boundary invariably disrupt the wild-type 4bslacZ stripe. Given the cell-autonomous stabilization and nuclear import of Ci in cos2 clones, it is inferred that the disruption of ptc and 4bslacZ expression in such clones is due to a lack of Ci activation (Wang, 2000).

The role of pka in Ci activation was assayed in the posterior compartment of discs, in which all cells are exposed to a high level of Hh ligand. An actin promoter was used to drive a low level of ci expression throughout the discs. In discs with ubiquitous ci, high-level ptc expression is observed in the posterior compartment cells (P cells) but not in the anterior compartment cells (A cells). pka mutant P cells in this background do not express ptc, indicating that the loss of pka function disrupts Hh signal transduction and compromises maximal Ci activity. Consistent with this result, loss of pka function also disrupts 4bslacZ expression in the Boundary Zone (Wang, 2000).

While the loss of ptc and 4bslacZ expression in pka clones could reflect a disruption of Ci activity, it could also be due to negative effects on other proteins that bind these enhancers. To distinguish between these two possibilities, wild-type Ci and a PKA site-mutant form of Ci, Ci(m1-3), were compared for their abilities to induce ectopic 4bslacZ in the posterior compartment. Wild-type Ci exhibits a sensitive response to Hh and, at a modest protein level, induces robust 4bslacZ expression in the posterior compartment but not in the anterior. In contrast, Ci(m1-3) shows no response to Hh and does not induce 4bslacZ in either compartment. Thus, the integrity of specific PKA sites within Ci is essential for it to respond to high levels of Hh and become fully active. Since pka is dispensable for the acceleration of Ci nuclear import in Hh-receiving cells, the requirement of PKA phosphorylation for maximal Ci activity presumably reflects a requirement for Ci activation (Wang, 2000).

Posttranslational modification of protein kinase A.

Both for Aplysia and Drosophila a key role in the molecular mechanism of learning and memory processes has been assigned to the cAMP cascade. In any learning process a short-time stimulus has to be translated into long-lasting cellular changes. The molecular correlate must be a cascade of biochemical reactions with different kinetics, functionally interlinked and operating in overlapping time ranges. Biochemical studies in Drosophila have led to the suggestion that one of these steps is a proteolytic modification of the regulatory subunits of protein kinase A. A quantitative analysis of the relaxation kinetics of a system of protein kinase A, phosphatases and a calcium-dependent protease can give an image of essential characteristics of learning behaviour in Drosophila (Spatz, 1995).

PKA interaction with the regulatory subunit Pka-R-II and indirectly with A kinase anchor protein 200

Protein kinase A (PKA) holoenzyme is anchored to specific subcellular regions by interactions between regulatory subunits (Pka-R) and A-kinase anchoring proteins (AKAPs). The functional importance of PKA anchoring during Drosophila oogenesis has been examined by analyzing membrane integrity and actin structures in mutants with disruptions in A kinase anchor protein 200 (Akap200), an AKAP. In wild-type ovaries, cAMP-dependent protein kinase R2 (Pka-RII) the regulatory subunit of cAMP-dependent protein kinase 1 (Pka-C1, also known as Protein kinase A) and Akap200 localize to membranes and to the outer rim of ring canals (actin-rich structures that connect germline cells). In Akap200 mutant ovaries, Pka-RII membrane localization decreases, leading to a destabilization of membrane structures and the formation of binucleate nurse cells. Defects in membrane integrity could be mimicked by expressing a constitutively active PKA catalytic subunit (Pka-C) throughout germline cells. Unexpectedly, nurse cells in Akap200 mutant ovaries also have enlarged, thin ring canals. In contrast, overexpressing Akap200 in the germline results in thicker, smaller ring canals. To investigate the role of Akap200 in regulating ring canal growth, genetic interactions with other genes that are known to regulate ring canal morphology were examined. Akap200 mutations suppress the small ring canal phenotype produced by Src64B mutants, linking Akap200 with the non-receptor tyrosine kinase pathway. Together, these results provide the first evidence that PKA localization is required for morphogenesis of actin structures in an intact organism (Jackson, 2002).

An approximate location for the PKAII tethering site in Drosophila A kinase anchor protein 200 (Akap200) was established by sequencing five cDNAs that directed synthesis of RII-binding, fusion proteins in plaques of a bacteriophage expression library. The sequence of the smallest cDNA (147 bp) encodes a region of the anchor protein bounded by Asp⁴⁹⁰ and Glu⁵³⁸. The same sequence was present in each of the larger (1.1-1.7 kbp) cDNAs. Tethering sites in mammalian AKAPs are composed of ~20 amino acids and contain a precisely spaced group of residues with large, aliphatic side chains (Leu, Val, Ile, and Thr) that cooperatively govern sequestration of RIIa and RIIß subunits. Residues 511-530 in Akap200 can be aligned with tethering regions of mammalian AKAPs, so that Ile⁵¹¹, Ile⁵¹⁸, Val⁵¹⁹, Thr⁵²³, and Val⁵³⁰ are in register with essential hydrophobic residues in previously characterized RII binding sites. A final conserved position is occupied by a smaller hydrophobic residue (Ala⁵²⁷) in the Drosophila anchor protein. RII binding domains of mammalian AKAPs are predicted to fold as an amphipathic a-helix that contains one markedly hydrophobic surface. This feature is also evident in the segment of Akap200 that encompasses residues 511-528. Folding algorithms predict that this partial polypeptide is organized into an a-helix in which 9 of 10 side chains on one surface have hydrophobic character. Thus, amino acids 511-530 constitute a candidate RII binding site in Akap200 (Li, 1999).

To further characterize the tethering domain, a fragment of Akap200 cDNA (nucleotides 1577-2314) that encodes amino acids 475-753 was amplified by polymerase chain reaction. The soluble His tagged fusion protein (named p-DAKAP70) was purified to near homogeneity by affinity chromatography on Ni²⁺-chelate Sepharose 4B resin. Small amounts of immobilized p-DAKAP70 avidly bind ³²P-RIIß (human) in an overlay assay performed with a low concentration of labeled ligand. Similar results were obtained with radiolabeled murine RIIa, thereby indicating that the tethering domain of Akap200 complexes both RII isoforms with high affinity. A caveat is that both binding studies and functional screening of cDNA expression libraries were designed and executed on the basis of a logical but unproved assumption, that mammalian RII isoforms are interchangeable substitutes for authentic Drosophila RII (RII_DR) subunits (Li, 1999).

To verify this assumption and demonstrate more directly the physiological relevance of the novel fly anchor protein, it was essential to examine the ability of Akap200 to bind RII_DR. The gene encoding the 376-residue RII_DR polypeptide has been cloned and sequenced. Structural features that govern subunit dimerization and create a docking surface for AKAPs are located near the amino terminus (residues 1-50) of mammalian RII isoforms. Alignment of the N termini of RII_DR and RIIa reveals that the two sequences are quite divergent (only 44% identity). However, groups of aromatic residues (Phe and Tyr) and amino acids with large aliphatic side chains (Leu, Val, and Ile) contribute the essential functional properties of dimerization and AKAP binding regions in RIIa and RII. Amino acids with these characteristics are conserved at all corresponding positions within residues 1-50 of RII _DR (Li, 1999)

Like mammalian RII subunits, RII_DR contains the PKA phosphorylation site sequence (RRXSX) in a linker region between the dimerization-AKAP binding domain and the cAMP binding sites. Thus, RII_DR was labeled by incubation with Mg-gamma-³²P ATP and the catalytic (C) subunit of PKA. ³²P-Labeled RII_DR binds with low levels of p-DAKAP70 and also forms a stable complex with bovine AKAP75. Thus, structural features that mediate interactions between RII subunits and AKAPs have co-evolved and are conserved from flies to humans. Binding interactions between RIIa, RIIß, or RII_DR and various AKAPs are sufficiently similar to enable their interchangeable use. Since recombinant mammalian RII isoforms are available in plentiful supply and these proteins are more thoroughly characterized than RII_DR, they were employed for most of the studies presented in this study. Repetition of experiments with RII_DR yielded similar results in all instances (Li, 1999).

The physiological relevance of Akap200 was investigated by testing the ability of the anchor protein to bind an endogenous ligand, RII_DR. Several domains in Akap200 may contribute to the targeting of tethered PKAII. Two Pro-rich regions (residues 328-332 and 468-479) are potential binding sites for cytoskeleton/organelle-associated proteins that contain Src homology 3 domains. Amino acids 2-7 constitute an acceptor site for N-myristoyltransferase. Myristoylation of Akap200 would provide a long saturated aliphatic chain that inserts into the hydrocarbon interior of phospholipid bilayers. A segment of Akap200 (residues 118-148) includes a PSD-like cluster of 13 Lys, five Ser, and five large hydrophobic residues. Positive charge in PSD2S promotes electrostatic binding with negatively charged head groups of membrane phospholipids. Intercalation of PSD hydrophobic side chains into the apolar interior of a bilayer further stabilizes association of PSD-containing proteins with membranes. N-terminal myristate and a PSD are critical features of MARCKS proteins, which mediate interactions between plasma membrane and F-actin. The MARCKS PSD is phosphorylated in situ by diacylglycerol-activated protein kinase C isoforms. Phosphorylation inhibits binding with membrane phospholipids, enables translocation of MARCKS from cell surface to cytoplasm, and promotes cytoskeleton remodeling. The nonphosphorylated PSD sequesters calmodulin in a calcium-dependent manner. Binding of Ca²⁺-calmodulin diminishes the ability of MARCKS to cross-link and bundle actin filaments. By analogy, the fly anchor protein may be involved in integrating signals propagated by three critical second messenger molecules: cAMP, diacylglycerol, and calcium. Moreover, the PSD region of Akap200 may enable phosphorylation-controlled shuttling of tethered PKA between two or more intracellular sites (Li, 1999).

Drosophila A kinase anchor protein 200 (Akap200), is predicted to be involved in routing, mediating, and integrating signals carried by cAMP, Ca²⁺, and diacylglycerol. Experiments designed to assess this hypothesis establish (1) the function, boundaries and identity of critical amino acids of the protein kinase AII (PKAII) tethering site of Akap200; (2) demonstrate that residues 119-148 mediate binding with Ca²⁺-calmodulin and F-actin; (3) show that a polybasic region of Akap200 is a substrate for protein kinase C; (4) reveal that phosphorylation of the polybasic domain regulates affinity for F-actin and Ca²⁺-calmodulin, and (5) indicate that Akap200 is myristoylated and that this modification promotes targeting of Akap200 to plasma membrane. DAkap200, a second product of the Akap200 gene, cannot tether PKAII. However, DAkap200 is myristoylated and contains a phosphorylation site domain that binds Ca²⁺-calmodulin and F-actin. An atypical amino acid composition, a high level of negative charge, exceptional thermostability, unusual hydrodynamic properties, properties of the phosphorylation site domain, and a calculated M_r of 38,000 suggest that DAkap200 is a new member of the myristoylated alanine-rich C kinase substrate protein family. Akap200 is a potentially mobile, chimeric A kinase anchor protein-myristoylated alanine-rich C kinase substrate protein that may facilitate localized reception and targeted transmission of signals carried by cAMP, Ca²⁺, and diacylglycerol (Rossi, 1999).

