BIOLOGICAL OVERVIEW

Complexes of D-type cyclins and cdk4 or 6 are thought to govern progression through the G₁ phase of the cell cycle. In Drosophila, single genes for Cyclin D and Cdk4 have been identified, simplifying genetic analysis. Drosophila Cdk4 has been shown to interact with Cyclin D and Retinoblastoma-family protein as expected, but is not absolutely essential for cell cycle progression or vitality. Flies homozygous for null mutations develop to the adult stage and are fertile, although only to a very limited degree. It is concluded that progression into and through the cell cycle can occur in the absence of Cdk4 (Meyer, 2000). However, the growth of cells and of the organism is reduced in Cdk4 mutants, indicating a role for D-type cyclin-dependent protein kinases in the modulation of growth rates. The cellular response to CycD-Cdk4-driven growth varies according to cell type. In undifferentiated proliferating wing imaginal cells, CycD-Cdk4 causes accelerated cell division (hyperplasia) without affecting cell cycle phasing or cell size. In endoreplicating salivary gland cells, CycD-Cdk4 causes excessive DNA replication and cell enlargement (hypertrophy). In differentiating eyes, CycD-Cdk4 causes hypertrophy in post-mitotic cells (Datar, 2000).

D-type cyclins, their kinase partners cdk4 and 6, the INK inhibitors and the kinase substrate retinoblastoma protein (Rb) are all known for their crucial importance in human tumorigenesis. At the cellular level, Rb has been shown to regulate progression through the G1 phase of the mammalian cell cycle, predominantly by binding to E2F transcription factors, which control a large number of genes involved in cell proliferation and DNA replication. Rb represses expression of E2F target genes by recruiting histone deacetylase activity and by inhibiting the E2F transcriptional activation domain. The ability of Rb to block progression through G1 is abolished by Rb hyperphosphorylation, which is initiated by D-type cyclin-dependent kinases during G1. In mammalian cells, the synthesis of D-type cyclins is controlled by extracellular signals. Mitogens induce a rapid accumulation of D-type cyclins. Conversely, antimitogens or withdrawal of mitogens result in a rapid decline. D-type cyclins are therefore thought to function as a functional link between extracellular signals and the cell cycle machinery. Accordingly, D-type cyclin-cdk complexes might be predicted to play an important role in the regulation of cell proliferation during development (Meyer, 2000 and references).

Given that Drosophila CycD-Cdk4 is not essential for cell cycle progression, how can these results be reconciled with the standard view that CycD-Cdk4 acts through Rb to control E2F activity? It is suggested that Cyclin D-Cdk4 and Cyclin E-Cdk2 might act as independent RBF kinases in Drosophila. Overexpression of either cyclin D-Cdk4 or Cyclin E results in an increase of hyperphosphorylated Rb. The specific Cyclin E-Cdk2 inhibitor Dacapo severely inhibits Rb hyperphosphorylation. Thus, Drosophila Cyclin E-Cdk2, which is known to phosphorylate RBF efficiently in vitro, might also phosphorylate Rb in Cdk4 mutants, thereby explaining why a decrease in Rb phosphorylation cannot be observed in Cdk4 mutants. The suggestion that Cyclin D-Cdk4 and Cyclin E-Cdk2 might act as functionally overlapping RBF kinases must remain tentative. The finding that Cdk4 mutants are extremely sensitive to a reduction of the Cyclin E+ and Cdk2+ gene dose further stresses the intimate functional relationship between Cyclin D-Cdk4 and Cyclin E-Cdk2 but does not address their interaction hierarchy. Even though the idea that Cyclin D-Cdk4 and Cyclin E-Cdk2 might act as overlapping RBF kinases in Drosophila is favored, it is emphasized that a number of phenotypic differences resulting from both loss- and gain-of-function mutations in the genes encoding the subunits of these different kinase complexes clearly demonstrate that they must also have distinct functions (Meyer, 2000 and references).

