Oncogene (2009) 28, 2925–2939 & 2009 Macmillan Publishers Limited All rights reserved 0950-9232/09 $32.00 www.nature.com/onc REVIEW Mammalian cell-cycle regulation: several Cdks, numerous cyclins and diverse compensatory mechanisms

A Satyanarayana1 and P Kaldis2

1Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute-Frederick, Frederick, MD, USA and 2Laboratory of Cell Division and Cancer, Institute of Molecular and Cell Biology (IMCB), Singapore, Republic of Singapore

After a decade of extensive work on gene knockout mouse cyclins (for example in S. pombe Cdc13, Cig1 and models of cell-cycle regulators, the classical model of Cig2). Besides Cdk1, yeast also expresses Cdks such as cell-cycle regulation was seriously challenged. Several PHO85 and Kin28 that may affect cell-cycle regulation unexpected compensatory mechanisms were uncovered in an indirect way (Morgan, 1997; Solomon et al., 1988; among cyclins and Cdks in these studies. The most Huang et al., 2007). Many functional homologues of astonishing observation is that Cdk2 is dispensable for the yeast Cdk1 have been identified in higher organisms. regulation of the mitotic cell cycle with both Cdk4 and These specialized G1,G1/S, S and G2/M phase-specific Cdk1 covering for Cdk2’s functions. Similar to yeast, it Cdks have replaced the major multifunctional Cdk1 of wasrecentlydiscoveredthatCdk1alonecandrivethe yeast. The discovery of approximately 20 Cdk-related mammalian cell cycle, indicating that the regulation of the proteins led to the concept that in higher eukaryotic mammalian cell cycle is highly conserved. Nevertheless, cells, cell-cycle events occur by complex combinations cell–cycle-independent functions of Cdks and cyclins such as of Cdks (Cdk1, Cdk2, Cdk3, Cdk5, Cdk4, Cdk6, in DNA damage repair are still under investigation. Here Cdk7, Cdk8 and so on) and cyclins (A1, A2, B1, B2, we review the compensatory mechanisms among major B3, C, D1, D2, D3, E1, E2, F and so on) in different cyclins and Cdks in mammalian cell-cycle regulation. phases of cell cycle, which in turn provide additional Oncogene (2009) 28, 2925–2939; doi:10.1038/onc.2009.170; control to the cell-cycle machinery (Figure 1). Cyclins published online 29 June 2009 were thought to confer substrate specificity and regula- tion (activation, inactivation, localization, binding of Keywords: cell cycle; cyclin; Cdk; DNA damage; subunits and others) to Cdk/cyclin complexes. In meiosis; knockout mouse accordance with this hypothesis, extensive research was conducted in eukaryotic cells and the classical model of cell-cycle regulation was established. According to this model, in early G1 phase Cdk4 and/ Introduction or Cdk6 are activated by D-type cyclins and initiate phosphorylation of the retinoblastoma protein (Rb) Cell division is a precisely regulated process and cells of family (Rb, p107 and p130; Sherr and Roberts, 1999, living organisms are required to divide and to produce 2004). This leads to the release of E2F transcription daughter cells. The ability of the cells to divide in turn is factors and results in the activation and transcription of mainly attributed to the presence of two classes of E2F responsive genes required for cell-cycle progression molecules, cyclin-dependent kinases (Cdks), a family of (Weinberg, 1995; Dyson, 1998). Early E2F responsive serine/threonine kinases and their binding partners, genes include E- and A-type cyclins (Lundberg and cyclins (Morgan, 1997). In yeast, only one major Cdk Weinberg, 1998; Sherr and Roberts, 1999). In the late is expressed (designated Cdk1, equivalent to p34Cdc28 in G1 phase, Cdk2 is activated by binding to cyclin E (for Saccharomyces cerevisiae and p34Cdc2 in Schizosaccharo- review see Sherr and Roberts, 1999, 2004) and completes myces pombe). Cdk1 activity oscillates during cell-cycle the phosphorylation of Rb (pocket proteins) leading to progression and is capable of regulating diverse cell- further activation of E2F mediated transcription. This leads to passage through the restriction point at the cycle transitions (G1/S, S and G2/M phases) by associating with different cell-cycle stage-specific boundary of the G1/S phase, and to S phase initiation. Later Cdk2 plays an important role in S phase progression by complexing with cyclin A. A-type cyclins Correspondence: Professor P Kaldis, Laboratory of Cell Division and are synthesized at the onset of the S phase and Cancer, Institute of Molecular and Cell Biology (IMCB), 61 Biopolis Drive, Proteos, 3-10B, Singapore 138673, Republic of Singapore. phosphorylate proteins involved in DNA replication E-mail: [email protected] or Dr A Satyanarayana, Mouse (Petersen et al., 1999; Coverley et al., 2000). During Cancer Genetics Program, Center for Cancer Research, National the G2/M transition, Cdk1/cyclin A activity is required Cancer Institute-Frederick, Bldg. 560/22-69, 1050 Boyles Street, for the initiation of prophase (Furuno et al., 1999). Frederick, MD 21702-1201, USA. E-mail: [email protected] Finally, Cdk1/cyclin B complexes actively participate in Received 11 March 2009; revised 17 May 2009; accepted 23 May 2009; and complete mitosis (Riabowol et al., 1989). The published online 29 June 2009 activities and functions of Cdk/cyclin complexes under Cyclins and Cdks in mammalian cell-cycle regulation A Satyanarayana and P Kaldis 2926 Compensatory mechanisms among cyclins