A role for Drosophila LKB1 in anterior-posterior axis formation and epithelial polarity; LKB1 is targeted by PAR-1 and PKA

The PAR-4 and PAR-1 kinases are necessary for the formation of the anterior-posterior (A-P) axis in Caenorhabditis elegans. PAR-1 is also required for A-P axis determination in Drosophila. The Drosophila par-4 homologue, lkb1, is required for the early A-P polarity of the oocyte, and for the repolarization of the oocyte cytoskeleton that defines the embryonic A-P axis. LKB1 is phosphorylated by PAR-1 in vitro, and overexpression of LKB1 partially rescues the par-1 phenotype. These two kinases therefore function in a conserved pathway for axis formation in flies and worms. lkb1 mutant clones also disrupt apical-basal epithelial polarity, suggesting a general role in cell polarization. The human homologue, LKB1, is mutated in Peutz-Jeghers syndrome and is regulated by prenylation and by phosphorylation by protein kinase A. Protein kinase A phosphorylates Drosophila LKB1 on a conserved site that is important for its activity. Thus, Drosophila and human LKB1 may be functional homologues, suggesting that loss of cell polarity may contribute to tumour formation in individuals with Peutz-Jeghers syndrome (Martin, 2003).

Drosophila LKB1 also has a conserved RKLS consensus phosphorylation site near its C terminus. In mammalian LKB1, this site is phosphorylated in vitro and in vivo by protein kinase A (PKA) and is required for its ability to suppress cell growth in culture. Like its vertebrate counterparts, Drosophila LKB1 is phosphorylated in a PKA-dependent manner. Coexpression of wild-type LKB1 and PKA in S2 cells induces a phosphatase-sensitive mobility shift of LKB1 on Western blots, which is abolished when serine 535 is mutated to alanine. To assay the significance of phosphorylation of this conserved site, transgenes were generated in which the serine was mutated to either alanine (S535A) to prevent phosphorylation, or to glutamic acid (S535E) to mimic the presence of a charged phosphate group. Expression of the transgenes with arm-GAL4 allows the recovery of lkb1 mutant flies expressing low amounts of either wild-type or mutant proteins in the germ line. GFP-LKB1^S535A does not rescue the localization of Staufen to the posterior of the oocyte, whereas GFP-LKB1^S535E rescues even more efficiently than the wild-type control, strongly suggesting that LKB1 is positively regulated by phosphorylation at this site. Because S535 is phosphorylated by PKA in vivo and PKA is required in the germ line to polarize the oocyte, it is speculate that PKA regulates Drosophila LKB1 in the germ line. Additional signals must regulate LKB1, however, because the lack of phosphorylation on S535 does not abolish LKB1 activity completely. Tenfold overexpression of S535A in lkb1 germline clones partially rescues the localization of Staufen to the posterior of the oocyte, albeit less efficiently than the wild-type transgene, whereas a kinase-dead version (K174M) shows no rescuing activity on overexpression. Thus, LKB1 may be regulated by both PAR-1 and PKA, and may function to integrate the two signalling pathways during the polarization of the oocyte (Martin, 2003).

PKA-R1 spatially restricts Oskar expression for Drosophila embryonic patterning

Targeting proteins to specific domains within the cell is central to the generation of polarity, which underlies many processes including cell fate specification and pattern formation during development. The anteroposterior and dorsoventral axes of the Drosophila embryo are determined by the activities of localized maternal gene products. At the posterior pole of the oocyte, Oskar directs the assembly of the pole plasm, and is thus responsible for formation of abdomen and germline in the embryo. Tight restriction of oskar activity is achieved by mRNA localization, localization-dependent translation, anchoring of the RNA and protein, and stabilization of Oskar at the posterior pole. The type 1 regulatory subunit of cAMP-dependent protein kinase (Pka-R1) is crucial for the restriction of Oskar protein to the oocyte posterior. Mutations in PKA-R1 cause premature and ectopic accumulation of Oskar protein throughout the oocyte. This phenotype is due to misregulation of PKA catalytic subunit activity and is suppressed by reducing catalytic subunit gene dosage. These data demonstrate that PKA mediates the spatial restriction of Oskar for anteroposterior patterning of the Drosophila embryo and that control of PKA activity by PKA-R1 is crucial in this process (Yoshida, 2004).

To isolate new factors involved in axis formation during Drosophila oogenesis, an FRT-based genetic screen was performed for maternal-effect mutants defective in anteroposterior patterning of the embryo. Embryos derived from germline clones of one line, 18304, showed anterior patterning defects ranging from deletion of the head to complete mirror-image duplication of posterior structures (abdominal segments and filzkörper material), the bicaudal phenotype. oskar plays a central role in abdomen and germline formation. At the posterior pole, oskar assembles the pole plasm, and recruits and activates translation of nanos RNA, which encodes the abdominal determinant. To address whether 18304 affects oskar or downstream factors, flies doubly mutant for 18304 and oskar were generated, and the phenotype of their progeny analyzed. Such embryos show a phenotype indistinguishable from that of the oskar single mutant. This indicates that the 18304 locus is required in the germline to control oskar activity (Yoshida, 2004).

These results reveal that Pka-R1 is an essential gene in Drosophila and that it is specifically required during oogenesis for post-transcriptional regulation of oskar. PKA-R1 forms a complex with the PKA catalytic subunit and controls its activity in response to cAMP. In Drosophila, the DC0 locus was identified as the catalytic subunit gene with the highest homology to its mammalian counterparts, and it serves as the major source of PKA catalytic activity. DC0 mutant germline clones show defective actin structures in the nurse cells and oocyte, indicating a role of PKA in organization of the actin cytoskeleton. Analysis of DC0 loss-of-function alleles in the germline has also revealed a requirement for PKA catalytic activity in the establishment of microtubule polarity along the anteroposterior axis of the oocyte, a prerequisite for oskar RNA localization. The effect of loss of DCO activity on microtubule polarity has prevented analysis of the role of PKA in regulation of Oskar protein expression, because oskar RNA is translationally repressed before its localization, and translation is not activated if the RNA fails to localize to the posterior pole (Yoshida, 2004).

This analysis demonstrates a requirement for precise modulation of PKA activity in the Drosophila germline for correct spatial distribution of Oskar protein. In the Pka-R1¹⁸³⁰⁴ mutant, where PKA activity is upregulated, Oskar protein is overexpressed and accumulates ectopically thoughout the oocyte, although oskar RNA localization and levels are normal. Dorsoventral patterning is correctly established. Anterior patterning also appears normal in the mutant, as revealed by the fact that Pka-R1¹⁸³⁰⁴, oskar double mutants develop a normal head and thorax. In addition, over-expression of the regulatory subunit causes a modest reduction in PKA activity, without affecting oskar RNA localization in the ovary. In such egg chambers, Oskar is underexpressed, suggesting that PKA activity is indeed required for Oskar expression. Taken together, these results suggest that, in addition to its role in the establishment of microtubule polarity and actin cytoskeleton integrity, PKA has a positive role in the regulation of Oskar protein expression at the posterior pole (Yoshida, 2004).

In addition to defects in oogenesis, Pka-R1 alleles have reduced viability, indicating that the control of PKA activity by PKA-R1 is required in several developmental processes. It has been shown that Pka-R1 is expressed throughout the cell-body layer of the central brain and optic lobes, and strongly accumulates in mushroom bodies. Analysis of hypomorphic alleles has revealed that Pka-R1 is involved in olfactory learning and courtship conditioning. The novel Pka-R1 mutants described in this study appear to be stronger alleles, and their analysis should prove useful for investigating the role of Pka-R1 in brain development and function. In addition, the role of PKA in different process of development has been investigated by making use of mutants in DC0, PKA-R2, and factors that modulate cAMP levels such as dunce and rutabaga. For example, it has been demonstrated that PKA antagonizes hh signaling by phosphorylating and inactivating the downstream transcription factor Cubitus interruputus. In addition, PKA-mediated signaling was shown to be involved in learning and behavior, and drug responses. Regulation of PKA by PKA-R1 is likely to be crucial in these processes as well (Yoshida, 2004).

The increase in PKA activity in Pka-R1¹⁸³⁰⁴ mutant extracts and the suppression of both the semi-lethality and the maternal-effect bicaudal phenotype by reduction of a functional copy of the DC0 catalytic subunit reveals that the phenotype of Pka-R1¹⁸³⁰⁴ is due to its failure to repress PKA activity in the mutant. Release of active PKA catalytic subunits from the inactive PKA holoenzyme is controlled by cAMP levels. It is also known that free catalytic subunits are more susceptible to proteolytic degradation than are catalytic subunits in the holoenzyme complex (Park, 2000). An excess of PKA catalytic activity is observed both in the absence and the presence of exogenous cAMP in the mutant extract, suggesting that upregulation of PKA catalytic subunit activity in Pka-R1¹⁸³⁰⁴ is due to a defect of mutant PKA-R1 in inhibiting catalytic subunit activity. In addition, the mutant extract still shows an increase in PKA activity in response to cAMP, which suggests the existence of a holoenzyme complex in the mutant. This is likely to be the case, as the point mutation in Pka-R1¹⁸³⁰⁴ is in a conserved arginine in the 'inhibitory domain' that acts as a catalytic unit pseudosubstrate. However, it is also possible that an autoregulatory feedback loop controlling expression or stability of catalytic subunits contributes to the increase in total PKA activity (Yoshida, 2004).

The premature and ectopic accumulation of Oskar in Pka-R1¹⁸³⁰⁴ suggests a role for PKA-R1 in oskar localization-dependent translation. An alternative explanation is that PKA-R1 is involved in the control of Oskar protein stability. In the case of C. elegans, some germ plasm components are excluded from the somatic cells by cullin-dependent degradation. A similar process might operate to restrict Oskar to the posterior pole. This assumes the existence of a mechanism whereby precociously and ectopically translated Oskar is degraded, and that this process requires PKA-R1 and its inhibition of PKA activity. However, there is no evidence to date of translation of oskar RNA prior to its posterior localization, or of active degradation of mislocalized Oskar (Yoshida, 2004).

Oskar degradation has been shown to be inhibited by phosphorylation. Both the protection and the degradation machineries operate throughout the oocyte and not just at the posterior pole. Because nucleotide substitutions in the 3' UTR of oskar lead to its ectopic translation and detectable accumulation, under normal circumstances, oskar translational regulation seems fairly tight, and is responsible for the specific accumulation of the protein at the posterior. Therefore it is speculated that the ectopic accumulation of Oskar in PKA-R1 mutants reflects a role of PKA in activation of oskar translation at the posterior pole, and that phosphorylation of translation regulatory proteins by PKA might cause the release of oskar mRNA from translational repression outside of the posterior domain (Yoshida, 2004).

