InteractiveFly: GeneBrief

SKP1-related A: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

Skp1 proteins function in protein degradation as a core component of the SCF (SKP1, cullin/CDC53, F-box protein) complex to link the substrate-recognition subunit (F-box protein) to a cullin (see Drosophila Cullin1) that in turn binds the ubiquitin-conjugating enzyme. Centrosome duplication must be coupled to the main cell cycle to ensure that each cell has precisely two centrosomes at the onset of mitosis. Supernumerary centrosomes are commonly observed in cancer cells, and may contribute to tumorigenesis. Drosophila SkpA, the Skp1 component of Drosophila SCF ubiquitin ligases, regulates the link between the cell and centrosome cycles. Lethal skpA null mutants exhibit dramatic centrosome overduplication and additional defects in chromatin condensation, cell cycle progression and endoreduplication. Surprisingly, many mutant cells are able to organize pseudo-bipolar spindles and execute a normal anaphase in the presence of extra functional centrosomes. SkpA mutant cells accumulate higher levels of cyclin E than wildtype cells during S and G2, suggesting that elevated cdk2/cyclin E activity may account for the supernumerary centrosomes in skpA^- cells. However, centrosome overduplication still occurs in skpA^-; cycE^- mutant animals, demonstrating that high cyclin E levels are not necessary for centrosome overduplication. These data suggest that additional SCF targets regulate the centrosome duplication pathway and that Drosophila SkpA regulates centrosome duplication independently of cyclin E accumulation (Murphy, 2003).

The centrosome serves as the major microtubule-organizing center in animal cells, helping to create polarity and organization within the cell. Each G1 cell contains a single centrosome, consisting of pericentriolar material organized around a pair of centrioles; the centrosome nucleates microtubule polymerization. During cell division, the centrosome must be duplicated precisely once and the resulting two centrosomes help to organize the bipolar spindle, which segregates the chromosomes (Murphy, 2003).

Recent studies have begun to elucidate how the cell initiates centrosome duplication. In most cell types, centriole duplication begins near the onset of S phase, suggesting that it may be controlled by part of the pathway that initiates DNA synthesis, such as cyclin E bound to cyclin-dependent kinase-2 (Cdk2-E). In somatic cells, levels of cyclin E rise in late G1 and the resulting rise in Cdk2-E kinase activity is necessary and sufficient to drive cells into S phase. Centrosome duplication is blocked by inhibitors of Cdk2 activity, and constitutive expression of cyclin E results in centrosome duplication beginning prematurely in early G1. In Swiss 3T3 cells, Cdk2-E phosphorylates nucleophosmin, a component of unduplicated centrosomes, and expression of a nonphosphorylatable form of nucleophosmin blocks centrosome duplication. Thus, Cdk2-E activity is necessary to initiate centrosome duplication, in part through the phosphorylation of nucleophosmin (Murphy, 2003 and references therein).

Little is known about the regulatory mechanism which ensures that centrosome duplication occurs only once in each cell cycle. Cells apparently lack a cell cycle checkpoint to detect the presence or production of excess centrosomes. Conversely, it is not known if cells will efficiently proceed into mitosis in the absence of centrosome duplication. Thus, the fidelity of centrosome production relies largely on regulating the duplication process itself, rather than by using checkpoints to monitor the fidelity of the process afterwards. The observation of supernumerary centrosomes (>3 centrosomes in a cell) has frequently been used as evidence for misregulation of centrosome duplication, suggesting that genes such as p53, Brca1, Brca2, p21, ATR and others are part of the pathway that regulates centrosome duplication. However, recent studies suggest that many instances of supernumerary centrosomes, including those in p53^-/- cells, arise through failed cell division resulting in tetraploid cells with twice the normal number of centrosomes. Consequently, an understanding of the pathway controlling centrosome duplication remains murky (Murphy, 2003).

Many diverse cellular processes are regulated by the SCF family of ubiquitin ligases, which target specific proteins for proteolysis (reviewed by Deshaies, 1999). SCF complexes are found in all eukaryotes and consist of an invariant core containing Skp1, Cul1 (See Drosophila Lin-19-like/Cul-1) and Rbx1/Roc1 complexed with one member of a large family of F-box proteins. Substrate recognition typically occurs through a protein interaction motif in the F-box protein, and the rest of the complex acts to recruit a ubiquitin-conjugating enzyme that catalyzes the assembly of a polyubiquitin chain on the substrate, thus targeting it for degradation by the proteasome. These biochemical studies suggest that mutations in SCF complex genes will disrupt the regulated degradation of many substrates in the cell. Several SCF components have been localized to centrosomes in vertebrate cells (Freed, 1999; Gstaiger, 1999), and supernumerary centrosomes have been reported in cells mutant for the F-box proteins skp2 (mouse: Drosophila homolog - CG9772) and slimb. However, many of the mammalian studies are confounded by high frequencies of polyploidy that have made it difficult to ascribe a direct role for SCF function in regulating centrosome duplication (Murphy, 2003).

Null mutations in Drosophila skpA, a homolog of Skp1, are shown to result in centrosome overduplication and defective endoreduplication, chromatin condensation and cell cycle progression. SkpA mutant cells accumulate elevated levels of cyclin E after entering S phase; however, genetic epistasis experiments demonstrate that high cyclin E levels are not necessary for centrosome overduplication to occur. Thus, the accumulation of other SCF substrates probably accounts for centrosome overduplication. One of these targets may function as a centrosome-licensing factor to restrict centrosome duplication to once per cell cycle (Murphy, 2003).

Null mutations in skpA were generated by imprecise excision of a P-element localized to the first intron of skpA. Four alleles were recovered with deletions of either the skpA ORF or promoter. The skpA¹ deletion completely removes the P element and 1782 bp 3' of the original insertion, including the entire skpA ORF, and is therefore a null allele (Murphy, 2003).

All four skpA alleles are homozygous lethal when crossed to skpA¹ or larger deficiencies. This lethality was completely rescued in transgenic flies expressing skpA, indicating that the lethality results from loss of skpA function. SkpA mutant embryos develop normally and hatch at wildtype frequencies, potentially because of perdurance of maternally loaded mRNA and protein. Most mutant larvae die within four days after hatching, and surviving mutant animals proceed through larval development but fail to pupate and grow significantly slower than wildtype. These results indicate that skpA function is required for larval growth and viability (Murphy, 2003).

SkpA^- larvae show pronounced defects in all proliferating tissues. The imaginal discs are rudimentary or absent, and the central nervous system (CNS) shows little increase in size past three days after egg deposition (AED). To further investigate these defects, various cell cycle parameters in the CNS from mutant larvae with wildtype controls were compared (Murphy, 2003).

SkpA^- cells exhibit a dramatic decrease in cell proliferation. The proportion of mitotic cells is comparable to wildtype shortly after hatching, but is dramatically reduced as early as 3.5 days AED and continues to decrease in surviving older animals. The proportion of cells in S phase is similarly reduced. These data suggest that skpA^- cells have a lengthened G1 and/or G2 phase of the cell cycle. To measure this more directly, the DNA content of individual nuclei was quantified to determine if they were in G1, S or G2 phase. No change was observed in the ratio of G1 to G2 cells in the CNS from young larvae; however, older mutant animals showed a dramatic increase in the proportion of G1 cells. Taken together, loss of skpA function results in a lengthening of the cell cycle by approximately twofold and ultimately a delay or arrest in G1 (Murphy, 2003).

Cell cycle defects may ultimately induce mutant cells to undergo programmed cell death; therefore, skpA^- cells were tested for changes in apoptosis by TUNEL-labeling. No increase in apoptotic cells was observed; in fact, significantly fewer cells were undergoing apoptosis, a condition that may result from disruption of the normal schedule of programmed cell death in the CNS. In contrast, virtually all cells in the few rudimentary imaginal discs observed were undergoing apoptosis; this probably accounts for the lack of imaginal discs in most mutant larvae (Murphy, 2003).

Larval growth occurs primarily through increasing cell size supported by nuclear endoreduplication; consequently, many cell proliferation mutants do not cause lethality until the beginning of pupation. To test if skpA lethality may result from a defect in endoreduplicating tissues, endoreduplication was assayed in larval salivary glands and fat bodies by BrdU incorporation. Comparable levels and frequencies of endoreduplication were observed in wildtype and skpA^- larval salivary glands; however, mutant fat body nuclei contain less DNA than wildtype and rarely undergo endoreduplication. Similarly, gut nuclei contain less DNA and have an abnormal morphology in skpA^- larvae. Thus, skpA is required for endoreduplication in some larval tissues, perhaps by regulating promoters or inhibitors of S phase (Murphy, 2003).

Previous studies have asserted roles for SCF components in regulating the separation or duplication of centrosomes (Freed, 1999; Nakayama, 2000; Wojcik, 2000). To determine if skpA plays a role in controlling centrosome duplication, centrosomes were stained in wildtype and skpA^- neuroblasts with antibodies against gamma-tubulin or centrosomin, two components of the pericentriolar matrix. As expected, nearly all mitotic wildtype cells contain two centrosomes that label with both gamma-tubulin and centrosomin. In contrast, three or more centrosomes were frequently observed in mitotic skpA^- cells. Supernumerary centrosomes were found in 4% of cells as early as 1.5 days AED, and in most mitotic cells in older animals with as many as 17 centrosomes observed in a single diploid cell. SkpA^- interphase (phospho-histone H3 negative) nuclei also frequently show aberrant chromatin condensation, which is especially pronounced in CNS cells from older animals. Clonal analyses demonstrate that the supernumerary centrosomes, delayed cell cycle and abnormally condensed chromatin are caused by cell autonomous defects in skpA function (Murphy, 2003).

Supernumerary centrosomes may arise from any of four mechanisms: (1) failed cytokinesis, (2) segregation of both centrosomes to the same daughter cell, (3) aberrant centriole splitting or fragmentation, or (4) formation of additional centrosomes in a single cell cycle, either de novo or from reduplication of the existing centrosomes. The extra centrosomes observed in skpA^- cells are unlikely to occur by the first three mechanisms for several reasons: (1) few skpA^- cells are polyploid, indicating that most cells complete cytokinesis; (2) all skpA^- anaphase cells have centrosomes at both poles, suggesting that skpA^- cells do not assemble acentrosomal spindles that would allow both centrosomes to (randomly) segregate to the same daughter cell; (3) most of the centrosomes observed in skpA^- cells are of uniform size and morphology, suggesting that they have not arisen from centrosome fragmentation. Furthermore, serial section electron microscopy of the CNS from skpA^- larvae found one cell with at least four pairs of centrioles, three of which had incomplete daughter centrioles, suggesting that they were undergoing assembly and that extra centrosomes arise from normal centriole duplication. Taken together, these data suggest that loss of skpA function results in the formation of extra centrosomes through multiple rounds of centrosome duplication in the same cell cycle (Murphy, 2003).

Cells with three or more centrosomes typically form multipolar spindles that ultimately lead to chromosome missegregation and aneuploidy. Surprisingly, all of the skpA^- anaphase cells with supernumerary centrosomes were segregating their chromosomes to only two poles, suggesting that the additional centrosomes may not be completely functional. This possibility was also raised for the supernumerary centrosomes observed in Drosophila slimb mutants, which encode another SCF component. Therefore, a detailed analysis of the functional properties of skpA^- supernumerary centrosomes was performed (Murphy, 2003).

Confocal analyses of skpA^- cells stained for centrosomes, microtubules and chromosomes reveal that skpA^- supernumerary centrosomes are competent to nucleate microtubules and attach to chromosomes. All of the centrosomes in skpA^- prophase and prometaphase neuroblasts appear to nucleate similar numbers of microtubules and are equally spaced around the nuclear periphery, suggesting that the microtubule arrays actively position the centrosomes relative to one another. However, once the chromosomes have attached to the spindle, most of the centrosomes are typically found clustered into two poles and form a pseudo-bipolar spindle with the chromosomes positioned at a normal metaphase plate. Anaphase cells retain a bipolar configuration, with the majority of centrosomes clustered at the two poles. Three-dimensional quantification of centrosome positioning in young larval CNSs reveal that supernumerary centrosomes are 2.5-fold more likely to be within 2 µm of another centrosome in metaphase and anaphase than earlier in the mitotic cycle, even though progression through the mitotic cycle is not significantly altered and similar numbers of centrosomes are seen at all stages. Therefore, the extra centrosomes are dynamically repositioned during mitosis allowing formation of pseudo-bipolar spindles and progression to anaphase (Murphy, 2003).