A decade of extensive work on gene knockout mouse models of cyclins disclosed that there is little significant impact on cell-cycle regulation or survival of the organisms in the absence of several individual cyclins, because each family of cyclins consists of numerous members. Four major classes of cyclins were discovered in mammalian cells: D-, E-, A-, B-type cyclins, which display dynamic localization patterns. In mammalian cells, cyclin B resides mainly in the cytoplasm, whereas cyclins A, D and E are nuclear (Sherr, 1993; Pines and Hunter, 1994; Ohtsubo et al., 1995). D-type cyclins (D1, D2 and D3) are expressed in a variety of cell types and tissues with cyclin D1 being the most ubiquitously expressed (Waclaw and Chatot, 2004).

D-type cyclins Cyclin D knockout mouse models uncovered an intricate situation where different members of the Cdk/D-type cyclin families play cell type-specific roles (Fantl et al., 1995; Sicinski et al., 1995). Mice lacking individual D-type cyclins were viable, developed nor- mally and did not show any severe impairment of cell proliferation (Kozar et al., 2004) but displayed several cell type-specific abnormalities. Cyclin D1À/À mice had reduced body size, displayed hypoplastic retinas and neurological abnormalities. The mammary glands of females failed to undergo normal lobuloalveolar devel- opment during pregnancy (Fantl et al., 1995; Sicinski et al., 1995). The cyclin D2À/À mice displayed defects in B-lymphocyte proliferation (Sicinski et al., 1996; Solva- Figure 1 Mammalian Cdk family members and their interacting son et al., 2000), pancreatic b-cell proliferation, cere- cyclin partners. Although more than 20 Cdk proteins have been identified in mammalian cells, here we are presenting only those bellar development (Huard et al., 1999) and adult Cdks whose cell-cycle and cell-cycle-independent functions are neurogenesis (Kowalczyk et al., 2004). Contrary to extensively studied. cyclin D2À/À mice, cyclin D3 deletion led to a defect in the T-lymphocyte development (Sicinska et al., 2003). It appears that loss of one of the D-type cyclins could normal as well as extreme conditions, such as stress, simply be substituted by one of its siblings in the DNA damage, telomere dysfunction and others, are majority of the cell types to prevent severe consequences regulated by two families of Cdk inhibitors; the INK4 on cell proliferation and survival (Figure 2). Never- family (p16INK4a,p15INK4b, p18INK4c, p19INK4d) specifically theless, it is clear that individual D-type cyclins still play bind to Cdk4 and Cdk6 and prevent D-type cyclin cell type-specific roles such as D2 has a specific role in activity and the Cip/Kip family (p21Cip1/Waf1/Sdi1, p27Kip1, B lymphocytes whereas D3 has a unique role in p57Kip2) inhibits Cdk2/cyclin E, Cdk2/cyclin A, Cdk1/ T lymphocytes. These minor cell type-specific defects cyclin A, as well as Cdk1/cyclin B activity (Toyoshima in single knockouts might be due to a lack of expression and Hunter, 1994; Aprelikova et al., 1995; O’Connor, or difference in the timing of expression of each of the 1997). D-type cyclins in that particular cell type. In support to The presence of multiple Cdks and cyclins in higher this view, it was demonstrated that cyclin D2 rescues the eukaryotic cells led to the speculation that each Cdk/ loss of cyclin D1 when expressed from the D1 locus cyclin complex harbors unique functions restricted (Carthon et al., 2005). This indicates that under normal to a particular phase of the cell cycle. In yeast, profound circumstances D2 can compensate for the loss of D1 but redundancies between the many cyclin molecules a slight difference in the timing of expression between were uncovered but there are only few Cdks present. D1 and D2 renders D2 unable to substitute for the loss In contrast, mammals express approximately 20 Cdks of D1 in certain cell types. and the specific function of each Cdk was not clear. Therefore, mouse models represented a useful approach to study the functions of Cdks in vivo. Cyclin D compound mice It was found that among the mammalian cyclins, The speculation that compensatory mechanisms operate widespread compensation was detected confirming the only among one type of cyclins proved wrong as cells findings in yeast. lacking two of the three or all three D-type cyclins can