It has been demonstated PKA is involved in the reception of the signal from the posterior follicle cells for the establishment of oocyte polarity at mid-oogenesis, specifically for the destabilization of the posterior microtubule organizing center. The signal from the posterior follicle cells might activate PKA at the posterior pole of the oocyte, and this local activation might in turn be responsible for the localized activation of oskar expression. It has been shown that the PKA holoenzyme can be targeted to specific subcellular domains by association with A-kinase anchoring proteins (AKAPs), a mechanism that has been proposed to regulate the spatial distribution of PKA activity. It is tempting to speculate that, in wild-type egg chambers, PKA holoenzyme complexes are targeted to specific AKAPs through PKA-R1, which blocks phosphorylation of specific targets, thus preventing ectopic expression of Oskar outside of the posterior domain. However, an alternative explanation is possible, whereby it is not PKA, but rather a PKA target involved in oskar activation that is asymmetrically distributed, with an enrichment at the posterior pole -- as is the case for oskar mRNA. Uniformly moderate levels of PKA activity throughout the oocyte would result in phosphorylation/activation of the target exclusively at the posterior pole; over-activation of PKA throughout the oocyte would result in activation of the target protein throughout the oocyte, which would be sufficient for ectopic activation of Oskar expression outside of the posterior pole. To address these possibilities, and to address directly the mechanism by which PKA controls the spatial restriction of oskar, it will be important to visualize the localization of the kinase activity, and to identify and determine the subcellular localization of the targets of PKA in this process (Yoshida, 2004).

Nervy links protein kinase a to plexin-mediated semaphorin repulsion

Cyclic nucleotides regulate axonal responses to a number of guidance cues through unknown molecular events. Drosophila nervy, a member of the myeloid translocation gene family of A kinase anchoring proteins (AKAPs), regulates repulsive axon guidance by linking the cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) to the Semaphorin 1a (Sema-1a) receptor Plexin A (PlexA). Nervy and PKA antagonize Sema-1a-PlexA-mediated repulsion, and the AKAP binding region of Nervy is critical for this effect. Thus, Nervy couples cAMP-PKA signaling to PlexA to regulate Sema-1a-mediated axonal repulsion, revealing a simple molecular mechanism that allows growing axons to integrate inputs from multiple guidance cues (Terman, 2004). Subsequent analysis has shown that Nervy is a member of the MTG protein family and probably functions in the nucleus as transcriptional corepressor. Although a cytoplasmic function for Nervy, described in this section of The Interactive Fly, cannot be ruled out, it is suggested that the axonal migration phenotypes observed in nervy mutant Drosophila embryos may be due to alterations in gene expression rather than a failure to anchor PKA to the plasma membrane (Ice, 2005). Nervy, like PlexA, is highly expressed in the Drosophila embryonic central nervous system (CNS), including in motor neurons (Feinstein, 1995) and their axons. An antibody to a conserved region of mammalian MTG proteins also identified Drosophila Nervy within CNS and motor axons. Immunoprecipitation of hemagglutinin (HA) epitope–tagged neuronal PlexA from Drosophila embryonic lysates revealed associated Nervy, and neuronal HA-PlexA was detected in immunoprecipitates of Nervy, which suggests that nervy and PlexA interact in neurons. Nervy also immunoprecipitates with PKA RII in Drosophila embryos, and an epitope (Myc) tagged neuronal nervy immunoprecipitated with Drosophila PKA RII, which indicates that nervy is a neuronal AKAP (Terman, 2004).

If Nervy serves to tether PKA to the PlexA receptor, then type II PKA should associate in a complex with PlexA. An antibody specific for PKA RII decorates embryonic Drosophila CNS and motor axons, and PKA RII coimmunoprecipitated (co-IP) with HA-PlexA expresses in neurons, showing that type II PKA is associated with the PlexA receptor complex. pka RII LOF mutant embryos also exhibit highly penetrant axon guidance defects that closely resemble the guidance defects observed in nervy LOF, PlexA GOF, and MICAL GOF mutants. In addition, pka RII LOF mutants, like nervy LOF mutants, enhance the repulsive effects of Sema-1a, which suggests that type II PKA antagonizes Sema-1a repulsive axon guidance (Terman, 2004).

To test the necessity of nervy-type II PKA interactions in regulating Sema-1a-PlexA signaling, a single amino acid substitution of a proline for a valine residue was made in Nervy (nervy^V523P) that was analogous to a mutation that disrupts MTG16-PKA RII interactions. Transgenic flies were generated expressing epitope (myc)-tagged nervy^V523P, but unlike neuronal expression of wild-type nervy in a nervy LOF mutant background, neuronal nervy^V523P failed to rescue the nervy LOF mutant phenotypes. Therefore, it was reasoned that nervy^V523P might function in a dominant-negative manner by retaining its ability to bind to PlexA but blocking the coupling of PKA to PlexA. Indeed, expression of myc-nervy^V523P in all neurons in a wild-type background results in axon guidance phenotypes similar to those seen in nervy or pka RII LOF mutants. These phenotypes are the opposite of those seen when wild-type Nervy is expressed in all neurons and are indicative of increased Sema-1a-PlexA repulsion because they resemble MICAL and PlexA GOF mutants. These results suggest that nervy's ability to bind type II PKA is critical for the modulation of Sema-1a-PlexA repulsive guidance (Terman, 2004).

The AKAP Yu is required for olfactory long-term memory formation in Drosophila

Extensive neurogenetic analysis has shown that memory formation depends critically on cAMP-protein kinase A (PKA) signaling. Details of how this pathway is involved in memory formation, however, remain to be fully elucidated. From a large-scale behavioral screen in Drosophila, the yu mutant was found to be to be defective in one-day memory after spaced training. The yu mutation disrupts a gene encoding an A-kinase anchoring protein (AKAP). AKAPs comprise a family of proteins, which determine the subcellular localization of PKAs and thereby critically restrict cAMP signaling within a cell. Further behavioral characterizations revealed that long-term memory (LTM) was disrupted specifically in the yu mutant, whereas learning, short-term memory and anesthesia-resistant memory all appeared normal. Another independently isolated mutation of the yu gene failed to complement the LTM defect associated with the yu mutation, and this phenotypic defect could be rescued by induced acute expression of a yu ⁺ transgene, suggesting that yu functions physiologically during memory formation. AKAP Yu is expressed preferentially in the mushroom body (MB) neuroanatomical structure, and expression of a yu ⁺ transgene to the MB, but not to other brain regions, is sufficient to rescue the LTM defect of the yu mutant. These observations lead to the conclusion that proper localization of PKA by Yu AKAP in MB neurons is required for the formation of LTM (Lu, 2007).

Studies in several species have revealed a time-dependent process of memory consolidation with distinct temporal phases of memory. After Drosophila olfactory learning, behavioral, pharmacological, and genetic manipulations have 'dissected' memory formation into four distinct but interdependent phases: STM, MTM, ARM, and LTM. The current study focuses on the role of a specific AKAP gene, yu, in formation of LTM. This study shos that (1) LTM specifically was abolished in yu mutants, whereas other memory phases appeared normal, (2) this LTM defect was produced by (independent) mutations in the yu transcription unit, CG3249, and (3) the LTM defect of the yu mutant could be rescued via acute induction of a yu ⁺ transgene. These data clearly define a specific, physiological function for Yu AKAP during memory formation (Lu, 2007).

Immunostaining of adult brain has revealed that Yu is expressed preferentially in the MB anatomical region, which many studies have established plays a key role in olfactory memory formation. Using multiple 'enhancer trap' GAL4-drivers, with overlapping patterns of expression in MB or with preferential expression in other potentially critical anatomical regions [i.e., antennal lobes and central complex, the expression of a UAS-yu ⁺ transgene was spatially restricted in the yu mutant. LTM was normal only when Yu was expressed in the MB. Finally, a classically designed genetic epistasis experiment has shown that Yu AKAP interacts with PKA-C1 during LTM formation. This synergistic effect supports the idea that Yu functions as an AKAP in mediating LTM formation (Lu, 2007).

Although genetic dissection has revealed distinct memory phases and gene disruption experiments have identified several components of cAMP signaling to be important for this process, few studies have determined whether these molecules actually function together during memory formation. Rut-AC and PKA-C1, for instance, are critical not only for learning and STM or MTM but also for LTM. But, do they function together in the same place during each memory phase? When considering AKAPs, more specifically, proteins of this family bind regulatory subunits of PKA, and thereby may serve to localize PKA to different subcellular compartments. But specifically which AKAP is involved in which cellular function in Drosophila? This work on the Yu AKAP presents the hypothesis that localization of Yu helps to define a function of cAMP signaling specific to LTM formation. As a corollary, it is speculated that other AKAPs (in the MB) may introduce specificities for cAMP signaling during earlier memory phases by means of differential localization of PKA to other subcellular compartments (Lu, 2007).

Although both STM and LTM are localized in the MB, these two memory components may not reside in the same population of MB neurons. There are evidences that the horizontal lobe is especially important for Rut-dependent STM formation (Zars, 2000; Akalal, 2006), whereas vertical (α and α') lobes are critical for LTM formation (Pascual, 2001). Previous studies have shown that Drosophila α/fl neurons not only function in the memory retrieval (McGuire, 2001; Krashes, 2007) but also form a branch-specific LTM trace after spaced training (Yu, 2006). Functional analysis of subsets of MB neurons show that α'/β' neurons are required to acquire and stabilized an olfactory memory (Krashes, 2007). To that end, this study also shows that Yu is expressed preferentially in α' and β' lobes (Lu, 2007).

Signaling at A-kinase anchoring proteins organizes anesthesia-sensitive memory in Drosophila

The ubiquitous cAMP-protein kinase A (PKA) signaling pathway exhibits complex temporal requirements during the time course of associative memory processing. This directly raises questions about the molecular mechanisms that provide signaling specificity to this pathway. This study used Drosophila olfactory conditioning to show that divergent cAMP signaling is mediated by functionally distinct pools of PKA. One particular pool is organized via the PKA regulatory type II subunit at the level of A-kinase anchoring proteins (AKAPs), a family of scaffolding proteins that provides focal points of spatiotemporal signal integration. This AKAP-bound pool of PKA is acting within neurons of the mushroom bodies to support a late phase of aversive memory. The requirement for AKAP-bound PKA signaling is limited to aversive memory, but dispensable during appetitive memory. This finding suggests the existence of additional mechanisms to support divergence within the cAMP-PKA signaling pathway during memory processing. Together, these results show that subcellular organization of signaling components plays a key role in memory processing (Schwaerzel, 2007).