Although all centrosomes are equally competent to nucleate microtubules in prophase and prometaphase, only a subset of centrosomes are associated with the bulk of the spindle microtubules in later mitotic stages and some spindle poles appeared to be detached from any centrosomes, suggesting that some centrosomes may be inactivated or have decreased microtubule retention capacity. Centrosome inactivation is a normal characteristic of wildtype ganglion mother cells (Bonaccorsi, 2000), which are descended from neuroblasts, suggesting that the reduced microtubule nucleation/retention of some supernumerary centrosomes may result from the normal developmental switch to ganglion mother cell characteristics. Nevertheless, all supernumerary centrosomes are associated with at least a few microtubules, even in mitotic ganglion mother cells, and some form functional kinetochore attachments that either displace chromosomes from the metaphase plate or generate multipolar spindles in a few cases. Furthermore, anaphase cells are increasingly rare and have fewer centrosomes than metaphases in older animals, suggesting that cells with many centrosomes delay or arrest in metaphase. These cells may ultimately forgo cytokinesis and account for the small increase in polyploid cells in older animals (Murphy, 2003).

In conclusion, the supernumerary centrosomes in skpA^- cells can act as functional microtubule organizing centers, but neuroblasts can partially compensate for this aberrant microtubule nucleation by either clustering extra centrosomes together or partially inactivating them in later mitotic stages. These compensation mechanisms are sufficient to allow some cells to divide normally, although older skpA^- cells delay or arrest in metaphase. These mitotic defects may ultimately induce the observed delay or arrest in G1; alternatively, the defect in progression into S phase may be independent of the accumulation of extra centrosomes (Murphy, 2003).

Thus, SkpA performs essential roles in regulating centrosome duplication, endoreduplication, chromatin condensation, cell cycle progression and cyclin E accumulation. Undoubtedly, these functions represent only a subset of the processes regulated by skpA. Three lines of evidence suggest that SkpA primarily acts as part of multiple SCF ubiquitin ligase complexes: (1) SkpA is highly similar to human and yeast Skp1, which form multiple SCF complexes in vitro and in vivo (Deshaies, 1999); (2) SkpA interacts with the Drosophila SCF homologs Cullin1 (Cul1), Supernumerary Limbs (Slimb) and Partner of Paired (Ppa) by in vitro or yeast two-hybrid assays (Bocca, 2001; Raj, 2000), indicating that it can form at least two types of SCF complexes; (3) mutations in the Drosophila F-box genes archipelago (ago) and slimb induce elevated cyclin E levels and centrosome overduplication, respectively (Moberg, 2001; Wojcik, 2000), similar to portions of the skpA mutant phenotype reported in this study. Thus, SkpA probably functions as a core component of SCF^ago, SCF^slimb, SCF^ppa and potentially other SCF complexes in mediating the poly-ubiquitination and subsequent degradation of specific target proteins (Murphy, 2003).

SkpA mutant cells accumulate dramatic numbers of supernumerary centrosomes from multiple rounds of centrosome duplication in each cell cycle. Supernumerary centrosomes are first observed in some cells within one day after hatching, soon after the maternal supply of SkpA protein has been exhausted and before any growth defects or lethality are detected. Furthermore, centrosome overduplication occurs in mitotic clones, demonstrating that it results from a cell autonomous function of skpA. Thus, extra centrosomes most probably accumulate directly from loss of SCF function and not as a secondary consequence of another skpA function such as cell cycle progression (Murphy, 2003).

Several groups have proposed that centrosome overduplication in cancer cells may arise from aberrant accumulation of cyclin E. This hypothesis was attractive because centrosome duplication requires cdk2 function, activated by cyclin E or in some cells cyclin A. Furthermore, overexpressed cyclin E associates with and is ubiquitinated by an SCF complex in human and Drosophila cells. Constitutive cyclin E overexpression in cultured mammalian cells induces little or no centrosome overduplication; however, the immortal cell lines used in the studies may have accumulated mutations which suppress aberrant centrosome duplication, as is seen in p53^-/- mouse epithelial cells in late passages (Murphy, 2003 and references therein).

This study has directly tested the role of cyclin E in centrosome overduplication by genetically manipulating cyclin E levels in wildtype and skpA^- cells. Strikingly, drastically reducing cyclin E levels with a near-null allele does not suppress centrosome overduplication in cycling skpA^- cells. One possibility is that cyclin E is not required for centrosome duplication in Drosophila. This seems unlikely, because Drosophila cdk2 does not associate with cyclin A and lacks in vitro kinase activity when immunoprecipitated from cyclin E-deficient embryos, and other functions of cdk2 are conserved between Drosophila and vertebrates. In any case, centrosome overduplication occurs independently of SCF control of cyclin E accumulation (Murphy, 2003).

How do SCF components regulate centrosome duplication? One possibility is that simply lengthening the cell cycle introduces enough time for multiple cycles of centrosome duplication to occur. Although this model cannot be ruled out, it seems unlikely given that a centrosome must duplicate in as little as 55 minutes in a cycling neuroblast but does not reduplicate in the 12-hour cycle of an imaginal wing disc cell. Furthermore, slowing the cell cycle in abdominal histoblasts by overexpressing the Drosophila retinoblastoma-family protein RBF is not sufficient to induce centrosome overduplication (Murphy, 2003).

Instead, the idea is favored that a target of SCF-mediated degradation acts as a Centrosome Licensing Factor (CLiF) that limits centrosome duplication to once per cell cycle. CLiF would be expressed early in the cell cycle, loaded onto centrosomes, and excess CLiF would be targeted to the proteasome by an SCF complex. One cycle of centrosome duplication could then be triggered by Cdk2-E activity, but the daughter centrosomes would not be relicensed until the next cell cycle. SCF mutants would fail to degrade excess CLiF, allowing duplicated centrosomes to relicense and reduplicate in the course of a single cell cycle. One candidate CLiF is nucleophosmin/B23, which is phosphorylated by Cdk2-E and associates specifically with unduplicated centrosomes (Okuda, 2000). Future experiments will need to determine if nucleophosmin/B23 or other candidate CLiFs are targeted for degradation by an SCF complex (Murphy, 2003).

In Xenopus, antibody-addition experiments using an in vitro assay suggest that Skp1 is required for centriole separation (Freed, 1999). The results presented here clearly demonstrate that centrioles can separate and duplicate in the absence of Drosophila SkpA. These contrasting results may reflect a functional difference between Drosophila and Xenopus, potentially related to the difference in SkpA/Skp1 localization to the centrosome in these two organisms. Alternatively, Skp1 antibodies may block centriole separation in a way that does not reflect an in vivo requirement for SCF activity. This second possibility is favored because immunodepletion of Skp1 from Xenopus extracts does not block centriole separation (Freed, 1999). Determining if Skp1 serves an additional role in vertebrate centriole separation will require genetic analyses in a vertebrate model system (Murphy, 2003).

Remarkably, the large numbers of supernumerary centrosomes in skpA^- cells typically do not generate multipolar spindles in mitosis. The extra centrosomes are probably not defective, because most centrosomes can efficiently nucleate microtubules in prometaphase and one cell examined by electron microscopy had multiple centrioles apparently undergoing duplication. Furthermore, anaphase cells are increasingly rare and have fewer centrosomes than metaphases in older animals, suggesting that cells with many centrosomes delay or arrest in metaphase. This differs from many cell types in which extra centrosomes frequently lead to the formation of multipolar spindles, although mouse neuroblastoma (N115) cells and p53^-/- mouse embryonic fibroblasts with extra centrosomes typically form bipolar spindles. Also, one or two extra centrosomes in sea urchin zygotes or PtK₁ cells do not delay anaphase onset (Murphy, 2003 and references therein).

How do skpA^- neuroblasts form bipolar spindles with extra centrosomes? The centrosomes appear to be dynamically rearranged during the mitotic cycle so that the majority are clustered into two cooperative poles, potentially through the action of microtubule bundling proteins such as the nuclear mitotic apparatus protein (NuMa) and the kinesin Ncd. This ability to rearrange centrosomes into two poles does not require additional genetic mutations, as has been proposed for mammalian cells. Instead, it may reflect an inherent preference for Drosophila neuroblasts to form bipolar spindles; alternatively, loss of skpA function may result in the upregulation of compensatory proteins. It is unclear how the presence of many centrosomes delays anaphase onset. Further studies are needed to determine if this indicates a novel way to activate the spindle assembly checkpoint or the presence of another checkpoint governing anaphase onset (Murphy, 2003).

The roles of SCF complexes in governing centrosome duplication and the cell cycle may be important for understanding tumorigenesis. Many solid tumors accumulate supernumerary centrosomes, which are thought to contribute to cancer progression, suggesting that upregulation of the proposed centrosome-licensing factor may be oncogenic. Recently, the human homolog of the F-box gene ago, hCdc4, was reported to be mutated in several human breast and ovarian cancer cell lines with high cyclin E levels (Moberg, 2001; Strohmaier, 2001). Levels of the F-box protein Skp2 are upregulated in some oral carcinomas and inversely correlate with levels of the tumor suppressor p27 (Gstaiger, 2001). Future studies will need to determine if other human SCF components including Skp1 are also mutated in cancer cells. Further analyses of the functions of Drosophila skpA will help to elucidate how SCF-mediated protein degradation may be a key mechanism governing centrosome duplication, cell proliferation and cancer progression (Murphy, 2003 and references therein).

REGULATION

Protein Interactions

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

F-box protein Partner of paired protein interacts with Paired and Skp1 to coordinates Paired degradation

Selective spatial regulation of gene expression lies at the core of pattern formation in the embryo. In Drosophila, localized transcriptional regulation accounts for much of the embryonic pattern. Properties of a newly identified gene, partner of paired (ppa), suggest that localized receptors for protein degradation are integrated into regulatory networks of transcription factors to ensure robust spatial regulation of gene expression. The Ppa protein interacts with the Pax transcription factor Paired (Prd) and contains an F-box, a motif found in receptors for ubiquitin-mediated protein degradation. In normal development, Prd functions only in cells in which ppa mRNA expression has been repressed by another segmentation protein, Even-skipped (Eve). When ppa is expressed ectopically in these cells, Prd protein, but not mRNA, levels diminish. When ppa function is removed from cells that express PRD mRNA, Prd protein levels increase. It is concluded that Ppa coordinates Prd degradation and is important for the correct localization of expressed Prd. In the presence of Ppa, Prd protein is targeted for degradation at sites where its mis-expression would disrupt development. In the absence of Ppa, Prd is longer-lived and regulates downstream target genes (Raj, 2000).

To gain further insight into the combinatorial regulation by Prd and Eve, a yeast two-hybrid screen was performed for a cDNA library derived from 0-12 hour old embryos, using as bait a 140 amino-acid fragment of Prd that included its homeodomain. From a total of 2.4 x 10⁶ primary transformants, 22 classes of clones were identified by restriction analysis; mRNA in situ hybridization analysis of representatives from each class indicated that one of the cDNAs from the screen is expressed in a pair-rule pattern of stripes. This cDNA was named ppa. To test the specificity of the interaction of Ppa with Prd, the ppa cDNA was retransformed into yeast and tested using a mating assay against a panel of different baits, including Prd. Ppa interacts either with the original homeodomain-containing Prd fragment, or a fragment containing both the homeodomain and the Prd domain, but not with homeodomain-containing fragments of Ftz or Bicoid, or with several unrelated control baits. In addition, Ppa does not interact with Prd containing a Ftz homeodomain substitution, suggesting that the Prd homeodomain sequences are required for the protein interaction (Raj, 2000).

Sequence analysis of ppa provided insights into its possible functions. The Ppa open reading frame (ORF) contains 11 leucine-rich repeats (LRRs). These 20-29 amino-acid motifs have Leu residues at characteristic positions and have been implicated in protein-protein interactions. Indeed, the carboxy-terminal 92 amino acids of Ppa, encompassing three LRRs, is sufficient for the interaction with Prd in the two-hybrid screen. Sequence alignments indicate that the LRRs in Ppa are similar to those found in yeast glucose repression regulator 1 (Grr1), C. elegans CO2F5.7, an ORF of unknown function, and a human hypothetical ORF that has been named Ppa because it is 63% identical (78% similar) to the carboxy-terminal 392 amino acids of the Drosophila protein (Raj, 2000).