Oncogene Cyclins and Cdks in mammalian cell-cycle regulation A Satyanarayana and P Kaldis 2927

Figure 2 Array of compensations among cyclins. The wide range of intra- and interfamily compensatory mechanisms among different members of cyclins are shown. Under normal circumstances the functions of cyclins are limited to a particular phase of the cell cycle, but in the absence of one set of cyclins, another set of cyclins will no longer restrict their function to one phase but rather operate at multiple phases of the cell cycle. still divide and progress through different phases of cell mouse embryonic fibroblasts (MEFs) proliferated rela- cycle and the mice can survive at least until midgestation tively normally, though MEFs displayed higher depen- (Ciemerych et al., 2002; Kozar et al., 2004; Pagano and dence on serum than wild-type cells (Kozar et al., 2004). Jackson, 2004; Figure 3). The cyclin D1/D2 double The proliferative defects were mainly limited to hema- knockout mice survived up to 3 weeks after birth. topoietic cells and myocardial cells, indicating that these Similar to D1 and D2 single knockout mice, they cells critically require D-type cyclins (Kozar et al., 2004). displayed defects such as reduced body size, hypoplastic These studies suggest that either their function is not cerebella and others (Ciemerych et al., 2002; Pogoriler essential or other cyclins such as E- or A-type cyclins et al., 2006). In contrast, most of the cyclin D1/D3 can drive cell-cycle progression in the absence of D-type double mutant mice died immediately after birth due to cyclins in the majority of cell types. This was directly neurological defects (Ciemerych et al., 2002). D2/D3 proven in a study where genetic replacement of cyclin double knockout mice died at E17.5 due to severe D1 by human cyclin E1 rescued the phenotypes of cyclin megaloblastic anemia (Ciemerych et al., 2002; Table 1). D1 knockout mice, indicating that E-type cyclins could It seems that cyclin D3 in combination with D1 or D2 is be the primary compensators of D-type cyclins (Geng more important for the survival of the organism, et al., 1999; Table 2). although D3 single knockout mice have relatively fewer The lack of all three D-type cyclins presents an abnormalities compared to either D1 or D2. Otherwise, intricate situation with regard to translocation of Cdk4/ it could be possible that several of D3’s functions are not Cdk6 from the cytoplasm to the nucleus. Under normal yet fully explored in the D3 single knockouts. It appears circumstances cyclin D1 and Cdk4/Cdk6 assemble in the that D3 can partially take over most of the cell-cycle and cytoplasm and translocate to the nucleus. The lack of cell-specific functions of D1 and D2 as the D1/D2 nuclear localization signals (NLS) in both proteins is double knockout mice are viable and can survive up to 3 overcome by a direct interaction with p21 or p27, which weeks. These single and double cyclin D knockout contain NLS, and provide a possible mechanism for mouse studies disclosed that although deletion of two nuclear import although other translocation mechan- D-type cyclins does not effect the cell cycle, at least two isms remain possible (Cheng et al., 1999; Coqueret, of the three D-type cyclins are necessary for complete 2003). After import to the nucleus, Cdk4-6/cyclin D survival of the organism (Figure 2). Mice lacking all complexes sequester p27 from resident Cdk2/cyclin three D-type (D1, D2, D3) cyclins were generated and E/p27 complexes. The impact of Cdk2/cyclin E activa- the triple knockout mice died around E16.5 (Kozar tion is the initiation of targeted degradation of p27, due et al., 2004). The majority of the cell types including to phosphorylation at threonine 187 whereas p27 bound