This study pioneered the role of AKAPs as organizers of PKA signaling in Drosophila associative olfactory memory processing. First, it was shown that after aversive conditioning ASM can be separated into two phases; whereas an early phase of ASM is AKAP-independent, late ASM requires AKAP-bound PKA-RII signaling within the MBs. Second, it was shown that, after appetitive conditioning, ASM 'bypasses' this AKAP-bound pool of PKA. This shows that PKA signaling at AKAP complexes does not disrupt associative functions per se, but that functionally distinct pools of PKA must exist on a subcellular level to serve defined functions during memory processing. A functional model is suggested in which cAMP signaling is distributed on the subcellular level by involvement of AKAP-bound pools of PKA (Schwaerzel, 2007).

Mutants affecting different steps along the cAMP-PKA cascade have identified distinct anesthesia-sensitive memory phases in Drosophila olfactory conditioning. Mutants directly affecting cAMP (e.g., the adenylyl cyclase rutabaga or the cAMP-specific phosphodiesterase dunce) disturb formation of ASM. Further downstream within the pathway, a conditional allele of the PKA catalytic subunit DC0 X4 impairs memory performance around 20 min to 2 h after training. The amnesiac mutant has revealed a critical time window shortly after conditioning, when olfactory memory requires activity of the dorsal paired medial (DPM) neurons to act onto the mushroom body structure to stabilize ASM. In fact, it has been this temporal requirement for Rutabaga, Dunce, DC0 X4, and Amnesiac that lead to the separation of ASM into STM and MTM. This analysis now reveals that MTM can be further dissected into an early and a late phase. Although the early phase of MTM requires PKA signaling as revealed by the DC0 X4 allele, this study shows that it is independent of AKAP binding. However, a late phase of MTM requires AKAP-bound PKA-RII signaling and supports memory performance from 60 min after conditioning on. As performance of this late MTM becomes completely abolished, it is concluded that no redundancy exists on the molecular level to support this particular late phase. Moreover, early and late MTM must be supported by distinct pools of PKA that serve separable functions during the time course of processing (Schwaerzel, 2007).

The functional dissection of PKA into AKAP-bound and nonbound pools suggested here is derived from the eCOPR2 peptide that constitutes the AKAP binding site of endogenous Drosophila PKA-RII. Based on the conserved mechanism of this interaction across phyla, it is speculated that eCOPR2 disturbs the binding of PKA-RII to all potential AKAPs. Additional experiments are now required to (1) identify the particular AKAP(s) that are involved in late MTM stabilization, and (2) to formally prove that eCOPR2 does indeed disrupt interaction between AKAPs and PKA-RII. However, since it was shown that eCOPR2 expression affects very specific aspects of aversive ASM processing, rather than abolishing associative functions per se, provides strong arguments in favor of distinct PKA pools. A previous study in mammals has shown similar impairment of memory several hours after conditioning when using microinfusion of Ht-31, a specific competitor of PKA-RII-AKAP interaction (Moita, 2002). In Drosophila the effects of AKAP-bound PKA-RII on aversive ASM stabilization could be localized to 700 neurons of the MBs (i.e., to the same neurons that acquire aversive memory via a Rutabaga-dependent process of synaptic plasticity in the first place). It is speculated that processing aversive ASM from STM into early and late MTM takes place within these neurons (Schwaerzel, 2007).

In mammalian cell culture it has been shown that AKAPs are localized to specific subcellular foci of signal integration, including synaptic vesicles, ionotropic and metabotropic receptors, the cytoskeleton, or cellular organelles. Dependent on this localization, AKAPs can organize PKA signaling by selecting specific substrates to the AKAP complex. These findings can now guide research in identifying particular substrates that are involved in Drosophila aversive MTM processing (Schwaerzel, 2007).

Experiencing sugar reward or electric shock punishment during the training procedure gives rise to the formation of either appetitive or aversive olfactory memories. Both are dissociated on the level of catecholamines required, but display numerous similarities with respect to the neural and molecular networks involved in aversive and appetitive memory: both types of memory are acquired within the same subset of 700 MB neurons via a Rutabaga-dependent mechanism of cAMP-mediated plasticity. Moreover, secretion from DPM neurons onto the MBs is required within 30 min after conditioning to support stabilization of aversive and appetitive ASM, respectively. However, this study has shown that AKAP-bound PKA-RII dissociates between both forms, because neither pattern of competitor peptide expression affects appetitive ASM. This argues in favor of the hypothesis that different cAMP-dependent mechanisms support aversive and appetitive ASM; the remarkable stability of appetitive memory within the temporal domain strongly supports this hypothesis. Because the eCOPR2 peptide is believed to specifically block interactions between PKA-RII and appropriate AKAPs, additional pools of PKA might support appetitive ASM processing. Additional experiments can now probe soluble PKA pools or alternative anchoring mechanisms to AKAPs via the PKA regulatory subunit type I (Schwaerzel, 2007).

The finding that cAMP-signaling couples divergently to alternative downstream partners is a theme commonly observed throughout the animal kingdom. In Aplysia neurons, the temporal pattern of receptor activation is sufficient to trigger different cellular responses by threshold-dependent sorting mechanisms. Similar mechanisms might be in place in Drosophila olfactory conditioning, when either catecholamine receptors and/or DPM activity have the potential to trigger different threshold-dependent mechanisms of subcellular signal divergence within the MBs. In Drosophila, aversive olfactory conditioning dopaminergic and DPM signals exhibit complex temporal patterns that sustain the conditioning period. However, additional experiments will be required to substantiate this hypothesis (Schwaerzel, 2007).

Sequential phosphorylation of Smoothened transduces graded Hedgehog signaling

The correct interpretation of a gradient of the morphogen Hedgehog (Hh) during development requires phosphorylation of the Hh signaling activator Smoothened (Smo); however, the molecular mechanism by which Smo transduces graded Hh signaling is not well understood. This study shows that regulation of the phosphorylation status of Smo by distinct phosphatases at specific phosphorylated residues creates differential thresholds of Hh signaling. Phosphorylation of Smo was initiated by PKA and further enhanced by casein kinase I (CKI). Protein phosphatase 1 (PP1) directly dephosphorylates PKA-phosphorylated Smo to reduce signaling mediated by intermediate concentrations of Hh, whereas PP2A specifically dephosphorylates PKA-primed, CKI-phosphorylated Smo to restrict signaling by high concentrations of Hh. A functional link was established between sequentially phosphorylated Smo species and graded Hh activity. Thus, a sequential phosphorylation model is proposed in which precise interpretation of morphogen concentration can be achieved upon versatile phosphatase-mediated regulation of the phosphorylation status of an essential activator in developmental signaling (Su, 2011).

The conversion of a gradient of the morphogen Hh into distinct transcriptional responses is essential for cell-fate decisions and tissue patterning during development. This study has provided genetic and biochemical evidence to support a model in which sequential phosphorylation of Smo, which is established by distinct kinases and phosphatases on specific serines, transduces graded Hh signaling. A basal extent of Smo activity, regulated by as yet unknown kinases and phosphatases, was sufficient to transduce low-threshold Hh signaling. PKA and PP1 collaborate to sustain PKA-phosphorylated Smo to transduce intermediate-threshold Hh signaling, whereas CKI and PP2A facilitate high-threshold Hh signaling by maintaining PKA-primed, CKI-phosphorylated Smo (Su, 2011).

Wdb-PP2A directly and specifically acts on CKI-pSmo to restrict high-threshold Hh signaling. Apart from PP2A, another phosphatase, PP1, specifically dephosphorylated PKA-phosphorylated Smo. This collaborative regulation between different phosphatases on the same substrate also functions in other cellular processes. For example, PP1 and PP2A dephosphorylate Par-3 to regulate cell polarity in the specification of neuroblast cell fate. Similarly, PP2A and PP4 respond to different DNA damage signals to dephosphorylate γ-H2AX to facilitate the repair of DNA double-strand breaks (Su, 2011 and references therein).

The activity of several Hh signaling components, including Smo, Ci, and Cos2, is regulated by phosphorylation. For example, PKA- and CKI-mediated phosphorylation of Ci leads to its destabilization. PP2A is implicated in regulating Ci activity in flies. This study confirmed that the catalytic PP2A subunit Mts associates with both Smo and Ci in cl-8 cells. Consistent with the substrate specificity of PP2A being conferred by its obligate regulatory subunits, this study found that Wdb specifically regulates the signaling potential of Smo. Another regulatory subunit, Tws, may direct PP2A activity toward Ci, which may potentially promote the translocation of CiFL to the nucleus, thereby activating Hh signaling. The use of distinct PP2A regulatory subunits in the same developmental process was also observed in transforming growth factor-β (TGF-β) signaling. The effect of the regulatory Bα subunit on PP2A activity activates Smad2 signaling, whereas the Bδ subunit inhibits Smad2 activity. The elaborate regulation of these two signaling systems by PP2A highlights a potential paradigm in which differential PP2A activity plays an essential role in developmental signaling. PP2A is a strong tumor suppressor; thus, modulation of PP2A activity provides an additional route by which development and tumorigenesis might be controlled (Su, 2011).

Another phosphatase, PP4, may play a role in inhibiting Smo; however, inhibiting PP4 alone is not sufficient to promote constitutive cell surface localization of wild-type Smo, unless Hh protein is provided. The surface localization of Smo is tightly linked to PKA- and CKI-dependent enhanced phosphorylation of Smo. PP4-specific RNAi further increases the extent of constitutive surface localization of Smo mutants that mimic PKA- and CKI-mediated phosphorylation, which suggests that PP4 may act on sites other than those in the PKA-CKI clusters (Su, 2011).

To delineate mechanisms whereby PP2A and PP4 might act on Smo, the expression of Hh signaling components as well as of Hh targets were systematically examined in wing discs expressing pp4 RNAi. In addition to the Smo stabilization, it was found that pp4 RNAi reduced the abundance of Cos2 protein. This might be as a consequence of the increased Smo abundance, because smo RNAi stabilized Cos2. Alternatively, PP4 might regulate Cos2 directly, because phosphorylated Cos2 is not stable. To distinguish between these two possibilities, the genetic relationship between smo and pp4 was examined by monitoring the stabilization of Cos2. Cos2 abundance was still reduced in wing discs containing both pp4 and smo RNAi. This effect is similar to the effect of pp4 RNAi alone, thus placing pp4 downstream of smo in regulating Cos2. Consistent with this, reduced expression of pp4 compromised Ptc and Collier/Knot (Col) expression at the AP boundary. The expanded area containing Ptc, albeit at a reduced abundance, away from the AP boundary has been observed previously. These experiments are consistent with a positive role of Cos2 in mediating maximal activation of Hh signaling in cl-8 cells as well as in wing discs; Ptc and Col expression are reduced in cos2 clones abutting the AP boundary. These data, together with the observation of a direct interaction between Cos2 and PP4 (Jia, 2009), argue that PP4 might also directly affect the extent of Cos2 phosphorylation (Su, 2011).