Like GRR1, C. elegans CO2F5.7, and the human ORF, Drosophila Ppa also contains an F-box motif amino-terminal to the LRRs. Previously characterized F-box proteins, including Grr1 and yeast Cdc4, have been shown to be receptors that target their substrates for ubiquitin-mediated protein degradation. These proteins interact through their F-boxes with Skp1, which associates with Cdc53/Cullin, forming an SCF complex (Skp1/Cullin/F-box). The SCF complex functions as a ubiquitin ligase enzyme (E3), which facilitates transfer of ubiquitin from a ubiquitin conjugation enzyme (E2) to the substrate. The F-box proteins provide a vital link between this machinery and specific substrates to be degraded, the substrate interaction typically being mediated through WD40 or LRR protein-interaction motifs within the F-box protein. Thus, the F-box proteins provide for specificity of substrate choice. Unlike Grr1, CO2F5.7 or other described F-box proteins, Ppa also contains a region rich in Ala, His and Pro, which is similar to Ala-rich domains observed in previously identified transcriptional repressor proteins, including Kruppel, Knirps, Eve and En. The presence of the F-box and Ala/His/Pro motifs suggests that Ppa might function as a receptor for protein degradation, or as a transcriptional co-repressor, or both (Raj, 2000).

The ppa mRNA is not detected in unfertilized embryos, suggesting that ppa is not expressed maternally. Uniform expression throughout the embryo is first detected at nuclear cycle 10, and gradually increases in intensity during cycles 11-14. Ppa expression diminishes in the pole regions during cycle 13. During cycle 14 and early gastrulation, the expression of ppa transformed into a pair-rule striped pattern with the formation of interbands within which ppa expression is lost. This is followed during germ-band elongation by splitting of the ppa stripes to generate a one-segment-repeated pattern of reiterated interbands. The ppa stripes do not have sharp borders. Expression of ppa is lost throughout the ventral region of the embryo, which contributes to the ventral furrow during gastrulation, presumably as a result of dorsoventral regulators. The ppa mRNA is localized in the basal regions of cells, in contrast to the apical localization of most pair-rule gene mRNAs (Raj, 2000).

To assess the possible functional relationships with Prd and Eve, embryo fillets were double-stained for ppa mRNA and Prd or Eve protein. During the early stages of cycle 14, when ppa expression is being restricted to stripes, there are significant levels of ppa expression overlapping the stripes of Prd protein. As cycle 14 proceeds, the posterior regions of the forming ppa stripes transiently overlap the anterior regions of the primary Prd stripes but, by early gastrulation, the Prd and ppa stripes are almost distinct. This transient but limited overlap in the expression of ppa and Prd is consistent with the model that Ppa negatively regulates Prd protein function (Raj, 2000).

Comparison of ppa mRNA with Eve protein shows almost reciprocal expression of the two genes (ppa interbands coincide with Eve stripes), raising the possibility that Eve might repress ppa expression, thereby giving rise to the ppa interbands. This interpretation is supported by examination of eve mutant embryos, which have uniform instead of striped ppa expression during cycle 14 and germ-band elongation. Moreover, adding back a transgene (P[eve.2,3,7] that expresses eve stripes 2, 3 and 7 in an otherwise eve mutant background, results in ppa interbands corresponding to these three Eve stripes (Raj, 2000).

The spatial expression and sequence of ppa suggest that Ppa might negatively regulate Prd, either by transcriptional co-repression or degradation of the Prd protein. To test these possibilities, ppa was ectopically expressed in the Prd-expressing cells to determine whether activation of en transcription or levels of Prd protein are affected. A transgene with the full ORF of ppa driven by an hsp70 promoter (hs-ppa) was introduced into embryos. Heat treatment of hs-ppa embryos during cycle 14 has pronounced effects. The odd-numbered, Prd-dependent, en stripes are weakened or completely absent, suggesting that Prd activation of these stripes is repressed. This is not observed in heat-treated wild-type embryos processed in parallel (Raj, 2000).

Because the Drosophila embryo develops very quickly, the segmentation gene products are expected to be short lived. This is indeed the case for those products examined and is also likely to be true for the Prd protein, perhaps even in the absence of ppa function. Indeed, it is difficult to assess whether Prd protein levels are reduced in hs-ppa embryos because of the fairly broad range of immuno-staining signals observed between different embryos, a problem inherent to the detection technique. To overcome this problem, ppa was ectopically expressed over only part of the embryo, so that the effects of ectopic ppa could be assessed relative to regions of the same embryo where ppa expression is normal. eve mutant embryos with a transgene P[eve.2,3,7] that expresses only eve stripes 2, 3 and 7 have well-formed ppa interbands at these locations. Thus, it is possible to compare Prd protein expression at stripe 2, which overlaps the ppa interband at eve stripe 2, with Prd expression at stripe 4, where ppa is expressed ectopically. Examination of prd mRNA signals in eve-;P[eve.2,3,7] embryos reveals strong expression of stripe 4 when compared with stripe 2, consistent with previous observations that eve represses prd transcription, thereby contributing to refinement of Prd stripes. In contrast, Prd protein signal at stripe 4 is significantly lower than at stripe 2, correlating with the ectopic ppa expression at stripe 4, and suggesting that Ppa regulates Prd protein levels. Even though there is 50% more mRNA signal at stripe 4 than stripe 2 after ppa upregulation, there is 25% less protein. Note that it is formally possible that the reduced Prd protein levels result from changes in genes other than ppa that are regulated by eve. Nevertheless, these analyses of hs-ppa and ppa mutant embryos suggest that regulation by ppa is responsible (Raj, 2000).

The equivalent analysis of wild-type embryos shows similar mRNA signals at stripes 4 and 2, whereas the Prd protein signal at stripe 4 is somewhat reduced compared with stripe 2, correlating with the residual ppa expression normally still present at gastrulation at the ppa interband corresponding to Prd stripe 4. This decrease in Prd protein is less pronounced than in eve-;P[eve.2,3,7] embryos, presumably because the difference in ppa expression at stripes 2 and 4 is smaller. To confirm the interpretation that Ppa regulates Prd protein levels, the stripe 4 to stripe 2 ratios for mRNA and protein would be expected to be similar in heat-treated hs-ppa embryos, in which ppa is expressed ectopically at both stripes 2 and 4. This was indeed observed, supporting the conclusion that ppa regulates Prd protein levels. Because Ppa has an F-box, this regulation is most likely through targeted protein degradation rather than translational repression. Consistent with these data, Western analysis of embryo extracts indicates that Prd protein levels are reduced by approximately 50% in hs-ppa embryos, as compared with the wild type (Raj, 2000).

To confirm the role of Ppa in Prd degradation, a small chromosomal deletion was generated that removes the Ppa ORF, starting 345bp upstream and ending 304bp downstream of the ORF. Consistent with the observation that high levels of ppa are normally only observed in regions where Prd function is not required, the homozygous ppa mutants survive to adulthood but with reduced viability and abnormal nuclear cycling. To analyze the mutants, advantage was taken of the normal anterior-posterior progression of ppa stripe development in wild-type embryos: at the gastrulation stage, the more anterior Prd stripes (for example, stripe 2) has little overlap with ppa expression, whereas the more posterior stripes (for example, stripes 4 and 6) still have significant overlap. When prd mRNA and protein signals at stripes 4 and 6 are measured relative to stripe 2 in the same embryos, the protein signals at stripes 4 and 6 are found to be significantly lower than the corresponding mRNA levels in wild-type embryos. In ppa mutant embryos, however, the mRNA and protein signals are similar, indicating that ppa normally reduces Prd protein expression (Raj, 2000).

Supporting the conclusion that Ppa mediates Prd degradation, Ppa was found to interact with Drosophila Skp1, the component of the protein degradation machinery that is predicted to link Ppa to the ubiquitin-mediated degradation pathway. Expressed-sequence-tag (EST) cDNAs for Drosophila Skp1 were identified from the Berkeley Drosophila Genome Project; yeast two-hybrid assays show that the Skp1 protein interacts with Ppa (amino acids 131-538, which lacks the Ala/His/Pro-rich region). As expected, the smaller fragment of Ppa (amino acids 447-538) originally identified in the two-hybrid screen does not interact with Skp1, presumably because it has no F-box. The interaction between Ppa and Skp1 was confirmed by co-immunoprecipitation analysis of yeast cell extracts. Immunoprecipitation of a hemagglutinin (HA) epitope-tagged Ppa fragment (amino acids 131-538) using anti-HA antibody also brings down LexA-tagged Skp1, which was detected with anti-LexA antibody. The Ppa-Skp1 interaction was also observed by immunoprecipitation with anti-LexA antibody, and the interaction of Ppa with Prd was also verified in these experiments (Raj, 2000).

This analysis of ppa function indicates that, when it is expressed ectopically in Prd-expressing cells, the levels of Prd protein diminish about twofold. A similar change in substrate stability (2-4-fold) is observed when GRR1, the yeast gene most similar to ppa, is mutated. It is also possible that, in addition to reducing Prd protein levels, Ppa might function as a transcriptional co-repressor, interacting with Prd to reduce its activation of en. Together, these two repression functions would ensure robust negative regulation of Prd in the Ppa-expressing cells (Raj, 2000).

Ppa is the first example of an F-box receptor localized in stripes. Loss- and gain-of-function analyses show that, while the presence of ppa expression in stripes is important for embryo development, it is the absence of ppa expression in the interbands that is crucial. Homozygous ppa minus mutants survive to adulthood, but show 50%-70% lethality, consistent with the fact that Ppa works in conjunction with the transcriptional repressors slp and run to localize Prd function. The partial lethality may be due to altered Prd expression or abnormal nuclear cycling. In contrast, in embryos with a functional ppa gene, it is absolutely essential that its expression be spatially regulated (by eve). Even basal expression of one of the hs-ppa transgenes (two copies without heat treatment) causes complete lethality, whereas the same transformant is not lethal before removal of an FRT cassette that blocks transcription. This predicts that cis-regulatory mutations in ppa causing loss of spatial regulation will have profound detrimental effects on embryogenesis, and this could also apply to the vertebrate homologs of ppa, which have such striking sequence similarity (Raj, 2000).

With the recent cloning of F-box proteins and the realization that they provide specific links between substrates and the protein degradation machinery, it has been predicted that F-box proteins would play important roles in development. Because F-box-regulated degradation normally depends on phosphorylation of substrates, localized action of signal transduction systems can, in principle, lead to localized protein degradation. This is likely to be the case for the signal-dependent localized degradation of Drosophila Cactus, a homolog of vertebrate IkappaB, whose degradation is a prerequisite for nuclear import of the Dorsal transcription factor (a homolog of NFkappaB) in the ventral portion of the embryo. Degradation of Cactus is mediated by the F-box protein Slimb (a homolog of ß-TrCP), which is also implicated in Wingless and Hedgehog pathways. In contrast to these signal transduction systems, the localized protein degradation in the Ppa system depends on spatially regulated expression of the Ppa F-box protein itself. By having its transcription regulated by a segmentation protein (Eve), and by targeting other segmentation proteins for degradation (Prd), the Ppa F-box protein forms an integrated link in the segmentation protein regulatory cascade that serves to strengthen the spatial refinement required for pattern formation. It is predicted that integration into transcriptional cascades may be a property of an important subfamily of F-box proteins, which, as suggested above, may also have recruited transcriptional repression functions to optimize their negative regulation of targeted transcription factors (Raj, 2000).

Ppa is the first example of a localized F-box receptor for protein degradation that works alongside transcription factors to ensure localized gene expression in the Drosophila segmentation cascade. These analyses suggest that Ppa targets the Prd transcription factor for degradation in cell rows in which Prd function is inappropriate, and that it is crucial that ppa expression is removed, through repression by eve, from cell rows in which Prd function is required for normal embryonic development (Raj, 2000).