Drosophila Cdk4 is clearly required for normal growth of cells and the organism. Size regulation at the level of cells, as well as at the level of organs and organism is an important but poorly understood process. A number of observations in yeast and mammalian cells have indicated that cell cycle progression is dependent on a critical cell size. Many cell types delay the G₁-S transition (at a point called START in yeast and at restriction point in mammalian cells) until a critical size has been reached. Slow growth due to nutrient or growth factor limitations therefore results primarily in an extension of the G₁ phase. In budding yeast, the Cln cyclins, and in particular Cln3p, have been implicated in this coupling of cell growth and cell cycle progression. Cln3p is a very unstable protein which might only accumulate to an effective concentration that triggers progression through START when ribosome numbers have increased sufficiently. Experimental uncoupling of growth rates and the rates of Cln3p accumulation affects primarily cell division size without a comparable change of cell cycle duration. D-type cyclin-cdk complexes have been proposed to regulate progression through the restriction point in response to growth factors and mitogens in a comparable manner. However, proliferating Cdk4 mutant cells in wing imaginal discs are of wild-type size and progress more slowly through the cell cycle with a proportionate extension of G₁, S and G₂ phases. This phenotype is incompatible with an exclusive role of Cdk4 in the coupling of cell size and progression through the restriction point. Cdk4 appears to regulate, rather than simply sense growth (Meyer, 2000).

Reduced growth has also been reported to result from inactivation of mouse cdk4 function (Rane, 1999; Tsutsui, 1999). Intriguingly, the phenotypic similarities observed in cdk4 mutant mice and flies extend further. Mutant mice are also infertile, particularly when female. In these females, infertility has been shown to be caused by deficits in the hypothalamic pituitary axis rather than by developmental abnormalities in reproductive organs (Rane, 1999). In addition, cdk4 mutant mice developed diabetes within 2 months after birth due to a loss of the insulin-expressing ß-islet cells (Rane, 1999). In Drosophila, a striking similarity of the phenotypic consequences of mutations in Cdk4 and in genes of the insulin signaling pathway is noted. Therefore, a future analysis of the relationship of Cdk4 and insulin signaling in Drosophila might reveal a striking evolutionary conservation (Meyer, 2000).

Apart from emphasizing functional homology, this analysis also reveals an apparent clear difference between the regulatory role of cyclin D-Cdk4 in Drosophila and in mammalian cells. In mammalian cells, D-type cyclin complexes do not only function catalytically as protein kinases. They also act stoichiometrically by titrating the cdk inhibitors p21^CIP1 and p27^KIP1. Thereby, they contribute to the activation of cyclin E-Cdk2 complexes. In contrast, the Drosophila cdk inhibitor Dacapo (the only detectable CIP/KIP family member in the known genome sequence) could not be detected in Cyclin D-Cdk4 complexes. This apparent inability of Drosophila Cyclin D-Cdk4 to titrate Dacapo, might explain why, to some extent, mammalian D-type cyclin complexes behave in a functionally distinct manner (Meyer, 2000).

In summary, these observations clearly implicate Cdk4 in the control of growth rates at the cellular and organismal level. Moreover, while these results confirm an involvement of Cdk4 in the regulation of cell proliferation, they also emphasize the importance of alternative pathways. In Drosophila, the developmental program of cell proliferation is surprisingly normal in the absence of Cdk4. The fact that the cyclin D-Cdk4 pathway is of paramount importance for tumorigenesis, therefore, should probably not lead to the view that it is the only pathway controlling entry into and exit from the cell cycle (Meyer, 2000).

The question arises: how does cyclin D/Cdk4 promote cellular growth in addition to cell cycle progression? To assess the function of Drosophila Cyclin D, the effects of overexpressing it were studied in actively proliferating wing imaginal disc cells. Cell clones overexpressing CycD and/or Cdk4 and marked with co-expressed green fluorescent protein (GFP) were induced in developing wings using the flip-out Gal4 system. Tissues were dissociated and analyzed by fluorescence activated cell sorting (FACS) 48 h post-induction (h.p.i.), to determine the proportion of cells in each phase of the cell cycle, and to assess effects on cell size. Expressing either CycD or Cdk4 alone has no detectable effects. Hence, both CycD and Cdk4 were overexpressed. CycD-Cdk4-overexpressing cells were compared with wild-type, non-expressing cells from the same discs. In parallel experiments, overexpressed Drosophila E2F/DP or cyclin E truncates the G₁ phase and decreases the cell size. However, no changes in cell cycle phasing or cell size were found in CycD-Cdk4-overexpressing cells. These data differ markedly from the effects of cyclin D1 or D2 overexpression demonstrated in cultured vertebrate cells (Jiang, 1993; Quelle, 1993; Resnitzky, 1994 and 1995) and suggest that the Drosophila CycD-Cdk4 complex does not promote G₁-S transitions directly in vivo (Datar, 2000).