Smo contains three PKA-CKI phosphorylation clusters, with one PKA and two CKI consensus serines in each cluster. A previous study compared the signaling potential of phosphorylation-defective Smo by mutating PKA consensus serines to alanines in one, two, or three of the PKA-CKI clusters and concluded that at least six serines in Smo are required to fully induce the expression of ptc-lacZ, whereas only three serines are needed for the expression of dpp-lacZ. Another study further demonstrated that PKA- and CKI-mediated phosphorylation, which results in the generation of negatively charged residues, counteracts the positive charges conferred by nearby arginine clusters, thus enabling Smo to adopt a conformational change required to activate Hh signaling. These two studies support a model of collective Smo phosphorylation such that the identity of the phosphorylated serines in the PKA-CKI consensus clusters is probably not important; rather, the resulting negative charges collectively carried by these residues after phosphorylation are critical to determine the signaling strength of Smo (Su, 2011).

On the basis of this model, a variant Smo (Smo^-CKI) in which the CKI, but not the PKA, consensus serines are mutated to alanines, thus rendering Smo^-CKI resistant to CKI-mediated phosphorylation, would be anticipated to have the same signaling potential as Smo^-PKA23, a variant containing a single intact PKA-CKI cluster, because both mutants contain three serines that can be phosphorylated. In smo loss-of-function clones, Smo^-PKA23 is sufficient to drive expression of dpp-lacZ; however, Smo^-CKI fails to rescue dpp-lacZ expression. The discrepancy between the effects of Smo^-PKA23 and Smo^-CKI on dpp-lacZ expression cannot be simply explained by the collective phosphorylation model. Moreover, these experiments reveal a functional distinction between different phosphorylated residues: Three PKA consensus serines in Smo^-CKI may have less signaling activity than the single PKA and two CKI consensus serines in Smo^-PKA23. The distinct signaling potentials of the two phosphorylation variants of Smo may be caused by different negative charge densities being carried by individual PKA-CKI clusters, or they may reflect intrinsic properties of sequential phosphorylation within each cluster (Su, 2011).

Indeed, the hierarchy of importance among individual PKA-CKI clusters in Smo has been revealed. Cluster 2 (also known as region V) is more prevalent than the other two clusters in activating ptc-luc reporter in smo-depleted cl-8 cells. Whether this functional distinction among PKA-CKI clusters also holds true in wing discs is unclear, because neither the hierarchical importance of individual clusters nor the relative importance of specific PKA and CKI phosphorylation events in neutralizing nearby arginine clusters has been directly studied. Nevertheless, when the relative importance of the three serines in cluster 2 was examined, the PKA-primed, CKI consensus sites (that is, sequential phosphorylation) were essential for Hh activation in cl-8 cells. The differential ability of Smo^CKI-SA and Smo^PKA-SA variants to activate dpp transcription in wing discs uncovered in this study is consistent with results obtained in cl-8 cells. Both observations challenge the model of collective Smo phosphorylation by arguing that the signaling potential of individual serines between each PKA-CKI cluster, as well as within a cluster, is most probably not equal. It is believed that regulated phosphorylation at specific serines may therefore contribute to graded Smo signaling (Su, 2011).

The collective phosphorylation model does not distinguish between the contributions of individual phosphorylated residues in PKA-CKI clusters. This study of phosphorylation-defective Smo variants revealed a Smo activity gradient in which phosphorylation at the PKA consensus sites and phosphorylation at the PKA-primed, CKI consensus sites were required for intermediate- and high-threshold Hh signaling, respectively. This activity gradient of Smo was directly visualized with the α-Smo-pS⁶⁶⁷ antibody. The abundance of PKA-phosphorylated Smo species, which is uncovered in Hh-stimulated fly cells by mass spectrometric analysis, increased initially but then declined sharply in response to Hh. As predicted from the model, phosphorylated Smo in response to intermediate-threshold Hh signaling was sensitive to dephosphorylation by PP1 but much less so to PP2A. Together, these data highlight the importance of sequential Smo phosphorylation to the transduction of graded Hh signaling. Sequential phosphorylation may be required to initialize graded Smo signaling activity. In addition, collective phosphorylation between different clusters may reinforce and maximize the Smo signaling potential to ensure the appropriate Hh signaling outcome (Su, 2011).

The presence of up to 26 serine or threonine residues in Smo that can be phosphorylated in response to Hh resembles the composition of residues found in the Kv2.1 potassium channel. Variable calcineurin-dependent dephosphorylation of Kv2.1 at 16 phosphorylated residues generates an activity gradient for channel gating and neuronal firing. The opposing actions of kinases and phosphatases on a multisite substrate are known through mathematical modeling to efficiently generate a range of stable phosphorylated forms. The spectrum of such distributions can be further increased with the number of phosphorylated sites. Two additional kinases, CK2 and G protein (heterotrimeric guanosine 5'-triphosphate-binding protein)-coupled receptor kinase 2 (GRK2), phosphorylate sites in Smo other than those targeted by PKA and CKI. Thus, the complex composition of phosphorylated residues in the cytoplasmic tail of Smo, coupled with versatile dephosphorylation by distinct phosphatases, provides an efficient and reliable mechanism to precisely convert the concentration thresholds of Hh into a graded signaling activity (Su, 2011).

Cellular site and molecular mode of synapsin action in associative learning

Synapsin is an evolutionarily conserved, presynaptic vesicular phosphoprotein. This study asked where and how synapsin functions in associative behavioral plasticity. Upon loss or reduction of synapsin in a deletion mutant or via RNAi, respectively, Drosophila larvae are impaired in odor-sugar associative learning. Acute global expression of synapsin and local expression in only the mushroom body, a third-order 'cortical' brain region, fully restores associative ability in the mutant. No rescue is found by synapsin expression in mushroom body input neurons or by expression excluding the mushroom bodies. On the molecular level, it was found that a transgenically expressed synapsin with dysfunctional PKA-consensus sites cannot rescue the defect of the mutant in associative function, thus assigning synapsin as a behaviorally relevant effector of the AC-cAMP-PKA cascade. It is therefore suggested that synapsin acts in associative memory trace formation in the mushroom bodies, as a downstream element of AC-cAMP-PKA signaling. These analyses provide a comprehensive chain of explanation from the molecular level to an associative behavioral change (Michels, 2011).

The associative defect in the syn⁹⁷-mutant (Michels, 2005) can be phenocopied by an RNAi-mediated knock-down of synapsin, and can be rescued by acutely restoring synapsin. In terms of site of action, locally restoring synapsin in the mushroom bodies fully restores associative ability, whereas restoring synapsin in the projection neurons does not. If synapsin is restored in wide areas of the brain excluding the mushroom bodies, learning ability is not restored either. Therefore, it was concluded that a synapsin-dependent memory trace is located in the mushroom bodies, and suggest that this likely is the only site where such a trace is established regarding odor-sugar short-term memory in larval Drosophila. In terms of mode of action, it was found that a synapsin protein that carries dysfunctional PKA sites cannot rescue the syn⁹⁷-mutant learning defect. It is therefore suggested that synapsin functions as a downstream element of AC-cAMP-PKA signaling in associative function (Michels, 2011).

Arguably, the Rutabaga type I adenylyl cyclase acts as a detector of the coincidence between an aminergic reinforcement signal (appetitive learning: octopamine; aversive learning: dopamine) and the odor-specific activation of the mushroom body neurons. Initially, this notion had been based on mutant and biochemical analyses in Drosophila and physiology in Aplysia. Indeed, activation of mushroom body neurons in temporal coincidence with dopamine application increases cAMP levels in wild-type, but not AC-deficient flies (rut²⁰⁸⁰), and a corresponding AC-dependence of PKA activation has been shown by mushroom body costimulation with octopamine. However, the downstream effects of the AC-cAMP-PKA cascade has remained clouded. This study suggests that, similar to the situation in snails, one of these PKA effectors is synapsin, such that synapsin phosphorylation allows a transient recruitment of synaptic vesicles from the reserve pool to the readily releasable pool. A subsequent presentation of the learned odor could then draw upon these newly recruited vesicles. This scenario also captures the lack of additivity of the syn⁹⁷ and rut²⁰⁸⁰ mutations in adult odor-shock associative function, and the selective defect of the syn⁹⁷-mutation in short- rather than longer-term memory (Michels, 2011).

Given that the memory trace established in the current paradigm likely is localized to few cells relative to the brain as a whole, given that these are transient, short-term memory traces, and given the possibility of dephosphorylation, it is not unexpected that Nuwal (2011) did not uncovered either predicted PKA site of synapsin as being phosphorylated in a biochemical approach, using whole-brain homogenates from untrained adult Drosophila. Given the likely spatial and temporal restriction of these events in vivo, immunohistological approaches are warranted to see whether, where, and under which experimental conditions synapsin phosphorylated at either of its PKA sites indeed can be detected (Michels, 2011).

Interestingly, the evolutionarily conserved N-terminal PKA-1 site undergoes ADAR-dependent mRNA editing, which despite the genomically coded RRFS motif yields a protein carrying RGFS. This editing event, as judged from whole-brain homogenates, occurs for most but not all synapsin and, as suggested by in vitro assays of an undecapeptide with bovine PKA, may reduce phosphorylation rates by PKA. Given that the successfully rescuing UAS-syn construct codes the edited RGFS sequence, it should be interesting to see whether this rescue is conferred by residual phosphorylation at PKA-1, and/or by phosphorylation of the evolutionarily nonconserved PKA-2 site. Last, one may ask whether an otherwise wild-type synapsin protein featuring a nonedited RRFS motif is also rescuing associative function (Michels, 2011).

In any event, the finding that the PKA consensus sites of synapsin are required to restore learning in the syn⁹⁷-mutant is the first functional argument to date, in any experimental system, to suggest synapsin as an effector of the AC-cAMP-PKA cascade in associative function (Michels, 2011).

Interaction between differentiating cell- and niche-derived signals in hematopoietic progenitor maintenance

Maintenance of a hematopoietic progenitor population requires extensive interaction with cells within a microenvironment or niche (see Hematopoetic progenitor maintenance in the Drosophila blood system). In the Drosophila hematopoietic organ, niche-derived Hedgehog signaling maintains the progenitor population. This study shows that the hematopoietic progenitors also require a signal mediated by Adenosine deaminase growth factor A (Adgf-A) arising from differentiating cells that regulates extracellular levels of adenosine. The adenosine signal opposes the effects of Hedgehog signaling within the hematopoietic progenitor cells and the magnitude of the adenosine signal is kept in check by the level of Adgf-A secreted from differentiating cells. These findings reveal signals arising from differentiating cells that are required for maintaining progenitor cell quiescence and that function with the niche-derived signal in maintaining the progenitor state. Similar homeostatic mechanisms are likely to be utilized in other systems that maintain relatively large numbers of progenitors that are not all in direct contact with the cells of the niche (Mondal, 2011).