A novel F-box protein is required for caspase activation during cellular remodeling in Drosophila

Terminal differentiation of male germ cells in Drosophila and mammals requires extensive cytoarchitectural remodeling, the elimination of many organelles, and a large reduction in cell volume. The associated process, termed spermatid individualization, is facilitated by the apoptotic machinery, including caspases, but does not result in cell death. From a screen for genes defective in caspase activation in this system, a novel F-box protein, which was termed Nutcracker, was isolated that is strictly required for caspase activation and sperm differentiation. Nutcracker interacts through its F-box domain with members of a Cullin-1-based ubiquitin ligase complex (SCF): Cullin-1 and SkpA. This ubiquitin ligase does not regulate the stability of the caspase inhibitors DIAP1 and DIAP2, but physically binds Bruce, a BIR-containing giant protein involved in apoptosis regulation. Furthermore, nutcracker mutants disrupt proteasome activity without affecting their distribution. These findings define a new SCF complex required for caspase activation during sperm differentiation and highlight the role of regulated proteolysis during this process (Bader, 2010).

Most F-box proteins also possess another protein-interaction domain, usually comprising WD40 or LRR motifs, that is responsible for binding the ubiquitylation substrate. Nutcracker belongs to the class of F-box proteins that do not contain a known protein-protein interaction domain, and differs topologically from most F-box proteins in that its F-box domain is at the very C-terminus (Kirk, 2008). Sequence alignments with several F-box-only proteins revealed that Nutcracker shares some limited amino acid similarity with the mammalian FBXO7 protein, which also contains the F-box domain at the C-terminus. Although the sequence conservation is limited primarily to the F-box domain, it is possible that these two proteins share functional properties, as do other proteins that are conserved only within limited regions. For example, the C. elegans p53 protein displays less than 20% overall primary sequence similarity to the human protein, mostly in the active sites, but has been demonstrated to function in related cellular processes. Since FBXO7 has been shown to regulate the stability of cIAP1 (BIRC2) (Chang, 2006), it is possible that these two E3 ligases have a conserved function in caspase regulation (Bader, 2010).

The ubiquitin-proteasome system is implicated in regulating caspase activity. Several studies have shown that the ubiquitylation and degradation of DIAP1 is a means of displacing it from caspases when apoptosis is favored. Also, ubiquitylation of caspases themselves contributes to their regulation by preventing a critical mass of full-length caspases from auto-activation in a normal setting. The wide variety of other ubiquitin-modifying proteins that regulate apoptosis and caspase activity, including Bruce, Morgue and Uba1, imply the existence of an elaborate regulatory network that is controlled by ubiquitylation (Bader, 2010).

In the screen isolated another ubiquitin ligase, a Cullin-3-based complex, was isolated, indicating that caspase activation in this system is tightly controlled by ubiquitin modifications. These two complexes could regulate the stability of the same substrate, as is the case for regulation of Cubitus interruptus (Ci) stability in Hedgehog signaling by both Cullin-1-based and Cullin-3-based complexes, or they might target multiple important substrates. Alternatively, the E3 ligases isolated in the screen might play non-degradative roles in controlling caspase activity. For example, mono-ubiquitylation affects the targeted localization of proteins and these ubiquitin ligases might control the proper localization of caspase regulators. Another possibility is that these E3 ligases mediate the non-classical Lys63 ubiquitin chain addition that is important for protein-protein interaction. Thus, instead of degradation, these proteins might actually control interactions between caspase regulators (Bader, 2010).

Although DIAP1 is the only Drosophila BIR-containing protein that has been shown to directly inhibit caspases in vivo, Bruce has been implicated in modifying apoptosis in several death paradigms, and mutations in its mammalian homolog cause defects associated with excess cell death. This study showed that Bruce can physically bind Nutcracker, and that this interaction is independent of the F-box domain. Therefore, Bruce might be a substrate of Nutcracker. However, it was not possible to determine the steady-state levels of Bruce in nutcracker mutants, so it is as yet unclear whether it is indeed a substrate or a complex partner. The fact that Bruce also binds to another E3 ligase isolated in the screen suggests that this protein is a common regulator of caspase activation during individualization (Bader, 2010).

nutcracker mutants cause a reduction in proteasome activity. This decreased activity does not seem to be due to proteasome mislocalization or a reduction in their numbers, suggesting that Nutcracker controls proteasome activity directly. It is possible that Nutcracker modifies proteasome regulators, which could include, for example, proteins of the regulatory particle of the proteasome. An attractive model is that Nutcracker functions through proteasomes to activate caspases. Caspase activity is tightly controlled by the ubiquitin proteasome system. Therefore, it is possible that local activation of proteasomes controls localized caspase activation (Bader, 2010).

Many questions remain regarding the non-lethal role of caspases in cellular remodeling. For instance, is it a specialized activation that is governed by dedicated proteins, and to what extent are known apoptotic regulators involved in this process? Another intriguing question is how cells tolerate a certain level of caspase activation and avoid destruction by these potentially deadly proteases. Answers to these questions will not only uncover novel caspase regulators, but might also help us to understand how diseased cells, such as cancer cells, manage to escape cell death (Bader, 2010).

Drosophila Morgue Associates with SkpA and Polyubiquitin In Vivo

Morgue (Modifier of rpr and grim, ubiquitously expressed) is a unique ubiquitination protein that influences programmed cell death and circadian rhythms in Drosophila (Hays, 2002; Wing, 2002). This study found that over-expression of wild-type Morgue results in organismal lethality. This over-expression phenotype was used as the basis for an in vivo functional assay to investigate the importance of the Morgue zinc finger, F box, Ubiquitin E2 Conjugase Variant (UEV) domain, and active site Glycine residue. Removal of the zinc finger or UEV domain reduced Morgue's ability to induce lethality and enhance cell death. In contrast, lack of the F box as well as several different substitutions of the active site Glycine did not alter Morgue-induced lethality or cell death enhancement. To further characterize Morgue functions, a Flag:Morgue protein was used to isolate Morgue-associated proteins from whole adult Drosophila. Mass spectrometry analysis of the Morgue-associated proteins identified SkpA as well as a ubiquitin multimer. The identification of SkpA is consistent with previous in vitro studies and further suggests Morgue acts in an SCF-type ubiquitin E3 ligase complex. The identification of poly-ubiquitin was unexpected and this interaction had not been previously identified. The associated poly-ubiquitin was found to exhibit a Lys-48 topology, consistent with distinct functions of Morgue in proteasome-mediated protein turnover. Multiple regions of Morgue were subsequently shown to be required for poly-ubiquitin binding. Overall, Morgue is a novel multi-functional ubiquitin-binding protein (Zhou, 2013).

Knockdown of SCF^Skp2 function causes double-parked accumulation in the nucleus and DNA re-replication in Drosophila plasmatocytes

In Drosophila, circulating hemocytes are derived from the cephalic mesoderm during the embryonic wave of hematopoiesis. These cells are contributed to the larva and persist through metamorphosis into the adult. To analyze this population of hemocytes, data was considered from a previously published RNAi screen in the hematopoietic niche, which suggested several members of the SCF complex play a role in lymph gland development. eater-Gal4;UAS-GFP flies were crossed to UAS-RNAi lines to knockdown the function of all known SCF complex members in a plasmatocyte-specific fashion, in order to identify which members are novel regulators of plasmatocytes. This specific SCF complex contains five core members: Lin-19-like (Cul-1), SkpA, Skp2, Roc1a and complex activator Nedd8. The complex was identified by its very distinctive large cell phenotype. Furthermore, these large cells stained for anti-P1, a plasmatocyte-specific antibody. It was also noted that the DNA in these cells appeared to be over-replicated. Gamma-tubulin and DAPI staining suggest the cells are undergoing re-replication as they had multiple centrioles and excessive DNA content. Further experimentation determined enlarged cells were BrdU-positive indicating they have progressed through S-phase. To determine how these cells become enlarged and undergo re-replication, cell cycle proteins were analyzed by immunofluorescence. This analysis identified three proteins that had altered subcellular localization in these enlarged cells: Cyclin E, Geminin and Double-parked. Previous research has shown that Double-parked must be degraded to exit S-phase, otherwise the DNA will undergo re-replication. When Double-parked was titrated from the nucleus by an excess of its inhibitor, geminin, the enlarged cells and aberrant protein localization phenotypes were partially rescued. The data in this report suggests that the SCF^Skp2 complex is necessary to ubiquitinate Double-parked during plasmatocyte cell division, ensuring proper cell cycle progression and the generation of a normal population of this essential blood cell type (Kroeger, 2013).

The generation of an eaterGal4; UAS-GFP strain allowed identification the functional importance of SCF complex members for the plasmatocyte blood cell lineage by a RNAi knockdown approach. Using this technique, several genes belonging to the core SCF complex were identified that, when knocked-down, caused a very distinctive giant cell phenotype. Importantly, as eater was bing used as a driver to identify complex components, it was confirmed that these enlarged cells were plasmatocytes by anti-P1 plasmatocyte-specific antibody staining. This suggested, as proof-of-principle, that knockdown of gene function in mature plasmatocytes could elicit aberrant phenotypes dependent on the functional requirement of an essential gene/gene complex (Kroeger, 2013).

Previous research has shown that there are several Drosophila genes that may be involved in SCF complexes in order to determine specificity for a substrate. The F-box is thought to convey specificity of this complex by recruiting the substrate, however activation of the Cullin by neddylation factors also plays a role in ubiquitation of the substrate. A comprehensive list of all known and predicted complex members was used to identify the remaining members of the specific SCF complex that function in Drosophila hematopoiesis, as knockdown of only one of each of the core components caused enlarged plasmatocytes. lin19, SkpA and Roc1a likewise play a role in the hematopoietic niche, the PSC of the larval lymph gland. Knockdown of these genes caused a decrease in the number of PSC cells, as well as an increase in the size of these cells. These data, along with the findings in this current study, suggest that the SCF complex has a significant role in multiple aspects of Drosophila larval hematopoiesis (Kroeger, 2013).

Using fluorescence microscopy, it was noted that the enlarged cells caused by the SCF knockdown had a significant excess of DNA in the nuclear region. To investigate the hypothesis that DNA re-replication was occurring in plasmatocytes with the SCF complex knockdown, anti-gamma- Tubulin staining of centrioles was performed. Previously, it was shown that knockdown of Gem elicits DNA re-replication, therefore this study used it as a positive control. It was evident that the lin19 knockdown had multiple centrioles in one giant plasmatocyte, similar to plasmatocytes from the gem RNAi samples. It was also clear that the DNA had replicated many times, without any cellular division as indicated by BrdU-positive, but phospho-Histone H3-negative enlarged cells. These data support the idea that plasmatocytes from SCF knockdown animals undergo DNA re-replication, thus the SCF complex is necessary for Dup degradation. Additionally, previous research had identified a number of proteins that when misexpressed or knocked-down cause an enlarged cell phenotype with excess DNA replication. Several papers have shown that misregulation of Cyclin E can cause aberrant DNA synthesis. Research has also suggested that knockdown of Gem can cause this excessive DNA phenotype. In the current experiments, antibody staining identified that the subcellular localization of both these proteins changed between control samples and the lin19 knockdown. Importantly, Dup is necessary for DNA replication, but it must be degraded to prevent re-replication. As the main role of Gem is to inhibit Dup, and Gem was no longer found in the nucleus in the knockdown, this is suggestive that Gem had complexed with Dup, removing it from the nucleus. Conversely, Cyclin E was found in the nucleus. This is notable because Cyclin E is known to phosphorylate Dup marking it for ubiquitination, leading to its nuclear localization. It is also known that SCF^Skp2 degrades Cyclin E. This is another explanation for the accumulation of Cyclin E in the nucleus of SCF knockdown hemolymph samples. These data suggest that Dup may be the target substrate for the SCF complex being studied, with a secondary target possibly being Cyclin E. Previous research in human cells has shown that SCF^Skp2 regulates the degradation of Cdt1 (the homolog of Drosophila Dup)(Li, 2003). It has also been shown that the activated SCF^Skp2 complex plays a role in murine hematopoiesis, by ubiquitinating proteins necessary for proper cell cycle, such as Cyclin E. There are still many questions to be answered about SCF regulation in blood cells, as some of these results are contradictory (Kroeger, 2013).

