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).

GENE STRUCTURE

cDNA clone length - 1134

(CG16983-PA)

Bases in 5' UTR - 339

Exons - 2

Bases in 3' UTR - 306

PROTEIN STRUCTURE

Amino Acids - 162

Structural Domains

Six Drosophila skp1-related genes were identified and named skpA through skpF. SkpA was chosen for further analysis because it is the most widely expressed and shares the highest identity with human and yeast Skp1 (76% and 45%, respectively). cDNA analyses reveal that the skpA transcript is alternatively spliced, but all of the splice forms encode the same 162 amino acid protein (Murphy, 2003).

date revised: 12 February 2004

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