The mammalian hematopoietic niche displays complex interactions between populations of HSCs and progenitors to maintain their numbers. The relative in vivo contributions of cues emanating from the microenvironment in regulating stem cell versus progenitor maintenance remains unclear. Several stem cell and progenitor populations demonstrate slow cell cycling and this property of 'quiescence' is critical for maintaining their integrity over a period of time (Mondal, 2011).

In vivo genetic analysis in Drosophila allows for the study of stem cell properties in their endogenous microenvironment (Losick, 2011). Drosophila blood cells, or hemocytes, develop within an organ called the lymph gland, where differentiating hemocytes, their progenitors, and the cells of the signaling microenvironment or niche, are found. Differentiated blood cells in Drosophila are all myeloid in nature and are located along the outer edge of the lymph gland, in a region termed the cortical zone (CZ. These arise from a group of progenitors located within an inner core of cells termed the medullary zone (MZ). The MZ cells are akin to the common myeloid progenitors (CMP) of the vertebrate hematopoietic system. They quiesce, lack differentiation markers, are multipotent, and give rise to all Drosophila blood lineage. MZ progenitors are maintained by a small group of cells, collectively termed the posterior signaling center (PSC), that function as a hematopoietic niche. Clonal analysis has suggested the existence of a niche-bound population of hematopoietic stem cells, although such cells have not yet been directly identified (Mondal, 2011).

The PSC cells express Hedgehog (Hh), which is required for the maintenance of the MZ progenitors. Cubitus interruptus (Ci) is a downstream effector of Hh signaling similar to vertebrate Gli proteins; it is maintained in its active Ci¹⁵⁵ form in the presence of Hh and degraded to the repressor Ci⁷⁵ form in the absence of Hh. PSC-derived Hh signaling causes MZ cells to exhibit high Ci¹⁵⁵ (Mondal, 2011).

Proliferation of circulating larval hemocytes is also regulated by Adenosine Deaminase Growth Factor-A (Adgf-A), which is similar to vertebrate adenosine deaminases (ADAs). Adgf-A is a secreted enzyme that converts extracellular adenosine into inosine by deamination. Two distinct adenosine deaminases, ADA1 and ADA2/CECR1, are found in humans. CECR1 is secreted by monocytes as they differentiate into macrophages. In Drosophila, mutation of Adgf-A causes increased adenosine levels and increase in circulating blood cells (Mondal, 2011 and references therein).

Extracellular adenosine is sensed by the single Drosophila adenosine receptor (AdoR) that generates a mitogenic signal through the G protein/adenylate cyclase/cAMP-dependent Protein Kinase A (PKA) pathway (Dolezelova, 2007). A target of PKA is the transcription factor Ci, which also transduces the Hedgehog signal. This study explored the potential link between adenosine and Hedgehog signaling, both through PKA mediated regulation of Ci, and a model was proposed that the niche signal and the CZ signal interact to maintain the progenitor population in a quiescent and undifferentiated state within the MZ of the lymph gland (Mondal, 2011).

The first cells that express differentiation markers appear stereotypically at the peripheral edge of the lymph gland. These differentiating cells will eventually populate an entire peripheral compartment that will comprise the CZ. The timing of the first signs of differentiation matches closely with the onset of quiescence among the precursor population, eventually giving rise to the medullary zone (MZ) (Mondal, 2011).

The close temporal synchronization of CZ formation and the quiescence of MZ progenitors raised the intriguing possibility that the onset of differentiation might regulate the proliferation profile of the progenitors. To test this hypothesis, cell death was induced by expressing the pro-apoptotic proteins Hid and Reaper in the differentiating hemocytes, and the effect of their loss was assayed in the progenitor population. Loss of CZ cells was found to induce proliferation of the adjacent progenitor cells, which are normally quiescent at this stage (Mondal, 2011).

Candidate ligands in the lymph gland were knocked down by RNA interference (RNAi) and monitored for a loss of progenitor quiescence. This survey identified Pvf1 as a signaling molecule that is required for the maintenance of quiescence within the lymph gland. Expressing Pvf1^RNAi using Gal4 drivers specific to either niche (PSC) cells using Antp-gal4, progenitor cells using dome-gal4, or differentiating cells using Hml-gal4 showed that PSC-specific knockdown is sufficient to induce progenitor proliferation, whereas Pvf1 knockdown in progenitors or differentiating cells has no effect on the lymph gland. These results indicate that Pvf1 synthesized in the PSC is required for progenitor quiescence (Mondal, 2011).

To determine the site of Pvf1 function, its receptor Pvr was knocked down in the lymph gland using a similar approach. Interestingly, it was found that Pvr^RNAi expressed under the control of drivers specific to differentiating cells (Hml-gal4 and pxn-gal4) causes a loss of progenitor quiescence. The BrdU incorporating cells do not express differentiation markers. Thus, differentiation follows the proliferative event. Lymph glands are not similarly affected when Pvr function is downregulated in the progenitors themselves. These results indicate that Pvf1 originates in the niche and activates Pvr in maturing hemocytes, and that this signaling system is important for the quiescence of MZ progenitors. These results did not explain, though, how maturing cells might signal back to the progenitors causing them to maintain quiescence (Mondal, 2011).

Given the previously known role of Adgf-A in the control of hemocyte number in circulation (Dolezal, 2005), whether this protein plays a similar role in the lymph gland was investigated. Remarkably, downregulation of the secreted Adgf-A protein in the differentiating hemocytes of the CZ, achieved by expressing Adgf-A^RNAi under Hml-gal4 control, induces loss of quiescence of MZ progenitors, similar to that seen with loss of Pvr in the CZ. This suggests that Adgf-A may act as a signal originating from differentiating hemocytes that is required for maintaining progenitor quiescence. In support of this idea, while overexpression of Adgf-A in differentiating hemocytes alone does not affect normal zonation, it suppresses the induced progenitor proliferation caused by downregulation of Pvr. For loss of signaling molecules, it is the break in the signaling network necessary for reducing adenosine that causes continued proliferation and eventual differentiation. For rpr/hid the signaling cell itself has been removed, thereby causing a lack in a backward signal. Quantitative analysis of the data is consistent with a role for Adgf-A downstream of Pvr (Mondal, 2011).

The role of a niche signal is well established in many developmental systems that involve stem cell/progenitor populations. In the Drosophila lymph gland the niche expresses Hh and maintains a group of progenitor cells (Mandal, 2007). This current study establishes an additional mechanism, parallel to the niche signal that originates from differentiating cells, which also regulates quiescence of hematopoietic progenitors (Mondal, 2011).

The cells of the lymph gland proliferate at early stages, from embryo to mid second instar. At this stage, cells farthest from the PSC initiate differentiation and the rest enter a quiescent phase defining a MZ. In wild-type, the cells of the MZ remain quiescent and in progenitor form throughout the third instar, and this process requires a combination of the PSC and CZ signals. If either signal is removed, the progenitor population will eventually be lost due to differentiation. In many different genetic backgrounds, if quiescence is lost, the progenitor population initially continues to incorporate BrdU during the second instar without expressing any maturation markers. The differentiation phenotype, characterized by the expression of such markers, follows this abnormal proliferation. The net result is that whenever the progenitors accumulate BrdU (but not express any markers of differentiation) in the second instar, all cells of the lymph gland are differentiated and no MZ remains in the third instar. While the nature of the signal that triggers hemocyte differentiation is not known, withdrawal of Wingless may play a role in this process (Mondal, 2011).

Experimental analysis has demonstrated a novel role for Pvr in maturing hemocytes and its ligand, Pvf1, in the cells of the PSC. Pvf1 expression increases at a stage when the lymph gland is highly proliferative. At this critical window in development, Pvf1 originating from the PSC is transported to the differentiating hemocytes, binds to its receptor Pvr, and activates a STAT-dependent signaling cascade. At this stage, Pvf1 is sensed by all cells but it is only in the differentiating hemocytes that it activates Adgf-A in an AdoR/Pvr-dependent manner. This secreted factor Adgf-A is required for regulating extracellular adenosine levels. High adenosine would signal through AdoR and PKA to inactivate Ci and reduce the effects of the niche-derived Hedgehog signal leading to differentiation of the progenitor cells. The function of the Adgf-A signal is to reduce this adenosine signal and therefore reinforce the maintenance of progenitors by the Hedgehog signal. Thus, the Adgf-A and Hh signals work in the same direction but Adgf-A does so by negating a proliferative signal due to adenosine. In wild-type, equilibrium is reached through a signal that does not originate from the niche that opposes this proliferative process. The attractive step in this model is that the CZ and niche (in this case Hh-dependent) signals both impinge on common downstream elements allowing for control of the progenitor population relative to the niche and the differentiated cells. Most importantly, this is a mechanism for maintaining quiescence within a moderately large population of cells that is not in direct contact with a niche. By the time the three zone PSC/MZ/CZ system is set up in the late second instar all the cells of the MZ express high levels of E-cadherin, become quiescent and are maintained as progenitors and are capable of giving rise to all blood cell lineages. Under such circumstances, the interaction between a niche-derived signal and an equilibrium signal originating from differentiating cells can maintain homeostatic control of the progenitor population. Several vertebrate stem cell/progenitor scenarios such as during bone morphogenesis and hematopoiesis or in the Drosophila intestine have progenitors and differentiating cells in close proximity that could pose an opportunity for a similar niche and differentiating cell-derived signal interaction. In fact, evidence for such interactions have recently been provided for vertebrate skin cells (Mondal, 2011).

The role of small molecules such as adenosine has not yet been adequately addressed in vertebrate progenitor maintenance. A small molecule such as extracellular adenosine is unlikely to form a gradient over the population of cells and maintain such a gradient over a developmental time scale. It is much more likely that this system operates similar to the 'quorum sensing' mechanisms described for prokaryotes. A critical level of adenosine is required for proliferation and by expressing the Adgf-A signal this threshold amount is lowered, causing quiescence in the entire population (Mondal, 2011).

This study describes a developmental mechanism that is relevant to the generation of an optimal number of blood cells in the absence of any overt injury or infection. However, a system that utilizes such a mechanism to maintain a progenitor population could potentially sense a disruption upon induction of various metabolic stresses to cause differentiation of myeloid cells. Various mitochondrial and cellular stresses can cause an increase in extracellular adenosine (Fredholm, 2007), but whether they are relevant to this system remains to be studied. In the past, dual use has been observed of reactive oxygen species (ROS) as well as Hypoxia Inducible Factor-a (HIF-a) in both development and stress response of the Drosophila hematopoietic. Responses to injury have been described in the Drosophila intestine, and in satellite cells that respond during injury, a stress related signal could be the initiating factor that overrides a maintenance signal. Thus, the equilibrium generated through developmental interactions is disrupted to promote a cellular response to stress signals (Mondal, 2011).