In addition to these data, protein localization in the knockdown of Cyclin E showed that Gem had been removed from the nucleus, again consistent with the notion that it was titrated away from the nucleus by binding Dup. This is plausible because the SCF complex can recognize its substrates due to phosphorylation state. Since Cyclin E was knocked-down, Dup was not properly phosphorylated, and it was not recognized as the substrate by the SCF complex, therefore never being ubiquitinated nor degraded. Furthermore, in the Cyclin E knockdown, Dup localized to the nucleus similar to its localization in the SCF knockdown. This would make it necessary for Gem to inhibit Dup, causing Gem to take on a non-nuclear localization, while Dup would have a nuclear localization, if Dup was in excess. Taken together, these lines of investigation support the hypothesis that Cyclin E is necessary to phosphorylate Dup, allowing the SCF complex to recognize and ubiquitinate it. Dup that remains in the nucleus after degradation must be bound by Gem for the cell cycle to progress properly. DNA re-replication will occur if Dup remains in the nucleus. It is highly suggestive that knockdown of Cyclin E or the SCF complex perturbs this mechanism, causing Dup accumulation in the nucleus, and the cells to re-initiate DNA replication. Furthermore, others have shown there must be a balance of Gem and Dup in the nucleus for proper progression through the cell cycle. This research shows that there is a lack of Gem and an accumulation of Dup in the nucleus, which leads to excessive DNA replication and additional centriole replication in five percent of the plasmatocyte population (Kroeger, 2013).

Although re-replication is one mechanism to explain the SCF loss-of-function phenotype, a similar non-canonical process, known as endoreplication, could also account for the over-replicative system in these cells. Endoreplication is a cycle in which cells undergo S phases that are separated only by gap phases but not an intervening mitosis. However, endoreplication is not known to occur in wild-type Drosophila plasmatocytes. Further, Drosophila plasmatocytes are most similar to mammalian macrophages, which also do not endoreplicate. Since several of the proteins studied in this paper have been implicated in re-replication with phenotypes including enlarged cells, increased DNA content, and multiple centriole replication, the hypothesis is favored that re-replication is triggered in plasmatocyte development in the absence of SCF complex activity (Kroeger, 2013).

It is intriguing that only five percent of the cells display the re-replication phenotype. One explanation is that the smaller cells have arrested. There are many intrinsic mechanisms to ensure proper cell cycle progression preventing re-replication and ultimately cancer. It is possible these enlarged cells have escaped these mechanisms, causing the cell to replicate their DNA many times without going through mitosis, while the smaller cells arrest, to prevent this phenotype. It is also possible that only five percent of these cells are going through cell division during the time the RNAi is functionally knocking down the gene. Previous research has suggested that during mid-to-late third instar larval stages, only one to two percent of cells are going through mitosis at a given time. eaterGal4 is activated during second instar, however there is likely a latent period between activation of Gal4 and protein knockdown by the RNAi. This is consistent with only five percent of cells having an active cell cycle, and becoming enlarged through re-replication. A final possibility is that there are partially redundant mechanisms for the regulation of Dup. As previously described, the SCF complex has been shown to be involved in the ubiquitination and subsequent degradation of Dup, and Gem will inhibit the remainder of the Dup that may be in the nucleus. There may be additional mechanisms which ubiquitinate or inhibit Dup, therefore avoiding re-replication. The smaller cells may have activated one of these mechanisms to aid the cell in proper cell cycle, ultimately avoiding cancer. The regulation of Dup is of vast importance, and there are several possibilities of alternate mechanisms to prevent the re-replication phenotype elicited by cells which have excess Dup in the nucleus (Kroeger, 2013).

To further implicate the necessity of Dup regulation in the proper cell cycle of plasmatocytes, a rescue experiment was performed by overexpressing the Dup inhibitor, Gem. By overexpressing this inhibitory protein, it was hypothesized that the nuclear localization of Gem would increase, the protein would bind Dup, and therefore decrease the re-replication that is observed in SCF complex knockdown. Performing immunohistochemistry experiments identified that there was an increase in nuclear Gem and a decrease in Dup. Additional experimental evidence supports this hypothesis as there is a decrease in size of plasmatocytes with genotype pxnGal4>UAS-Gem⁴³>UAS-lin19 RNAi^HM05197 compared with pxnGal4>UAS-lin19 RNAi^HM05197. There is a drastic decrease in the number of giant cells, which are larger than 25.1 μm, in pxn>UAS-Gem⁴³>UAS-lin19 RNAi^HM05197 (8/100) plasmatocytes compared with SCF knockdown hemocytes (45/100). It was also noted that there was a significant decrease in the average size of plasmatocytes in hemolymph samples from Gem overexpression in the SCF knockdown background (p<0.001). These lines of evidence are all suggestive that knockdown of the SCF complex increased nuclear Dup leading to re-replication. By over-expressing its inhibitor, Gem, it is possible to partially rescue this enlarged cell phenotype generated by excess nuclear Dup. These data suggest the regulation of Dup is important in the proper cell cycle progression of plasmatocytes. Furthermore, these data support the hypothesis that the SCF^Skp2 complex is responsible for the ubiquitination of Dup, allowing plasmatocytes to proliferate properly. Although this study provides substantial genetic evidence that the SCF^Skp2 complex is necessary to ubiquitinate Dup allowing for proper hematopoietic cell cycle progression, future studies using biochemical techniques to show physical interactions are needed to support the model proposed here (Kroeger, 2013).

Furthermore, there are two ubiquitin ligase complexes known to be involved in the ultimate degradation of Dup: The SCF^Skp2 complex, described in this manuscript, and the Cul4-DDB1-CDT2-PCNA (Cul4^CDT2) complex. To vastly decrease the possibility that the Cul4^CDT2 complex was responsible for the enlarged cell phenotype, both DDB1 and PCNA were knocked-down via RNAi and Cul4 mutants were also analyzed. Although DDB1 functional knockdown elicited a small number of enlarged cells, these cells had a different morphology than the SCF^Skp2 knockdown. Additionally, none of the other analyses elicited any enlarged cells as observed when the SCF^Skp2 complex was knocked-down. This further implicates the necessity of the SCF^Skp2 complex in the proper plasmatocyte cell cycle (Kroeger, 2013).

To summarize, this manuscript identifies the SCF ubiquitin ligase complex as a novel regulator of plasmatocytes. Genetic evidence is presented that suggests that Dup is the main target for the SCF^Skp2 complex. It is proposed that the SCF^Skp2 complex plays an integral role in Drosophila hematopoiesis by ubiquitinating Dup, which is necessary for proper cell cycle progression. Knockdown of the SCF complex causes an accumulation of Dup in the nucleus, inducing the cell to undergo multiple rounds of replication without an intervening mitosis or cytokinesis. This causes some plasmatocytes to become vastly enlarged, with multiple centrioles and excessive DNA content. Taken together, these findings provide evidence that the SCF complex is necessary for proper cell cycle progression during plasmatocyte development in Drosophila. As the SCF complex is conserved from Drosophila to humans, these findings implicate the importance of the roles of ubiquitin ligase complexes in the cell cycle and their potential malfunctions in blood cell cancers (Kroeger, 2013).

Signal transduction by the Fat cytoplasmic domain

The Drosophila protocadherin Fat (Ft) regulates growth, planar cell polarity (PCP) and proximodistal patterning. A key downstream component of Ft signaling is the atypical myosin Dachs (D). Multiple regions of the intracellular domain of Ft have been implicated in regulating growth and PCP but how Ft regulates D is not known. Mutations in Fbxl7 (CG4221), which encodes an F-box protein, result in tissue overgrowth and abnormalities in proximodistal patterning that phenocopy deleting a specific portion of the intracellular domain (ICD) of Ft that regulates both growth and PCP. Fbxl7 binds to this same portion of the Ft ICD, co-localizes with Ft to the proximal edge of cells and regulates the levels and asymmetry of D at the apical membrane. Fbxl7 can also regulate the trafficking of proteins between the apical membrane and intracellular vesicles. Thus Fbxl7 functions in a subset of pathways downstream of Ft and links Ft to D localization (Bosch, 2014).

The protocadherin Ft lies at the apex of multiple pathways that together regulate growth, several aspects of PCP, and proximodistal patterning. The mechanism by which Ft functions as a signaling molecule remains poorly understood. This study has identified the F-box protein Fbxl7 as an immediate effector of Ft, that functions to restrict the levels of the atypical myosin D at the apical membrane as well as its distribution around the perimeter of the cell. In addition, Fbxl7 can regulate levels of Ft at the apical membrane (Bosch, 2014).

Recent studies have revealed that Ft's effects on distinct pathways may be genetically separated, and that multiple effector domains can contribute to the same function. Indeed, the growth-suppressing function of Ft may occur via at least two regions of the Ft ICD. One or more regions between amino acids 4834 and 4899 in full-length Ft appear responsible for Ft's ability to regulate Hippo signaling. Several mutations within this region compromise this function of Ft and cause massive tissue overgrowth (Bossuyt, 2013). Intriguingly, an allele of ft, ft⁶¹, which harbors such a mutation, showed neither an effect on the recruitment of Fbxl7 to the apical membrane nor on the binding of Ft to Fbxl7. Thus, signaling via this region of the ICD appears to be independent of Fbxl7. A second, more C-terminal region of the Ft ICD (Region D) that extends between amino acids 4975 and 4993 of full-length Ft, is removed by the ftΔD deletion and also has a growth-suppressive function albeit weaker than that of HM. This second growth-suppressive pathway requires the function of Fbxl7, as the protein generated by the ftΔD allele cannot bind to Fbxl7 nor can it localize Fbxl7 to the apical membrane. Additionally, the phenotypic abnormalities of null alleles of ft rescued by ftΔD are very similar, if not identical to those of Fbxl7 mutants. Furthermore, like ftΔD, Fbxl7 mutations do not display overt abnormalities of hair orientation in the wingor abdomen (Bosch, 2014).

Hyperactivation of the 'weaker' Fbxl7-dependent pathway can overcome the absence of the ‘stronger' Fbxl7-independent pathway; overexpression of Fbxl7 can suppress the overgrowth of ft⁶¹. Thus, while these two pathways can be dissociated at the level of the Ft ICD, they nevertheless seem to converge further downstream. This point of convergence likely involves Dachs (D) since the overgrowth of ft mutant tissue can be suppressed completely by eliminating D function. Indeed, it has previously been suggested that Ft regulates growth by restricting the levels of apical D, and regulates PCP by influencing the planar asymmetry of apical D (Bosch, 2014).

Another key finding in these experiments is that Fbxl7 mutations perturb the distribution of D around the perimeter of the apical region of the cell. D is normally biased towards the distal edge of the cell; in Fbxl7 mutants, D is more evenly distributed around the cell perimeter. The asymmetric localization of D depends on at least two different regions of Ft (Pan, 2013). One is the region that binds to Fbxl7 (Region D) and the other is composed of the last three amino acids at the C-terminus of the protein (Region F), which is not necessary for Fbxl7 localization to the apical membrane. Thus, for the regulation of D asymmetry as well, there appears to be an Fbxl7-independent pathway. The existence of multiple downstream effector pathways that converge on common biological outcomes suggests that these pathways might function redundantly to some extent and thus provide robustness. This might also explain why the phenotypes elicited by overexpression of Fbxl7 are, in general, more severe than those observed in loss-of-function mutations (Bosch, 2014).

Previous observations of the localization of Ft, Ds, and D to vesicles are suggestive of trafficking events being involved in Ft signaling. It was therefore demonstrated that, in addition to the apical membrane, Fbxl7 localizes to vesicles. Moreover, FLAG-Fbxl7 vesicles can contain Ft, Ds and D, and these may be related to the apical puncta observed on cell edges. This localization is likely specific, since no Fbxl7 co-localization is seen with other cell surface proteins such as Crumbs, Notch, and E-cadherin. Currently very little is known about the role of each of these proteins in vesicles. However, there is an increasing appreciation that most transmembrane proteins, and even proteins that are associated with the inner leaflet of the cell membrane are maintained at the plasma membrane by a dynamic process involving endocytosis and vesicle recycling (Bosch, 2014).