To assess the effect of CycD-Cdk4 on rates of cell growth and proliferation, CycD-Cdk4-expressing cell clones were evaluated in situ in wing imaginal discs. Although overexpressed CycD-Cdk4 does not alter cell cycle phasing, it does affect the proliferative rate of cells in the developing wing. At 44 h.p.i. a significant increase in the number of cells in CycD-Cdk4-expressing clones, which had a median of 16 cells, compared with 12 cells in controls, was noted. This corresponds to a 10% decrease in the cell doubling time, from 12 h in controls to 10.8 h in cells overexpressing CycD-Cdk4. Since cells overexpressing CycD-Cdk4 are not reduced in size, it follows that they must acquire more mass than control cells over the same developmental period. Consistent with this it was observed that CycD-Cdk4-overexpressing clones encompass 150% of the area of controls at 44 h.p.i., and 180% of the area of controls at 67 h.p.i. It is concluded that CycD-Cdk4 can stimulate cellular and clonal growth. In the context of the developing wing this increased growth results in faster cell division without detectable changes in cell size (Datar, 2000).

It was next asked what effect CycD-Cdk4 overexpression would have on the development of adult tissues. Adult wings containing CycD-Cdk4-expressing clones show no gross abnormalities in overall shape, veination, bristle or trichome patterning. Optical sections through pupal wings failed to reveal significant changes in cell morphology or cell density within clones overexpressing CycD-Cdk4. However, a striking enlargement of ommatidiae was observed in adult eyes that clonally overexpress CycD-Cdk4. This phenotype requires expression of both CycD and Cdk4. Optical sections of pupal eyes confirmed that overexpressed CycD-Cdk4 leads to increased cell size (hypertrophy) and that this effect is cell autonomous. A mild disorganization in the regular arrangement of ommatidia was observed, but no specific defects in cell differentiation or overall patterning. Hypertrophy occurs in several cell types and structures, including primary pigment and cone cells, photoreceptors and interommatidial bristles. In ommatidiae overexpressing CycD-Cdk4, five cone cells were often observed instead of the normal four, suggesting that an extra cell division occurred (Datar, 2000).

CycD-Cdk4 was also induced in the eye using the GMR-Gal4 and sev-Gal4 drivers, which are expressed starting late in eye development posterior to the morphogenetic furrow (MF), as cells enter their ultimate or penultimate cell cycle and begin to differentiate. Using these drivers to induce CycD-Cdk4 expression it was found that all ommatidiae are enlarged, as is the entire eye, which bulges out of the head in an ominous fashion. Light and transmission electron microscopy of pupal and adult eyes from GMR-Gal4, UAS-CycD, UAS-Cdk4 flies reveal enlarged photoreceptor cell bodies and rhabdomeres, and excessive accumulations of actin, suggesting that CycD-Cdk4 increases cellular protein content as well as cell volume. Driving expression of two copies of CycD-Cdk4 with a single copy of GMR-Gal4 led to even larger ommatidiae and eyes, indicating dose dependence. No significant eye overgrowth was observed when CycD was expressed alone or when it was co-expressed with a kinase-impaired mutant form of Cdk4 (Cdk4^D175N). Thus, the bulging eye phenotype appears to require phosphorylation of CycD-Cdk4 substrates. In comparison, CycE or E2F does not cause eye overgrowth when expressed under GMR-Gal4 control, even though both factors promote ectopic DNA replication and cell proliferation in the eye (Datar, 2000).

To assess these phenotypes further FACS was performed on eye discs in which UAS-GFP was co-expressed with either UAS-CycD-Cdk4 or UAS-CycE under GMR-Gal4 control. FACS analysis shows that overexpression of either CycD-Cdk4 or CycE significantly increases the fraction of S- and G₂-phase cells posterior to the MF (GFP+ cells). Normally these cells are nearly all arrested in G₁, and thus both cyclins appear to perturb the normal program of cell cycle exit at differentiation. The effects of CycD-Cdk4 and CycE on cell size, however, were dramatically different. CycD-Cdk4 greatly increases the size of posterior eye cells, whereas CycE caused a slight decrease in cell size. This cell size effect is not phase specific: even G₁ cells overexpressing CycD-Cdk4 are much larger than controls. These results are consistent with findings in wing cells in that CycD-Cdk4 appears to promote both cell cycle progression and cellular growth, whereas CycE affects only cell cycle progression. In addition, neither CycE nor CycD-Cdk4 completely prevents the G₁ arrest that accompanies cell differentiation, but only CycD-Cdk4 appears to continue promoting growth in post-mitotic cells, leading to cellular hypertrophy (Datar, 2000).