Switch of PKA substrates from Cubitus interruptus to Smoothened in the Hedgehog signalosome complex

Hedgehog (Hh) signalling is crucial for developmental patterning and tissue homeostasis. In Drosophila, Hh signalling is mediated by a bifunctional transcriptional mediator, called Cubitus interruptus (Ci). Protein Kinase A (PKA)-dependent phosphorylation of the serpentine protein Smoothened (Smo) leads to Ci activation, whereas PKA-dependent phosphorylation of Ci leads to the formation of Ci repressor form. The mechanism that switches PKA from an activator to a repressor is not known. This study shows that Hh signalling activation causes PKA to switch its substrates from Ci to Smo within the Hh signalling complex (HSC). In particular, Hh signalling increases the level of Smo, which then outcompetes Ci for association with PKA and causes a switch in PKA substrate recognition. A new model is proposed in which the PKA is constitutively present and active within the HSC, and in which the relative levels of Ci and Smo within the HSC determine differential activation and cellular response to Hh signalling (Ranieri, 2014).

14-3-3ε couples protein kinase A to semaphorin signaling and silences plexin RasGAP-mediated axonal repulsion

The biochemical means through which multiple signaling pathways are integrated in navigating axons is poorly understood. Semaphorins are among the largest families of axon guidance cues and utilize Plexin (Plex) receptors to exert repulsive effects on axon extension. However, Semaphorin repulsion can be silenced by other distinct cues and signaling cascades, raising questions of the logic underlying these events. This study uncovers a simple biochemical switch that controls Semaphorin/Plexin repulsive guidance. Plexins are Ras/Rap family GTPase activating proteins (GAPs) and this study finds that the PlexA GAP domain is phosphorylated by the cAMP-dependent protein kinase (PKA). This PlexA phosphorylation generates a specific binding site for 14-3-3ε, a phospho-binding protein that is necessary for axon guidance. These PKA-mediated Plexin-14-3-3ε interactions prevent PlexA from interacting with its Ras family GTPase substrate and antagonize Semaphorin repulsion. These results indicate that these interactions switch repulsion to adhesion and identify a point of convergence for multiple guidance molecules (Yang, 2012).

Axons rely on the activation of guidance receptors for correct navigation but receptor inactivation is also thought to be a means through which growth cones integrate both attractive and repulsive guidance signals. The current results indicate that such a mechanism plays a critical role in Sema/Plex-mediated repulsive axon guidance. PlexA was found to use its GAP activity to specify axon guidance but this activity is antagonized by a PKA-mediated signaling pathway. PKA directly phosphorylates the GAP domain of PlexA and this phosphorylation provides a binding site for 14-3-3ε. 14-3-3ε is critical for axon guidance and disrupts the ability of PlexA to interact with its Ras GTPase substrate. These interactions effectively switch PlexA-mediated axonal repulsion to Integrin-mediated adhesion and provide a simple biochemical mechanism to integrate antagonistic axon guidance signals (Yang, 2012).

Genetic experiments identify a critical role for 14-3-3ε proteins in directing axon guidance events during development. The 14-3-3 proteins are a phylogentically well-conserved family of cytosolic signaling proteins including seven mammalian members that play key roles in a number of cellular processes. Interestingly, 14-3-3 family proteins were first identified because of their high level of expression in the brain, but despite considerable interest in their functions, their roles in the nervous system are still incompletely understood. For instance, 14-3-3 proteins are highly expressed in growing axons and have been found to modulate neurite extension and growth cone turning in vitro in a number of contexts. However, their necessity for directing axonal growth and guidance events in vivo are unknown as is the functional role of each family member in these neurodevelopmental processes. This study found that one of the two Drosophila 14-3-3 family members, 14-3-3ε, is required in vivo for axon guidance and plays specific roles in the pathfinding of motor and CNS axons. Moreover, previous mutant analysis has revealed that the other 14-3-3 family member in Drosophila, 14-3-3ζ (Leonardo), does not exhibit significant motor axon guidance or innervation defects but plays a critical role in synaptic transmission and learning and memory. These results indicate that individual 14-3-3 family members play specific roles in the development of the nervous system and in light of the requirement of 14-3-3ε in mammalian brain development and neuronal migration, and potential roles for 14-3-3ε (YWHAE) in human neurological disease, future work will determine if 14-3-3ε's role in axon guidance is phylogenetically conserved (Yang, 2012).

Genetic and biochemical experiments also identify a specific role for 14-3-3ε in regulating Sema/Plex-mediated repulsive axon guidance. Sema/Plex-mediated repulsive axon guidance is antagonized by increasing cAMP levels, but the mechanisms underlying these cAMP-mediated effects are poorly understood. Interestingly, Plexins associate with the cAMP-dependent protein kinase (PKA) via MTG/Nervy family PKA (A kinase) anchoring proteins (AKAPs). AKAPs position PKA at defined locations to allow for the spatially and temporally specific phosphorylation of target proteins in response to local increases in cAMP and this study now finds that PKA phosphorylates the cytoplasmic portion of PlexA. Genetic and biochemical results suggest that this phosphorylation provides a binding site for a specific 14-3-3 family member, 14-3-3ε. 14-3-3 proteins are well known as phosphoserine/threonine-binding proteins and have been found to utilize this ability to regulate the activity of specific enzymes. This study found that mutating the 14-3-3ε binding site on PlexA generates a hyperactive PlexA receptor, providing a better understanding of the molecular and biochemical events through which cAMP signaling regulates Sema/Plex repulsive axon guidance. Future work will focus on identifying the upstream extracellular signal that increases cAMP levels, although it is interesting that the axonal attractant Netrin is known to increase cAMP levels and antagonize Sema-mediated axonal repulsion (Yang, 2012).

The results also indicate that the GAP activity of PlexA is critical in vivo for repulsive axon guidance and that cAMP/PKA/14-3-3ε signaling regulates this Plexin RasGAP-mediated repulsion. Plexins are GAPs for Ras family proteins and in vitro work has revealed that the GAP activity of Plexin is important for its signaling role. This study now finds that RasGAP activity is required in vivo in neurons for Plex-mediated repulsive axon guidance. Moreover, the results indicate that 14-3-3ε binds to a single phosphoserine residue within the PlexA GAP domain and antagonizes PlexA RasGAP-mediated axon guidance. Interestingly, positive regulation of GTPase signaling may be a conserved function for 14-3-3ε since it also increases the efficiency of Ras signaling during Drosophila eye development and 14-3-3 turns off the activity of other known GAPs and enhances Ras signaling. Therefore, the results suggest a model in which Sema/Plex interactions activate PlexA GAP activity, which inactivates Ras/Rap and disables Integrin-mediated adhesion. However, these Sema/Plex-mediated effects are subject to regulation, such that increasing cAMP levels activates PlexA-bound PKA to phosphorylate PlexA and provide a binding site for 14-3-3ε. These PlexA-14-3-3ε interactions occlude PlexA GAP-mediated inactivation of Ras family GTPases and restore Integrin-dependent adhesion (Yang, 2012).

In conclusion, this study has identified a simple mechanism that allows multiple axon guidance signals to be incorporated during axon guidance. Neuronal growth cones encounter both attractive and repulsive guidance cues but the molecular pathways and biochemical mechanisms that integrate these antagonistic cues and enable a discrete steering event are incompletely understood. One way in which to integrate these disparate signals is to allow different axon guidance receptors to directly modulate each other's function. Another means is to tightly regulate the cell surface expression of specific receptors and thereby actively prevent axons from seeing certain guidance cues. Still further results are not simply explained by relatively slow modulatory mechanisms like receptor trafficking, endocytosis, and local protein synthesis but indicate that interpreting a particular guidance cue is susceptible to rapid intracellular modulation by other, distinct, signaling pathways. The results now indicate a means to allow for such intracellular signaling crosstalk events and present a logic by which axon guidance signaling pathways override one another. Given this molecular link between such key regulators of axon pathfinding as cyclic nucleotides, phosphorylation, and GTPases, the observations on silencing Sema/Plex-mediated repulsive axon guidance also suggest approaches to neutralize axonal growth inhibition and encourage axon regeneration (Yang, 2012).

AKAPs act in a two-step mechanism of memory acquisition

Defining the molecular and neuronal basis of associative memories is based upon behavioral preparations that yield high performance due to selection of salient stimuli, strong reinforcement, and repeated conditioning trials. One of those preparations is the Drosophila aversive olfactory conditioning procedure where animals initiate multiple memory components after experience of a single cycle training procedure. This study explored the analysis of acquisition dynamics as a means to define memory components and revealed strong correlations between particular chronologies of shock impact and number experienced during the associative training situation and subsequent performance of conditioned avoidance. Analyzing acquisition dynamics in Drosophila memory mutants revealed that rutabaga (rut)-dependent cAMP signals couple in a divergent fashion for support of different memory components. In case of anesthesia-sensitive memory (ASM) this study identified a characteristic two-step mechanism that links rut-AC1 to A-kinase anchoring proteins (AKAP)-sequestered protein kinase A at the level of Kenyon cells, a recognized center of olfactory learning within the fly brain. It is proposed that integration of rut-derived cAMP signals at level of AKAPs might serve as counting register that accounts for the two-step mechanism of ASM acquisition (Scheunemann, 2013).

Conditioned odor avoidance is subject to a general dichotomy since multiple memory components are engaged in control of behavior. This is usually analyzed at two time points, i.e., 3 min and 3 h after training. At 3 min, basal and dynamic STM are separable by genetic means as revealed by opposing phenotypes of rut¹ and dnc¹ mutants. Moreover, those components are also separable due to characteristic differences in their acquisition dynamics as revealed by different effects of shock number. A similar dichotomy applied to 3 h memory when ASM and ARM were separable by means of amnestic treatment. It was striking that basal STM and consolidated ARM were instantaneously acquired, resulting in a front line of protection by eliciting conditioned avoidance after a singular experience of a CS/US pairing. Interestingly, consolidated ARM (as defined by means of resisting amnestic treatment) was installed quickly after training. However, it remains to be addressed at the genetic and molecular level, whether 3 h ARM linearly results from the functionally similar basal SMT component (Scheunemann, 2013).

In contrast, the two components of dynamic STM and labile ASM acquire in a dynamic fashion but are clearly dissociated from each other by characteristic chronologies of CS/US pairings required for their acquisition. However, either component contributes to behavioral performance in addition to the appropriate instantaneous component, and hence, increases avoidance probability during the test situation. Considering a potential benefit from avoiding aversive situations this overall dichotomy of behavioral control seems plausible and is also reflected at the genetic level since rut-dependent cAMP signals are limited to support of dynamic but not instantaneous memory components. Rut-dependent STM and ASM, however, are dissociated by means of shock impact and discontinuous formation of ASM is limited to situations where animals repeatedly experience high-impact CS/US pairings within a predefined time window. Experience that does not meet this criterion, however, is not discounted but adds to continuously acquired dynamic STM. By functional means these two components are thus clearly separated but commonly dependent on rut-derived cAMP signals within the KC layer, forming ties between genetically and functionally defined memory components (Scheunemann, 2013).