Oncogene Cyclins and Cdks in mammalian cell-cycle regulation A Satyanarayana and P Kaldis 2928

Figure 3 Consequence of loss of cyclins and Cdks on the survival of mice. The impact of loss of one or more cyclins and Cdks on the survival of the animals is indicated. Solid arrows indicate that the mice are viable and survived similar to wild-type mice. Dotted lines indicate that the deletion of cyclin and Cdks led to embryonic lethality, death of neonates or premature death of adult mice.

to Cdk4-6/cyclin D is stable (Coqueret, 2003). p27 are rescued, the embryos reached birth. Impairment in bound to Cdk4-6/cyclin D is released when cyclin D is the endoreplication of megakaryocytes was also degraded, and can subsequently bind new complexes. observed in the absence of cyclin E (Geng et al., 2003; The export of p27 from the nucleus to the cytoplasm for Parisi et al., 2003). The double mutant cyclin E degradation is also tightly regulated and recently it was embryonic fibroblasts displayed defects in cell-cycle discovered that cyclin D2 facilitates the translocation of reentry from quiescence although the cells proliferate p27 from the nucleus into the cytoplasm for its KPC- normally in continuous culture (Geng et al., 2003). This dependent degradation (Susaki et al., 2007). However, might be due to the timing and the expression levels of defects were not observed in cell-cycle initiation and cyclin E function as a rate limiting step in G1 progression of D1/D2/D3 triple knockout cells indicat- progression and G1/S transition and overexpression of ing the presence of alternative mechanisms. Recent cyclin E results in shortening of G1 phase and studies indicate that cyclin D1 might also have a specific accelerated S phase entry (Ohtsubo et al., 1995). The cytoplasmic function in transformed cells but its exact studies of cyclin E mouse models indicate that E-type functions are still not known (Dhar et al., 1999; Alao cyclins are not essential for cell proliferation and D- and et al., 2006). Similarly, cyclin D3 was identified to be A-type cyclins substitute for the absence of E-type present exclusively in the cytoplasm of germ cells cyclins (Figure 2). although these functions need to be explored further (Kang et al., 1997). A-type cyclins Two A-type cyclins, A1 and A2, have been discovered in E-type cyclins mammalian cells (Girard et al., 1991; Zindy et al., 1992; The next class of cyclins is the E-type cyclins and mice Sweeney et al., 1996; Ravnik and Wolgemuth, 1999) and deficient of cyclins E1 or E2 are viable and develop mouse knockouts of A1 and A2 were generated. Cyclin normally (Geng et al., 2003), suggesting that there is a A1 knockout mice are viable and develop normally. In functional redundancy between E1 and E2. However, contrast, deletion of cyclin A2 results in early embryonic deletion of both E1 and E2 led to embryonic lethality at lethality and the mice die at E5.5 (Figure 3). Cyclin A2À/À E11.5 (Figure 3), indicating that at least one E-type embryos did not show any impairment until the cyclin is essential to complete embryogenesis and other preimplantation blastocyst stage but died immediately cyclins cannot substitute for E-type cyclins during mid after implantation (Murphy et al., 1997; Liu et al., and late embryogenesis (Parisi et al., 2003). Analysis of 1998). This suggests that cyclin A2 is essential for early extra embryonic tissues in cyclin E double mutants embryogenesis and none of the other cyclins are able to revealed placental defects and when the placental defects compensate for its loss. During early preimplantation it