Many studies have indicated that the major substrates for vertebrate D-type cyclin-Cdk complexes are the Rb family proteins. A Drosophila homolog of Rb, RBF, inhibits cell division when overexpressed in the wing imaginal disc. RBF does not immediately block cell growth, and as a result there is a large increase in cell size. To test interactions between CycD-Cdk4 and RBF, all three genes were co-expressed together in wings, salivary glands or eyes. Using the flip-out Gal4 method in the wing, it was found that cells co-expressing CycD-Cdk4 and RBF cycle more rapidly than cells expressing RBF alone, and thus that CycD-Cdk4 attenuates the inhibitory effects of RBF on cell cycle progression. Interestingly, although cell clones co-expressing CycD-Cdk4 and RBF have fewer cells than wild-type controls, they encompassed substantially more area. Flow cytometry and in situ cell size measurements confirm that the increased mass of these clones is due to increased cell size. The large size of cells co-expressing CycD-Cdk4 and RBF, despite a nearly normal division rate, suggests that CycD-Cdk4 promotes extra growth even while RBF slows cell cycle progression. A logical inference is that CycD-Cdk4 promotes growth via targets other than RBF. This effect is less evident at a later time-point (67 h.p.i.), perhaps because cell cycle suppression by RBF eventually throttles even the growth of CycD-Cdk4-expressing clones (Datar, 2000).

RBF-CycD interaction tests were performed in the larval salivary gland, a differentiated tissue in which cell growth is accomplished by cycles of DNA endoreplication. To express UAS-linked target genes, the F4-Gal4 driver was used, that commences its expression late in embryogenesis after cell proliferation in the salivary primordium is complete and stays active throughout the larval stages. Results obtained in the salivary glands are consistent with those described above for the wing. F4-Gal4-driven expression of CycD-Cdk4 results in oversized salivary glands with nuclei containing excessively endoreduplicated polytene chromosomes. Co-expression of cyclin D with the kinase impaired variant Cdk4^D175N or Cdk2 does not induce this size increase, suggesting that the protein kinase activity of cyclin D-Cdk4 complexes is required for the stimulation of salivary gland DNA endoreplication and growth. Although expression of Cdk4^D175N alone has little effect on salivary growth, overexpressed RBF strongly inhibits both growth and DNA endoreplication. Even stronger growth suppression is observed when RBF is co-expressed with Cdk4^D175N. As in wings and eyes, the growth-inhibitory effects of RBF are attenuated by simultaneous co-expression of CycD-Cdk4 (Datar, 2000).

Similar results are obtained when RBF and CycD-Cdk4 are co-expressed in the eye using ey-Gal4, which is expressed throughout the eye primordium beginning very early in eye development. Expression of RBF under ey-Gal4 control causes a dramatic reduction of the adult eye. This loss of eye tissue is suppressed virtually completely when CycD-Cdk4 is co-expressed with RBF, providing further evidence that CycD-Cdk4 can functionally inactivate RBF. Interestingly, suppression of RBF is also observed when CycD is expressed without the Cdk4 kinase subunit or to a lesser extent when CycD is co-expressed with the kinase-impaired variant Cdk4^D175N. This suggests that CycD may suppress RBF function by utilizing an endogenous Cdk, or in a kinase-independent fashion (Datar, 2000).

Although ectopic RBF suppresses growth in developing wings, salivary glands and eyes, growth inhibition by RBF is secondary to its effects on cell cycle progression (Neufeld, 1998). The tests described above are consistent with this interpretation, since in all cases both cell cycle progression (DNA replication) and growth are affected coincidentally. However, these tests cannot rule out the possibility that RBF inhibits cellular growth directly. Therefore the late acting eye specific drivers, GMR-Gal4 and sev-Gal4, were used to test whether RBF could suppress growth in non-cycling cells. Expression of RBF alone using these drivers has little effect on eye size, suggesting that RBF cannot suppress the post-mitotic growth that normally occurs in the eye. Moreover, co-expressed RBF does not substantially suppress eye overgrowth caused by GMR-Gal4- or sev-Gal4-driven CycD-Cdk4. Given this, a parsimonious interpretation of all these interaction tests is that whereas CycD-Cdk4 can promote cellular growth in both proliferating and non-cycling cells, RBF can suppress growth only in cells that are undergoing cycles of DNA replication. In this case, growth suppression by ectopic RBF most likely stems from its ability to inhibit cell cycle progression (Datar, 2000).