Genetic dissection of memory has a long-standing history in Drosophila and provided a powerful means to define molecular, cellular, and neuronal networks involved in regulation of conditioned odor avoidance. Among others, the cAMP-signaling cascade has been identified as one central tenet of aversive odor memory foremost by means of two single-gene mutants affecting either a Ca²⁺-sensitive type 1 adenylyl cyclase (AC1) and/or a cAMP-specific type 4 phosphodiesterase (PDE4) affected in the Drosophila learning mutants rutabaga (rut-AC1) and dunce (dnc-PDE4). Although originally isolated due to poor performance in the aversive odor learning paradigm, a general dichotomy has been recently revealed that separates memory components by their dependency on either rut-AC1 or dnc-PDE4 function, and the view was established that two different types of cAMP signals are engaged during the single-cycle training procedure (Scheunemann, 2012). A similar dichotomy is observed at level of acquisition dynamics and suggests that rut-dependent cAMP signals are limited to formation of dynamically acquired memory components, i.e., dynamic STM and ASM. Interestingly, rut-dependent cAMP is also required for long-term memory (LTM), which acquires after spaced and repeated training sessions. Downstream the signaling cascade, however, appropriate cAMP signals are differently channeled to either support LTM in a CREB-dependent manner, ASM via tomosyn-dependent plasticity, or basal STM via synapsin-dependent regulation of synaptic efficacy. It appears that the chronology of CS/US pairings is an important determinant of which downstream effect is triggered and hence molecular mechanisms must be installed that are sensitive to the temporal order of training (Scheunemann, 2013).

At the level of molecular scaffolds, literature suggests that AKAPs serve the integration of cAMP with other signaling processes and are crucially involved in the control of a plethora of cellular functions in any organ. For example, AKAP79 coordinates cAMP and Ca²⁺ signaling in neurons to control ion channel activities. The recognized design principle of AKAPs to serve as molecular switch is well in line with the recognized two-step register mechanism involved in ASM formation. An increasing body of evidence shows that AKAPs are involved in memory processing across phyla and accordantly those studies revealed a contribution for support of matured, but not immediate memories. Communality among all those AKAP-dependent memories is the need for repeated and temporally organized training sessions, i.e., only spaced training sessions are effective to induce protein synthesis-dependent LTM in flies and mammals. Similarly, ASM requires the precise timing of two training sessions and mechanistically acts via an 'activated' state generated by the initial CS/US pairing that persists within the brain for ~5 min. Such temporal integration might well take place at level of AKAPs within the KC layer to operate rut-AC1-dependent cAMP signals finally onto phosphorylation of tomosyn. Identification of the particular AKAPs involved in two-step ASM formation will require further analysis of appropriate mutants. To date, only four Drosophila AKAPs are characterized, i.e., rugose, a 550 kDa protein that impacts on STM performance probably via molecular domains other than its AKAP function; yu/spoonbill that supports LTM; and Nervy and AKAP²⁰⁰ have not been tested for their impact on aversive odor memory (Scheunemann, 2013).

Together, the benchmarking of Drosophila aversive odor memory performance by means of acquisition dynamics that were demonstrated in this study will provide a valuable tool since dynamic aspects of acquisition are obviously informative and add to the steady-state condition determined by the single-cycle training procedure (Scheunemann, 2013).

PDF and cAMP enhance Per stability in Drosophila clock neurons

The neuropeptide PDF is important for Drosophila circadian rhythms: pdf⁰¹ (pdf-null) animals are mostly arrhythmic or short period in constant darkness and have an advanced activity peak in light-dark conditions. PDF contributes to the amplitude, synchrony, as well as the pace of circadian rhythms within clock neurons. PDF is known to increase cAMP levels in PDR receptor (PDFR)-containing neurons. However, there is no known connection of PDF or of cAMP with the Drosophila molecular clockworks. This study discovered that the mutant period gene per^S ameliorates the phenotypes of pdf-null flies. The period protein (Per) is a well-studied repressor of clock gene transcription, and the per^S protein (PerS) has a markedly short half-life. The result therefore suggests that the PDF-mediated increase in cAMP might lengthen circadian period by directly enhancing Per stability. Indeed, increasing cAMP levels and cAMP-mediated protein kinase A (PKA) activity stabilizes Per, in S2 tissue culture cells and in fly circadian neurons. Adding PDF to fly brains in vitro has a similar effect. Consistent with these relationships, a light pulse causes more prominent Per degradation in pdf⁰¹ circadian neurons than in wild-type neurons. The results indicate that PDF contributes to clock neuron synchrony by increasing cAMP and PKA, which enhance Per stability and decrease clock speed in intrinsically fast-paced PDFR-containing clock neurons. It is further suggested that the more rapid degradation of PerS bypasses PKA regulation and makes the pace of clock neurons more uniform, allowing them to avoid much of the asynchrony caused by the absence of PDF (Li, 2014).

Since the original observation that pdf⁰¹ flies have a highly reliable 1-2 h advanced activity phase in LD and short period in DD before they become arrhythmic, it has been assumed that PDF functions at least in part to lengthen the period of at least some brain oscillators that run too fast in its absence. Indeed, there is evidence in favor of this notion, and it is likely that the pdf⁰¹ strain arrhythmicity results from conflicts between neuronal oscillators that run too fast and others that maintain a ∼24-h pace or may even run more slowly without PDF. The substantial improvement of pdf⁰¹ rhythmicity by the per^S gene therefore suggests that per^S endows all oscillators with such a short period that they have a more uniform pace and substantially reduced oscillator asynchrony without PDF (Li, 2014).

Although there was no information on how PDF might function to lengthen the period of the fast oscillators, the effect of per^S implicates Per as a candidate molecular target. Because PerS is known to disappear rapidly in the nighttime, this further suggests that the Per degradation rate might be the biochemical target of PDF period lengthening. An even more specific version of this notion follows from the PDF-mediated increases in cAMP levels in PDFR-expressing clock neurons. Because PDFR is expressed in many clock neurons, including subsets of LNvs, LNds, and DN1s, this increase in cAMP may slow the pace of Per degradation in intrinsically fast-paced PDFR-expressing clock neurons. Indeed, the data indicate that increasing cAMP levels and PKA activity inhibits Per degradation in cell culture as well as in fly brains. Although these increases are probably in excess of what normally occurs in response to PDF, addition of PDF to brains in vitro has a similar effect. Because the additions of kinase inhibitors Rp-cAMPS and PKI increased the rate of Per degradation in S2 cells as well as in brains, it is suggested that PDF-induced up-regulation of cAMP level and PKA activity likely affect Per stability (Li, 2014).

A light pulse at night caused more prominent Per degradation in pdf⁰¹ mutant flies than in wild-type flies. As nighttime light also causes premature Tim degradation and a consequent advance in Per degradation in many clock neurons, some of these neurons could be the intrinsically fast (22- to 23-h period) oscillators that are impacted by PDF and experience enhanced cAMP levels to slow their rate of Per degradation and clock pace. These probably include the s-LNvs and the DN1s, many of which are PDFR-positive. Based on the behavioral phenotype of pdf⁰¹ flies in LD and DD, the effect of PDF on Per degradation probably occurs in the late night–early morning in a LD cycle and at the same (subjective) time in DD. This is also the time when Per degradation is most prominent (Li, 2014).

Interestingly, the firing rate of PDF-containing neurons, the l-LNvs as well as the s-LNvs, is also maximal near the beginning of the day, in DD as well as LD; this is also the likely time of maximal PDF release from s-LNv dorsal projections. In addition, the l-LNvs promote light-mediated arousal, also mediated at least in part by PDF. Taken together with the fact that light has been shown to increase the firing rate of l-LNvs in a CRY-dependent manner, it is likely that lights on in the morning also potentiates the PDF-cAMP system. Note that the end of the night-beginning of the day is the time in the circadian cycle dominated by clock protein turnover, i.e., this is when there is little per or tim RNA or protein synthesis. This further supports a focus on clock protein turnover regulation at these times (Li, 2014).

Because the mammalian neuropeptide VIP contributes to oscillator synchrony within the SCN in a manner that resembles at least superficially the contribution of PDF to oscillator synchrony within the fly brain circadian network, VIP might function similarly to PDF. However, VIP probably connects differently to the mammalian clock system. For example, morning light almost certainly up-regulates clock protein transcription in mammals, for example, per1 transcription. Therefore, VIP-mediated up-regulation of cAMP levels probably activates CREB and clock gene transcription through CRE sites in mammalian clock gene promoters rather than influencing clock protein turnover like in flies (Li, 2014).

The stabilization effect of PDF and cAMP on Per requires PKA activity within circadian neurons. The effect could be indirect, through unknown PKA targets including other clock proteins. However, Per is known to be directly phosphorylated by multiple kinases; they include Nemo, which stabilizes Per. In addition, a study in Neurospora shows that PKA directly phosphorylates and stabilizes FRQ. Because FRQ and Per have similar roles, protein turnover in the two clock systems may be similar beyond the shared role of the CK1 kinase. Based also on the S2 cell experiments, it is suggested that PKA directly phosphorylates Per and enhances its stability. This could occur by inhibiting a conformational switch to a less stable structure, a possibility that also applies to NEMO-mediated Per stabilization. PKA could also phosphorylate other clock proteins; this is by analogy to the known Per kinases Nemo and Doubletime (Dbt), which also phosphorylate Clk (Li, 2014).

The more rapid intrinsic degradation of PerS may at least partially bypass the effect of PKA phosphorylation and therefore PDFR stimulation. This may endow all circadian neurons with a more uniform period, which can maintain synchrony and therefore rhythmicity without PDF. The fact that PerS is less sensitive than Per to increases in cAMP levels is consistent with this interpretation, although an earlier phase of PerS degradation might also influence this result (Li, 2014).

One further consideration is the 0.5-h period difference between the per^S and the per^S;;pdf⁰¹ strains. A residual period-lengthening effect of PDF suggests that per^S does not endow all oscillators with the identical period, i.e., that there is still some asynchrony between different per^S neurons without PDF. This may reflect an incomplete bypass of PKA by PerS or an additional effect of cAMP or PKA on other clock proteins. Nonetheless, several per^S neuronal oscillators maintain a strong amplitude without PDF. Although this is commonly taken to reflect an effect on synchrony, another possibility is based on data indicating that PDF normally enhances oscillator amplitude as well as synchrony; weak amplitudes may then be the more proximal cause of behavioral arrhythmicity. With this notion in mind, it is suggested that PerS-containing oscillators are not only short period but also more robust, i.e., that the more rapid turnover of PerS makes the clock stronger. More robust rhythmicity is also apparent in the behavioral records of all per^S-containing strains. In this view, the stronger degradation 'drive' of PerS makes these oscillators more cell autonomous and therefore less dependent on neuronal mechanisms like firing and PDF release, which enhance oscillator synchrony and amplitude. The general notion is that discrete differences in clock molecule properties can change the relationship of the transcriptional cycle to the circadian brain network (Li, 2014).

back to cAMP-dependent protein kinase 1: Protein Interactions part 1/2

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