Evidence is provided that Fbxl7 regulates Ft apical localization, but how this regulation relates to the Fbxl7 phenotypes is not clear. Since Fbxl7 overexpression increases Fat signaling, and rescues the overgrowth-inducing ft⁶¹ allele, perhaps this is due to the increased levels of Ft protein at the apical membrane. However, Ft levels are slightly elevated in Fbxl7 mutants, which display mild overgrowth. Therefore the mutant phenotype cannot be explained by the effect on Ft. Another known regulator of apical Ft levels is lowfat (lft). Fbxl7 and Lft appear to regulate Ft in different ways. Lft overexpression, like Fbxl7, increases Ft levels. However, while Ft levels are decreased in lft mutant cells, Ft levels are increased in Fbxl7 mutant cells, though less so compared to Fbxl7 overexpression. Interestingly, for many proteins that regulate cellular trafficking, similar phenotypic abnormalities are observed with gain-of-function and loss-of-function mutations, since the normal execution of the process requires the protein to shuttle efficiently between two states. Thus dynamic aspects of the localization of Ft, Ds and D clearly merit more attention (Bosch, 2014).

The interactions observed between Fbxl7 and the adapter protein Cindr may provide clues for how Fbxl7 regulates D localization. Fbxl7-associated vesicles show almost complete overlap with GFP-Cindr and Fbxl7 can re-localize Cindr from the apical membrane to the interior of the cell. This finding, together with the observed increase in basal levels of D upon Fbxl7 overexpression, suggests that Fbxl7 may function to regulate D trafficking in a similar manner. Cindr and its mammalian orthologues Cin85 and CD2AP are thought to regulate interactions between membrane proteins and actin cytoskeleton. D is an atypical myosin with a predicted actin binding domain in its conserved head domain. Therefore, the vesicles which Fbxl7 associates with D and Cindr may be linked to the actin cytoskeleton. In addition, the finding of partial colocalization of Fbxl7 with retromer components further supports the possibility that Fbxl7 may have a role in protein trafficking (Bosch, 2014).

Many F-box proteins associate with Skp1 and Cul1 to form an SCF E3 ubiquitin ligase complex. Recruitment of specific substrates results in their poly-ubiquitylation and degradation, or mono-ubiquitylation, which can have non-degradative signaling roles. In addition, some F-box proteins have SCF-independent roles. Fbxl proteins are thought to recruit substrates to the SCF complex through the interaction with their LRR domains, and substrates have been identified for several Fbxls such as Skp2 (Fbxl1), which degrades p27. However many, like Fbxl7, are still uncharacterized as 'orphan' F-box proteins with no known substrates (Bosch, 2014).

Since this study found that Fbxl7 associates with Skp1 and Cul1, its potential substrates may be involved in Ft signaling. Fbxl7 has one described substrate in mice, Aurora A. However it is not believed Aurora A is a relevant substrate in Drosophila, as no Ft signaling defects are observed when Aurora A is knocked down or overexpressed. The identification of F-box protein substrates has mainly been accomplished by unbiased approaches. Similarly, a combination of unbiased approaches, involving proteomics, genetic interaction screens, and identifying proteins that co-localize with Fbxl7 in vesicles could be used to identify Fbxl7 substrates (Bosch, 2014).

DEVELOPMENTAL BIOLOGY

Embryonic

Skp1 is localized to centrosomes throughout the cell cycle in vertebrate cells (Freed, 1999; Gstaiger, 1999), suggesting that it may act directly at the centrosome to regulate duplication. To determine if SKPa shows a similar localization pattern in Drosophila cells, two polyclonal antibodies were raised against recombinant SKPa. Both antisera predominantly recognize a single 24 kDa band in embryo, larval and adult extracts which is absent from skpA^- larval extracts, indicating that the antisera specifically recognize SKPa (Murphy, 2003).

Immunofluorescence analyses reveal that SKPa is localized throughout the cytoplasm and nucleus in diploid tissues such as the CNS and during embryogenesis. Triple labeling cells for SKPa, DNA and centrosomes suggests that SKPa does not preferentially associate with centrosomes or chromosomes at any point during the cell cycle. SKPa is slightly more concentrated in the nucleus of some diploid cells, and is predominantly nuclear in salivary gland and fat body cells. No signal was observed in skpA^- cells from mosaic or homozygous mutant larvae, demonstrating that the staining pattern specifically represents the localization of SKPa in CNS cells (Murphy, 2003).

The relatively uniform distribution of Drosophila SKPa in diploid tissues is dramatically different from the centrosomal localization observed in vertebrate cells. In Drosophila, SKPa may regulate centrosome duplication by transiently associating with the centrosome; alternatively, it may function indirectly by acting on cytoplasmic or nuclear proteins. In contrast, the pronounced localization of SKPa to the nucleus of polyploid cells suggests that it may function directly on nuclear proteins involved in endoreduplication (Murphy, 2003).

EVOLUTIONARY HOMOLOGS

Evolution of the Skp1 family of proteins

Skp1 (S-phase kinase-associated protein 1) is a core component of SCF ubiquitin ligases and mediates protein degradation, thereby regulating eukaryotic fundamental processes such as cell cycle progression, transcriptional regulation, and signal transduction. Among the four components of the SCF complexes, Rbx1 and Cullin form a core catalytic complex, an F-box protein acts as a receptor for target proteins, and Skp1 is an adaptor between one of the variable F-box proteins and Cullin. Whereas protists, fungi and some vertebrates have a single SKP1 gene, many animal and plant species possess multiple SKP1 homologs. It has been shown that the same Skp1 homolog can interact with two or more F-box proteins, and different Skp1 homologs from the same species sometimes can interact with the same F-box protein. Multiple Skp1 homologs from the same species have evolved at highly heterogeneous rates. Parametric bootstrap analyses suggest that the differences in evolutionary rate are so large that true phylogenies are not recoverable from the full data set. Only when the original data set is partitioned into sets of genes with slow, medium, and rapid rates of evolution and analyzed separately, are better resolved relationships observed. The slowly evolving Skp1 homologs, which are relatively highly conserved in sequence and expressed widely and/or at high levels, usually have very low dN/dS values, suggesting that they have evolved under functional constraint and serve the most fundamental function(s). In contrast, the rapidly evolving members are structurally more diverse and usually have limited expression patterns and higher dN/dS values, suggesting that they may have evolved under relaxed or altered constraint, or even under positive selection. Some rapidly evolving members may have lost their original function(s) and/or acquired new function(s), or become pseudogenes, as suggested by their expression patterns, dN/dS values, and amino acid changes at key positions. In addition, these analyses revealed several monophyletic groups within the SKP1 gene family, one for each of protists, fungi, animals and plants, as well as nematodes, arthropods and angiosperms, suggesting that the extant SKP1 genes within each of these eukaryote groups shared only one common ancestor (Kong, 2003).

Skp1 genes in plants

The yeast and human SKP1 genes regulate the mitotic cell cycle but are not yet known to be required for meiosis. Nine Arabidopsis SKP1 homologs have been uncovered and were named ASK1 through ASK9. A male sterile Arabidopsis mutant has been isolated and characterized that was caused by a Ds transposon insertion into the ASK1 gene. In the ask1-1 mutant, abnormal microspores exhibit a range of sizes. Furthermore, during mutant male meiosis, although homologous chromosome pairing appears normal at metaphase I, chromosome segregation at anaphase I is unequal, and some chromosomes are abnormally extended. Therefore, in ask1-1, at least some homologs remain associated after metaphase I. In addition, immunofluorescence microscopy indicates that the mutant spindle morphology at both metaphase I and early anaphase I is normal; thus, the abnormal chromosome segregation is not likely caused by a spindle defect. Because the yeast Skp1p is required for targeting specific proteins for ubiquitin-mediated proteolysis, it is proposed that ASK1 controls homolog separation by degrading or otherwise removing a protein that is required directly or indirectly for homolog association before anaphase I (Yang, 1999).

The infection of plants by Agrobacterium tumefaciens leads to the formation of crown gall tumors due to the transfer of a nucleoprotein complex into plant cells that is mediated by the virulence (vir) region-encoded transport system. In addition, A. tumefaciens secretes the Vir proteins, VirE2 and VirF, directly into plant cells via the same VirB/VirD4 transport system, and both assist there in the transformation of normal cells into tumor cells. The function of the 22 kDa VirF protein is not clear. Deletion of the virF gene in A. tumefaciens leads to diminished virulence and can be complemented by the expression of the virF gene in the host plant. This finding indicates that VirF functions within the plant cell. The VirF protein is the first prokaryotic protein with an F box; it can interact via its F box with plant homologs of the yeast Skp1 protein. The presence of the F box turns out to be essential for the biological function of VirF. F box proteins and Skp1p are both subunits of a class of E3 ubiquitin ligases referred to as SCF complexes. Thus, VirF may be involved in the targeted proteolysis of specific host proteins in early stages of the transformation process (Schrammeijer, 2001).

The yeast Skp1 protein is a component of the SCF complex, an E3 enzyme involved in the specific protein degradation pathway via ubiquitination. Skp1 binds to F-box proteins to trigger specific recognition of proteins targeted for degradation. SKP1-like genes have been found in a variety of eukaryotes including yeast, man, Caenorhabditis elegans and Arabidopsis thaliana. The Arabidopsis genome contains 20 SKP1-like genes called ASK (for Arabidopsis SKP1-like), among which only ASK1 has been characterized in detail. The analysis of the expression pattern of the ASK genes in Arabidopsis should provide key information for the understanding of the biological role of this family in protein degradation and in different cellular mechanisms. The expression profiles are described of 19 ASK promoter-GUS fusions in stable transformants of Arabidopsis, with a special emphasis on floral organ development. Four ASK promoters did not show any detectable expression in either inflorescences or seedlings. These results on the ASK1 expression profile are consistent with previous reports. Several ASK promoters show clear tissue-specific expression (for instance in the connective of anthers or in the embryo). Almost half (9/19) of ASK promoters direct a post-meiotic expression in the male gametophyte. Tight regulation of the expression of this gene family indicates a crucial role of the ubiquitin degradation pathway during development, particularly during male gametophyte development (Marrocco, 2003).

Ubiquitin-mediated proteolysis by the proteasome is a critical regulatory mechanism controlling many biological processes. In particular, SKP1, cullin/CDC53, F-box protein (SCF) complexes play important roles in selecting substrates for proteolysis by facilitating the ligation of ubiquitin to specific proteins. In plants, SCF complexes have been found to regulate auxin responses and jasmonate signaling and may be involved in several other processes, such as flower development, circadian clock, and gibberellin signaling. Although 21 Skp1-related genes, called Arabidopsis-SKP1-like (ASK), have been uncovered in the Arabidopsis genome, ASK1 is the only gene that has been analyzed genetically. As a first step toward understanding their functions, expression of 20 ASK genes was tested using reverse transcription-polymerase chain reaction experiments. Also, the expression patterns of 11 ASK genes were examined by in situ hybridizations. The ASK genes exhibit a spectrum of expression levels and patterns, with a large subset showing expression in the flower and/or fruit. In addition, the ASK genes that have similar sequences tend to have similar expression patterns. On the basis of the expression results, the expression of a few ASK genes was selectively suppressed using RNA interference. Compared with the ask1 mutant, the strong ASK1 RNA interference (RNAi) line exhibits similar or enhanced phenotypes in both vegetative and floral development, whereas ASK11 RNAi plants had normal vegetative growth but mild defects in flower development. The diverse expression patterns and distinct defects observed in RNAi plants suggest that the ASK gene family may collectively perform a range of functions and may regulate different developmental and physiological processes (Zhao, 2003).

Interactions and functions of yeast Skp1

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

SCF ubiquitin ligases are composed of Skp1, Cdc53, Hrt1 and one member of a large family of substrate receptors known as F-box proteins (FBPs). Using sequential rounds of epitope tagging, affinity purification and mass spectrometry, 16 Skp1 and Cdc53-associated proteins have been identified in budding yeast, including all components of SCF, 9 FBPs, Yjr033 (Rav1) and Ydr202 (Rav2). Rav1, Rav2 and Skp1 form a complex named here 'regulator of the (H+)-ATPase of the vacuolar and endosomal membranes' (RAVE), which associates with the V1 domain of the vacuolar membrane (H+)-ATPase (V-ATPase). V-ATPases are conserved throughout eukaryotes, and have been implicated in tumor metastasis and multidrug resistance; this study shows that RAVE promotes glucose-triggered assembly of the V-ATPase holoenzyme. Previous systematic genome-wide two-hybrid screens yielded 17 proteins that interact with Skp1 and Cdc53, only 3 of which overlap with those reported here. Thus, these results provide a distinct view of the interactions that link proteins into a comprehensive cellular network (Seol, 2001).