To further test RBF function, FLP/FRT-mediated mitotic recombination was used to generate cell clones homozygous for a null allele of rbf (rbf¹⁴). Areas of rbf^14/14 cell clones were measured in wing discs, and these were compared with the areas of their rbf^+/+ sister clones ('twin spots'). Cell clones mutant for rbf are slightly smaller than their wild-type twin spots, though the difference is not statistically significant. Thus, loss of RBF does not confer a growth advantage. FACS analysis of rbf^14/14 cells shows a reduced cell size and, surprisingly, an increased G₁ population. Microscopic examination reveals many pyknotic nuclei associated with rbf^14/14 mutant clones, suggesting elevated levels of apoptosis. Reduced cell size and poor viability have also been observed in hyperproliferative cell clones overexpressing E2F/DP, and similar characteristics have been described for mouse embryo fibroblasts lacking pRB, p107 or p130. One explanation for these phenotypes is that they arise when cell division rates outpace rates of cell growth (Neufeld, 1998). This may also be the case for rbf^14/14 cells (Datar, 2000).

Finally, large rbf^14/14 clones were generated in the eye using the Minute technique. Although these clones populate large fractions of the eye they do not exhibit the hypertrophic characteristics noted when CycD-Cdk4 is overexpressed. Instead, rbf^14/14 clones in the adult eye exhibit slight to moderate hypoplasia and mild defects such as missing or duplicated inter-ommatidial bristles and fused ommatidiae. Similar phenotypes have been noted in eyes overexpressing E2F or cyclin E, both of which promote extra cell division in the eye without increasing growth. These results are consistent with the interpretation that RBF functions to inhibit cell cycle progression, rather than to inhibit cellular growth directly. If this is the case, stimulation of cellular growth by CycD-Cdk4 must be, at least in part, independent of RBF (Datar, 2000).

Thus Drosophila CycD-Cdk4 does not have a specialized function in promoting G₁ progression, but instead promotes accumulation of cellular mass, i.e. growth. The effects of CycD-Cdk4 differ profoundly from those of its presumed targets, RBF, E2F and CycE. In numerous assays, RBF, E2F and CycE behave as specific cell cycle regulators that affect cellular growth only indirectly, via their effects on the cell cycle. While these findings differ from those in cultured mammalian cells, they are remarkably consistent with data from in vivo studies of the murine D-type cyclins. Just as overexpressing CycD-Cdk4 in the fly wing or eye causes hyperplasia, targeted overexpression of CycD1 in mice can promote epidermal, mammary and thymic hyperplasia, and mutational activation of Cdk4 can cause pancreatic hyperplasia (Rane, 1999). Likewise, overexpressed cyclin D3 can promote endomitosis in platelets, much as overexpressed Drosophila CycD-Cdk4 drives extra DNA endoreplication in salivary gland cells. CycD1 knockout mice are smaller than their littermates and display hypoplasia of the retina and mammary epithelium, tissues that normally express CycD1 at high levels. Mice lacking CycD2 display hypoplastic growth phenotypes. Cdk4 mutant mice, like Cdk4 mutant Drosophila, are also small (Datar, 2000 and references therein).

Although it is not yet clear whether these growth effects in other systems are cell autonomous, studies of mouse embryo fibroblasts (MEFs) suggest that some are. Proliferating MEFs lacking CycD1 or Cdk4 have normal G₁/S/G₂ profiles and so, like studies in flies, cast doubt upon a direct role for CycD-Cdk4 in G₁-S progression. However, CycD1 or Cdk4 mutant MEFs are delayed in activating DNA replication upon serum stimulation, an effect that could be attributed to impaired growth. MEFs from mice lacking p16^INK4A, a specific inhibitor of CycD-dependent kinases, also exhibit accelerated growth. Taken together, all of these observations fall into place to support the view that one cellular function of cyclin D-Cdk complexes is to stimulate cellular growth (Datar, 2000 and references therein).

D-type cyclins are believed to function primarily by suppressing the function of the 'pocket' proteins: pRb, p107 and p130. Numerous genetic interactions have been described between Drosophila CycD-Cdk4 and RBF (Datar, 2000) that indicate in vivo cross-regulation of these gene products. CycD-Cdk4 counteracts the growth-suppressive effects of RBF in wing and eye imaginal discs, and also in the endoreplicating salivary gland. As in vertebrates, these interactions might reflect direct phosphorylation and neutralization of RBF by CycD-Cdk4 kinase. However, these interactions could also be indirect consequences of the effects of CycD-Cdk4 on cellular growth. For instance, growth stimulation by CycD-Cdk4 may increase the activity of CycE-Cdk2, which is also a potent suppressor of RBF activity (Datar, 2000 and references therein).