Skp1p-cullin-F-box protein (SCF) complexes are ubiquitin-ligases composed of a core complex including Skp1p, Cdc53p, Hrt1p, the E2 enzyme Cdc34p, and one of multiple F-box proteins which are thought to provide substrate specificity to the complex. F-box protein Rcy1p is required for recycling of the v-SNARE Snc1p in Saccharomyces cerevisiae. Rcy1p localizes to areas of polarized growth, and this polarized localization requires its CAAX box and an intact actin cytoskeleton. Rcy1p interacts with Skp1p in vivo in an F-box-dependent manner, and both deletion of its F box and loss of Skp1p function impair recycling. In contrast, cells deficient in Cdc53p, Hrt1p, or Cdc34p do not exhibit recycling defects. Unlike the case for F-box proteins that are known to participate in SCF complexes, degradation of Rcy1p requires neither its F box nor functional 26S proteasomes or other SCF core subunits. Importantly, Skp1p is the only major partner that copurifies with Rcy1p. These results thus suggest that a complex composed of Rcy1p and Skp1p but not other SCF components may play a direct role in recycling of internalized proteins (Galan, 2001).

Binding of CBF3, a protein complex consisting of Ndc10p, Cep3p, Ctf13p, and Skp1p, to the centromere DNA nucleates kinetochore formation in budding yeast. This study investigates how the Ctf13p/Skp1p complex becomes competent to form the CBF3-centromere DNA complex. As revealed by mass spectrometry, Ctf13p and Skp1p carry two and four phosphate groups, respectively. Complete dephosphorylation of Ctf13p and Skp1p does not interfere with the formation of CBF3-centromere DNA complexes in vitro. Furthermore, deletion of corresponding phosphorylation sites results in viable cells. Thus, in contrast to the current view, phosphorylation of Ctf13p and Skp1p is not essential for the formation of CBF3-centromere DNA complexes. Instead, the formation of active Ctf13p/Skp1p requires Hsp90. Several lines of evidence support this conclusion: activation of heterologous Ctf13p/Skp1p by reticulocyte lysate is inhibited by geldanamycin and Hsp90 depletion. skp1 mutants exhibit growth defects on media containing geldanamycin. A skp1 mutation together with Hsp90 mutations exhibits synthetic lethality. An Hsp90 mutant contains decreased levels of active Ctf13p/Skp1p (Stemmann, 2002).

SCF complexes are a conserved family of ubiquitin-ligases composed of a common core of components and a variable component called an F-box protein that defines substrate specificity. The F-box motif links the F-box protein to the core components via its interaction with Skp1p. In yeast, the SCFMet30p complex contains the Met30p F-box protein and regulates Met4p, a transcription factor that mediates sulfur fixation and methionine biosynthesis. Although a nuclear protein, Met30p lacks a definable nuclear localization sequence. The entire amino terminal half of Met30p is required for its proper nuclear localization. Mutations in the F-box, but not mutations in Skp1p, affect Met30p nuclear localization, indicating that the F-box motif plays an important role in Met30p trafficking independent of its interaction with Skp1p binding. Met30p mutants that poorly localize to the nucleus display increased nuclear to cytoplasmic exchange, indicating that the amino terminus mediates nuclear retention in addition to nuclear import. The Met30p F-box motif, residues 180-225, is necessary and sufficient to bind Skp1p, however, mutations upstream of the Met30p F-box inhibit Skp1p binding. It is proposed that additional factors bind the amino terminal region of Met30p and mediate its nuclear localization and assimilation into an SCF complex (Brunson, 2003).

Skp1-related genes in C. elegans

The SCF ubiquitin-ligase complex targets the ubiquitin-mediated degradation of proteins in multiple dynamic cellular processes. A key SCF component is the Skp1 protein that functions within the complex to link the substrate-recognition subunit to a cullin that in turn binds the ubiquitin-conjugating enzyme. In contrast to yeast and humans, Caenorhabditis elegans contains multiple expressed Skp1-related (skr) genes. The 21 Skp1-related (skr) genes in C. elegans form one phylogenetic clade, suggesting that a single ancestral Skp1 gene underwent independent expansion in C. elegans. The cellular and developmental functions of the 21 C. elegans skr genes were probed by dsRNA-mediated gene inactivation (RNAi). The RNAi phenotypes of the skr genes fall into two classes: (1) the highly similar skr-7, -8, -9, and -10 genes are required for posterior body morphogenesis, embryonic and larval development, and cell proliferation; (2) the related skr-1 and -2 genes are required for the restraint of cell proliferation, progression through the pachytene stage of meiosis, and the formation of bivalent chromosomes at diakinesis. CUL-1 was found to interact with SKR-1, -2, -3, -7, -8, and -10 in the yeast two-hybrid system. Interestingly, SKR-3 could interact with both CUL-1 and its close paralog CUL-6. It is concluded that members of the expanded skr gene family in C. elegans perform critical functions in regulating cell proliferation, meiosis, and morphogenesis. The finding that multiple SKRs are able to bind cullins suggests an extensive set of combinatorial SCF complexes (Nayak, 2002).

The ubiquitin-proteasome pathway of proteolysis controls the abundance of specific regulatory proteins. The SCF complex is a type of ubiquitin-protein ligase (E3) that contributes to this pathway in many biological systems. In yeast and mammals, the SCF complex consists of common components, including Skp1, Cdc53/Cul1, and Rbx1, as well as variable components known as F-box proteins. Whereas only one functional Skp1 gene is present in the human genome, the genome of Caenorhabditis elegans has now been shown to contain at least 21 Skp1-related (skr) genes. The biochemical properties, expression, and function of the C. elegans SKR proteins were examined. Of the 17 SKR proteins examined, eight (SKR-1, -2, -3, -4, -7, -8, -9, and -10) were shown to interact with C. elegans CUL1 by yeast two-hybrid analysis or a coimmunoprecipitation assay in mammalian cells. Furthermore, SKR proteins exhibit diverse binding specificities for C. elegans F-box proteins. The tissue specificity of expression of the CUL1-interacting SKR proteins is also varied. Suppression of skr-1 or skr-2 genes by double-stranded RNA interference results in embryonic death, whereas that of skr-7, -8, -9, or -10 is associated with slow growth and morphological abnormalities. It is concluded that the multiple C. elegans SKR proteins exhibit marked differences in their association with Cullins and F-box proteins, in tissue specificity of expression, and in phenotypes associated with functional suppression by RNAi. At least eight of the SKR proteins may, like F-box proteins, act as variable components of the SCF complex in C. elegans (Yamanaka, 2002).

Protein interactions in the SCF complex

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

F-box proteins are members of a large family that regulates the cell cycle, the immune response, signalling cascades and developmental programs by targeting proteins, such as cyclins, cyclin-dependent kinase inhibitors, IkappaBalpha and beta-catenin, for ubiquitination. F-box proteins are the substrate-recognition components of SCF (Skp1-Cullin-F-box protein) ubiquitin-protein ligases. They bind the SCF constant catalytic core by means of the F-box motif interacting with Skp1, and they bind substrates through their variable protein-protein interaction domains. The large number of F-box proteins is thought to allow ubiquitination of numerous, diverse substrates. Most organisms have several Skp1 family members, but the function of these Skp1 homologs and the rules of recognition between different F-box and Skp1 proteins remain unknown. The crystal structure of the human F-box protein Skp2 bound to Skp1 is described. Skp1 recruits the F-box protein through a bipartite interface involving both the F-box and the substrate-recognition domain. The structure raises the possibility that different Skp1 family members evolved to function with different subsets of F-box proteins, and suggests that the F-box protein may not only recruit substrate, but may also position it optimally for the ubiquitination reaction (Schulman, 2000).

Selective protein degradation targeted by members of the F-box protein family plays pivotal roles in cell biology. It is widely accepted that an F-box protein directs substrate ubiquitination within a Skp1.CUL1.F-box protein.ROC1 (SCF-ROC1) E3 ubiquitin ligase complex. This assembly utilizes the CUL1 molecular scaffold, allowing the F-box protein to position its bound substrate for ubiquitination by a ROC1-recruited E2-conjugating enzyme. An alternative mechanism is described for assembling an F-box protein-based E3 complex through a previously uncharacterized cullin, CUL7, identified by mass spectrometry as a ROC1-interacting protein. CUL7 is a large polypeptide containing a cullin domain, which is responsible for ROC1 binding, and a DOC domain, which is also present in the anaphase-promoting complex. Remarkably, CUL7 assembles an SCF-ROC1-like E3 ubiquitin ligase complex consisting of Skp1, CUL7, the Fbx29 F-box protein, and ROC1. In contrast to CUL1 that binds Skp1 by itself, CUL7 interacts with the Skp1.Fbx29 complex, but not with Skp1 alone. Strikingly, CUL7 selectively interacts with Skp1.Fbx29 but not with Skp1.betaTRCP2 or Skp1.Skp2. Thus, CUL7 may define a previously uncharacterized, Fbx29-mediated, and ubiquitin-dependent proteolysis pathway (Dias, 2002).

SCF complexes are the largest family of E3 ubiquitin-protein ligases and mediate the ubiquitination of diverse regulatory and signalling proteins. The crystal structure of the Cul1-Rbx1-Skp1-F boxSkp2 SCF complex is presented; Cul1 is shown to be an elongated protein that consists of a long stalk and a globular domain. The globular domain binds the RING finger protein Rbx1 through an intermolecular beta-sheet, forming a two-subunit catalytic core that recruits the ubiquitin-conjugating enzyme. The long stalk, which consists of three repeats of a novel five-helix motif, binds the Skp1-F boxSkp2 protein substrate-recognition complex at its tip. Cul1 serves as a rigid scaffold that organizes the Skp1-F boxSkp2 and Rbx1 subunits, holding them over 100 Å apart. The structure suggests that Cul1 may contribute to catalysis through the positioning of the substrate and the ubiquitin-conjugating enzyme, and this model is supported by Cul1 mutations designed to eliminate the rigidity of the scaffold (Zheng, 2002).

Function of the Skp1-containing SCF complex in cell cycle and centrosome cycle regulation

Deregulation of cell proliferation is a hallmark of cancer. In many transformed cells, the cyclin A/CDK2 complex that contains S-phase kinase associated proteins 1 and 2 (SKP1 and SKP2) is highly induced. To determine the roles of this complex in the cell cycle regulation and transformation, the composition of this complex was analyzed. This complex contains an additional protein, human CUL-1, a member of the cullin/CDC53 family. The identification of CUL-1 as a member of the complex raises the possibility that the p19(SKP1)/p45(SKP2)/CUL-1 complex may function as the yeast SKP1-CDC53-F-box (SCF) protein complex that acts as a ubiquitin E3 ligase to regulate the G1/S transition. In mammalian cells, cyclin D, p21(CIP1/WAF1), and p27(KIP1) are short-lived proteins that are controlled by ubiquitin-dependent proteolysis. To determine the potential in vivo targets of the p19(SKP1)/p45(SKP2)/CUL-1 complex, the specific antisense oligodeoxynucleotides were used against either SKP1, SKP2, or CUL-1 RNA to inhibit their expression. Treatment of cells with these oligonucleotides causes the selective accumulation of p21 and cyclin D proteins. The protein level of p27 is not affected. These data suggest that the human p19(SKP1)/p45(SKP2)/CUL-1 complex is likely to function as an E3 ligase to selectively target cyclin D and p21 for the ubiquitin-dependent protein degradation. Aberrant expression of human p19(SKP1)/p45(SKP2)/CUL-1 complex thus may contribute to tumorigenesis by regulating the protein levels of G1 cell cycle regulators (Yu, 1998).

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

In Saccharomyces cerevisiae, the initiation of DNA replication and mitotic progression requires SKP1p function. SKP1p is an essential subunit of a newly identified class of E3 ubiquitin protein ligases, the SCF complexes, that catalyze ubiquitin-mediated proteolysis of key cell-cycle-regulatory proteins at distinct times in the cell cycle. SKP1p is also required for proper kinetochore assembly. Little is known about the corresponding human homolog, p19(SKP1), except that it is expressed throughout the cell cycle and that it too is a component of an S-phase-regulating SCF-E3 ligase complex. p19(SKP1) localizes to the centrosomes. Centrosome association occurs throughout the mammalian cell cycle, including all stages of mitosis. These findings suggest that p19(SKP1) is a novel component of the centrosome and the mitotic spindle, which, in turn, implies a physiological role of this protein in the regulation of one or more aspects of the centrosome cycle (Gstaiger, 1999).

Skp1 is involved in a variety of crucial cellular functions, among which the best understood is the formation, together with Cul1, of Skp1-cullin-F-box protein ubiquitin ligases. To investigate the role of Skp1, transgenic (Tg) mice were generated expressing a Cul1 deletion mutant (Cul1-N252) able to sequestrate and inactivate Skp1. In vivo interference with Skp1 function through expression of the Cul1-N252 mutant into the T-cell lineage results in lymphoid organ hypoplasia and reduced proliferation. Nonetheless, after a period of latency, Cul1-N252 Tg mice succumb to T-cell lymphomas with high penetrance (>80%). Both T-cell depletion and the neoplastic phenotype of Cul1-N252 Tg mice are largely rescued in Cul1-N252, Skp1 double-Tg mice, indicating that the effects of Cul1-N252 are due to a sequestration of the endogenous Skp1. Analysis of Cul1-N252 lymphomas demonstrates striking karyotype heterogeneity associated with c-myc amplification and c-Myc overexpression. The in vitro expression of the Cul1-N252 mutant causes a pleiotrophic phenotype, which includes the formation of multinucleated cells, centrosome and mitotic spindle abnormalities, and impaired chromosome segregation. These findings support a crucial role for Skp1 in proper chromosomal segregation, which is required for the maintenance of euploidy and suppression of transformation (Piva, 2002).

Visualizing developmentally programmed endoreplication in mammals using ubiquitin oscillators

The majority of mammalian somatic cells maintain a diploid genome. However, some mammalian cell types undergo multiple rounds of genome replication (endoreplication) as part of normal development and differentiation. For example, trophoblast giant cells (TGCs) in the placenta become polyploid through endoreduplication (bypassed mitosis), and megakaryocytes (MKCs) in the bone marrow become polyploid through endomitosis (abortive mitosis). During the normal mitotic cell cycle, geminin and Cdt1 are involved in 'licensing' of replication origins, which ensures that replication occurs only once in a cell cycle. Their protein accumulation is directly regulated by two E3 ubiquitin ligase activities, APC^Cdh1 and SCF^Skp2, which oscillate reciprocally during the cell cycle. Although proteolysis-mediated, oscillatory accumulation of proteins has been documented in endoreplicating Drosophila cells, it is not known whether the ubiquitin oscillators that control normal cell cycle transitions also function during mammalian endoreplication. In this study, transgenic mice were used expressing Fucci fluorescent cell-cycle probes that report the activity of APC^Cdh1 and SCF^Skp2. By performing long-term, high temporal-resolution Fucci imaging, it was possible to visualize reciprocal activation of APC^Cdh1 and SCF^Skp2 in differentiating TGCs and MKCs grown in custom-designed culture wells. TGCs and MKCs were found to both skip cytokinesis, but in different ways, and that the reciprocal activation of the ubiquitin oscillators in MKCs varies with the polyploidy level. Three-dimensional reconstructions were obtained of highly polyploid TGCs in whole, fixed mouse placentas. Thus, the Fucci technique is able to reveal the spatiotemporal regulation of the endoreplicative cell cycle during differentiation (Sakaue-Sawano, 2013).

SCF-dependent IkappaB degradation

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

SCF-dependent ß-Catenin degradation

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

Destruction of ß-catenin is regulated through phosphorylation-dependent interactions with the F box protein ß-TrCP. A novel pathway for ß-catenin degradation was discovered involving mammalian homologs of Drosophila Sina (Siah), which bind ubiquitin ß-conjugating enzymes, and Ebi, an F box protein that binds ß-catenin independent of the phosphorylation sites recognized by ß-TrCP. A series of protein interactions were identified in which Siah is physically linked to Ebi by association with a novel Sgt1 homolog SIP that binds Skp1, a central component of Skp1-Cullin-F box complexes. Expression of Siah is induced by p53, revealing a way of linking genotoxic injury to destruction of ß-catenin, thus reducing activity of Tcf/LEF transcription factors and contributing to cell cycle arrest (Matsuzawa, 2001).

A pathway linking the RING protein Siah-1 to the F box protein Ebi has been mapped and it has been shown that Ebi can bind ß-catenin. Unlike ß-TrCP, however, which requires GSK3ß-mediated phosphorylation of ß-catenin on serine 33 and serine 37, Ebi interacts with ß-catenin independently of these phosphorylation sites. Also, the Siah binding protein SIP associates with complexes containing Ebi but not ß-TrCP, suggesting differences compared to previously characterized E3 ubiquitin ligase complexes, where E2 enzymes are supplied via Cullin-mediated interactions with RING-containing proteins such as Rbx-1/Roc-1. Recent identification of interactions between Siah-1 and the ß-catenin binding protein APC suggest that this scaffold protein represents a point of common intersection of the Wnt and Siah-1 pathways for ß-catenin degradation (Matsuzawa, 2001).

Two alternative pathways for regulation of ß-catenin levels are presented, involving different F box proteins (Ebi versus ß-TrCP). One pathway is initiated by increases in the expression of Siah-family proteins, which can be induced, for example, by p53 in response to DNA damage, and involves sequential protein interactions with SIP, Skp1, and Ebi. Ebi binds ß-catenin, thus recruiting it to the Siah-1-SIP-Skp1 complex for polyubiquitination and subsequent proteosome-mediated degradation. Siah-1 binds the E2 UbcH5. The other pathway is regulated by Wnt signals (Dsh) and possibly PI3K/Akt. This pathway is phosphorylation dependent and involves GSK3ß-induced phosphorylation of Ser-33 and Ser-37 on ß-catenin, allowing ß-TrCP binding, resulting in recruitment of ß-catenin to Skp1-Cullin-1- ß-TrCP complexes (SCF). Cullin-1, in collaboration with other proteins, supplies this SCF complex with E2s, such as UbcH3. APC is required for both pathways as a scaffold protein, binding ß-catenin via one domain and also binding Siah-1 and GSK3ß (Matsuzawa, 2001).

In the fly, Sina recruits E2s to Phyllopod/Tramtrack complexes, targeting Tramtrack for ubiquitination. The ebi-gene product also binds Tramtrack and promotes its degradation in vitro and when expressed in insect cells in culture. Loss-of-function mutations of ebi cause Tramtrack accumulation and prevent R7 cell differentiation. Similar to ß-TrCP, the ebi gene of Drosophila encodes an F box/WD-40-repeat protein with sequence homology to Cdc4 (yeast), Sel-10 (C. elegans), and Slimb (Drosophila), suggesting that it provides a functional connection between a Sina-regulated pathway and SCF complexes. How this linkage between Sina and SCF complexes is achieved, however, has been unclear (Matsuzawa, 2001).

The finding that SIP functions as a molecular bridge between the human homologs of Sina and the SCF-component Skp1 provides evidence of a physical linkage between components of these two ubiquitin ligase systems, thus corroborating the genetic evidence from Drosophila that these two pathways for targeted protein degradation interact. The Drosophila ortholog of SIP is also capable of bridging the fly Skp1 and Sina proteins in three-hybrid experiments. Thus, an evolutionarily conserved network of protein interactions exists in which Siah-1 (Sina) binds to SIP, which in turn binds to Skp1, which binds Ebi (Matsuzawa, 2001).

p53 can induce expression of Siah-family genes in mammals, establishing p53 as one factor capable of invoking Siah-dependent pathways for protein degradation. Siah-family proteins are normally maintained at a relatively low level through ubiquitination-dependent protein turnover, where human Siah-1 and Siah-2 promote their own degradation through interactions of their RING domains with E2s. This therefore suggests that activation of p53 leads to a burst of Siah-1 mRNA and protein production, triggering the Siah/SIP/Skp1/Ebi pathway for ß-catenin degradation. In contrast to Siah-family proteins, it seems unlikely that SIP, Skp1, or Ebi are limiting components of this pathway, since overexpression of them has little effect on ß-catenin levels (Matsuzawa, 2001).

Though p53-mediated degradation of ß-catenin correlates with cell cycle arrest, it remains to be established whether these events are functionally linked. Activation of Tcf/LEF-family transcription factors by ß-catenin is known to induce expression of cyclin D1, c-myc, and other genes important for cell proliferation, making it plausible that ß-catenin degradation is linked to p53-mediated cell cycle arrest. However, given the role established for the cyclin-dependent kinase inhibitor p21^Waf1 in mediating G1 arrest induced by p53, it is unclear whether a parallel pathway for ß-catenin degradation would be required. Circumstances have been described where p53 fails to induce cell cycle arrest despite inducing p21^Waf1 expression, raising the question of whether p21^Waf1 is necessary but insufficient for p53-mediated G1 arrest. Recently, a genetic interaction between ebi and p21^Waf1 has been identified using an assay in Drosophila where flies are engineered to ectopically express human p21^Waf1 in the developing eye disc (Boulton, 2000). Specifically, mutant alleles of ebi abrogate inhibition of S phase entry by p21^Waf1, implying a need for Ebi in p21-mediated cell cycle arrest. Flies with mutant ebi also display ectopic S phases and overproliferation phenotypes (Boulton, 2000), further implying a role for ebi in growth suppression. Defects in cell cycle arrest in ebi mutants, however, do not necessarily implicate ß-catenin/Armadillo. For example, p53 can induce degradation of c-Myb through a proteosome-dependent mechanism partly mediated by Siah (Tanikawa, 2000). Thus, Ebi may have other targets in addition to ß-catenin that are relevant to mechanisms of p53-mediated cell cycle arrest. Future experiments should explore whether the fly homolog of p53 is linked to an ebi-dependent pathway for cell cycle arrest entailing degradation of Armadillo. In the M1 cell model, p53 induces both G1 arrest and apoptosis. Though Ebi(DeltaF)-expressing M1 cells may exhibit some delay in p53-induced apoptosis, this could result indirectly because of failed G1 arrest. Moreover, Siah-1 often fails to induce apoptosis when overexpressed in cells. However, links of Siah to apoptosis can occur under some circumstances, as demonstrated by the observation that coexpression of Siah-1 with a Siah binding protein Pw1/Peg3 causes apoptosis, whereas neither Siah-1 nor Pw1/Peg3 alone are sufficient. Mutations affecting components of the Wnt-signaling pathway are commonly observed in human cancers, resulting in aberrant accumulation of ß-catenin and activation of Tcf/LEF-target genes. Wnt-family ligands, frizzled-family receptors, and the signaling proteins downstream of these define one mechanism for regulating ß-catenin levels. However, additional inputs into pathways controlling ß-catenin turnover have recently been identified, including a mitogen-activated protein kinase pathway involving a Tak1 homolog and Nemo-like kinases in C. elegans and a cell adhesion-dependent pathway involving integrin-linked kinase. The findings reported here reveal yet another pathway for regulating ß-catenin levels that is linked at least in part to p53-dependent responses to genotoxic injury. It is speculated that loss of p53 or components of the Siah/SIP/Skp/Ebi pathway for ß-catenin destruction may contribute to aberrant ß-catenin accumulation in cancers (Matsuzawa, 2001).

The SCF ubiquitin ligases catalyze protein ubiquitination in diverse cellular processes. SCFs bind substrates through the interchangeable F box protein subunit, with the >70 human F box proteins, allowing the recognition of a wide range of substrates. The F box protein β-TrCP1 recognizes the doubly phosphorylated DpSGφXpS destruction motif, present in β-catenin and IκB, and directs the SCF^β-TrCP1 to ubiquitinate these proteins at specific lysines. The 3.0 Å structure of a β-TrCP1-Skp1-β-catenin complex reveals the basis of substrate recognition by the β-TrCP1 WD40 domain. The structure, together with the previous SCF^Skp2 structure, leads to the model of SCF catalyzing ubiquitination by increasing the effective concentration of the substrate lysine at the E2 active site. The model's prediction that the lysine-destruction motif spacing is a determinant of ubiquitination efficiency is confirmed by measuring ubiquitination rates of mutant β-catenin peptides, solidifying the model and also providing a mechanistic basis for lysine selection (Wu, 2003).

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date revised: 10 October 2014

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