Although the CycD-RBF interactions describe for Drosophila confirm current paradigms to some extent, they also highlight the differences between CycD-Cdk4 and RBF function. For instance, RBF’s ability to suppress growth is limited to cells actively undergoing DNA replication, such as proliferating cells in the imaginal discs and endoreplicating cells in the salivary gland. In the post-mitotic eye, where CycD-Cdk4 is a potent stimulator of growth, RBF is virtually inert. Moreover, loss of RBF does not enhance growth in either proliferating or post-mitotic cells, and thus does not phenocopy gain of CycD-Cdk4 function. Indeed, all the data on RBF suggest that it functions specifically to suppress cell cycle progression, and that its growth-suppressive effects are secondary consequences of this function. CycD-Cdk4, in contrast, promotes growth in both proliferating and post-mitotic cells. An important implication of this analysis is, therefore, that Drosophila CycD-Cdk4 must promote growth via targets other than RBF. These growth-regulatory targets remain unknown, but the requirement for a catalytically active Cdk4 subunit suggests that they are phosphorylation substrates. Potential candidates include pocket proteins other than RBF, or factors that affect biosynthesis directly, such as regulators of protein synthesis or turnover. Consistently, reports describe non-Rb targets for mammalian CycD1, and indicate that the transforming activity of CycD1 does not require Rb binding (Datar, 2000 and references therein).

How CycD-Cdk4 fits into the hierarchy of genes that regulate growth in Drosophila is unclear, and will remain obscure until mutations in the cycD gene are identified. It is tempting to speculate that CycD-Cdk4 might mediate the growth effects of the Wnt, Bmp and Egf signaling pathways, which orchestrate patterned growth in Drosophila and throughout the animal kingdom. However, there are presently scant data to support this hypothesis in flies. Recent work has revealed that insulin receptor/Pi3K/AKT signaling is also an important growth control pathway in Drosophila, and may respond to nutritional conditions. Like CycD-Cdk4, genes in this pathway appear to be growth specific and to have little role in tissue patterning or differentiation. But there are clear distinctions between CycD-Cdk4-driven growth and Pi3K-driven growth. In the wing for instance, CycD-Cdk4 accelerates growth in a balanced fashion such that cell cycle phasing and cell size remain normal, whereas Pi3K-driven growth is characterized by a truncated G₁ phase and increased cell size. The origin of these differences is presently unclear. One plausible explanation is that CycD-Cdk4 can promote G₂-M progression, perhaps by repressing RBF and activating Cdc25/Stg, whereas Pi3K cannot. In support of this idea it has recently been shown (Gabrielli, 1999) that human Cdk4 activity is required for the G₂-M transition in HeLa cells(Datar, 2000 and references therein).

There is an abundance of correlative evidence associating cyclin D with diverse cancers. It is telling that nearly all cases are associated with elevated levels of cyclin D function as a result of amplification or translocation of the cyclin D locus, or inactivation of the cyclin D-specific inhibitor, p16^INK4A. When cyclin D-Cdk4 activity is increased in Drosophila, overgrowth is induced as well. In thinking about how the human D-type cyclins are involved in the progression of cancer, it may prove fruitful to consider them, not as regulators of G₁ progression, but as promoters of cellular growth (Datar, 2000 and references therein).

PROTEIN STRUCTURE

Amino Acids - 317

Structural Domains

Analysis of full-length cDNAs indicates the presence of the characteristic pRb-binding motif LXCXL (Meyer, 2000)

In addition to the previously identified Drosophila cdc2 and cdc2c genes, four additional cdc2-related genes have been identified with low stringency and polymerase chain reaction approaches. Sequence comparisons suggest that the four putative kinases represent the Drosophila homologs of vertebrate cdk4/6, cdk5, PCTAIRE, and PITSLRE kinases. Although the similarity between human and Drosophila homologs is extensive in the case of cdk5, PCTAIRE, and PITSLRE kinases (78%, 58%, and 65% identity in the kinase domain), only limited conservation is observed for Drosophila cdk4 (47% identity) (Sauer, 1996).

date revised: 21 October 2000

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