Oncogene Cyclins and Cdks in mammalian cell-cycle regulation A Satyanarayana and P Kaldis 2929 Table 1 Consequence of deletion of Cdks and cyclins Gene Uncompensated phenotypes Survival/ Meiotic phenotypes Primary References deleted lethality cellular localization

Cdk1 None BE2.5 Not known Cytoplasm (Santamaria et al., 2007) Cdk2 Reduced body size, impaired neural Viable Both males and Nucleus (Berthet et al., 2003; progenitor cell proliferation females are sterile Ortega et al., 2003) Cdk4 Reduced body size, insulin deficient Viable Male and female Nucleus (Rane et al., 1999; diabetes due to decreased pancreatic infertility Tsutsui et al., 1999) b-cells Cdk6 Hypoplasia of thymus and spleen and Viable Fertile Nucleus (Malumbres et al., defects 2004) in hematopoiesis Cdk2/4 Heart defects E15.5 If viable, might be (Berthet et al., 2006) sterile similar to Cdk2À/À ? Cdk2/6 Reduced body size, hematopoietic defects Viable Both male and female (Malumbres et al., are sterile 2004) Cdk4/6 Severe anemia E14.5-E18.5 If viable, might be sterile (Malumbres et al., similar to Cdk4À/À ? 2004) Cdk2/4/6 Heart defects, hematopoietic defects E13.5 If viable, might be sterile (Santamaria et al., similar to Cdk2 & Cdk4 2007) Cdk3 No defects Viable Fertile Nucleus (Ye et al., 2001) Cdk5 Severe neurological defects Died immediately Not known Nucleus (Ohshima et al., 1996) after birth Cdk11 Mitotic defects E3.5 Not known Nucleus/ (Li et al., 2004) cytoplasm Cyclin A1 No abnormalities Viable Only males are sterile Nucleus (Liu et al., 1998) Cyclin A2 Not-known E5.5 Not-known Nucleus (Murphy et al., 1997) Cyclin B1 Not-known BE10.5 Not-known Cytoplasm (Brandeis et al., 1998) Cyclin B2 No abnormalities Viable Fertile Cytoplasm (Brandeis et al., 1998) Cyclin D1 Reduced body size, hypoplastic retina, Viable Fertile Nucleus (Fantl et al., 1995; neurological abnormalities, mammary Sicinski et al., 1995) gland defects Cyclin D2 Defects in B-lymphocyte proliferation, Viable Females sterile; males Nucleus (Huard et al., 1999; pancreatic b-cell proliferation, cerebellar fertile with hypoplastic Kowalczyk et al., 2004; development, adult neurogenesis and testes Sicinski et al., 1996; hypoplastic thymus Solvason et al., 2000) Cyclin D3 Defects in T-lymphocyte development Viable Fertile Nucleus (Sicinska et al., 2003) Cyclin D1/D2 Reduced body size, hypoplastic cerebella Viable but die If viable, might be sterile (Ciemerych et al., within 3 weeks similar to D2À/À ? 2002) after birth Cyclin D1/D3 Neurological abnormalities Die soon after birth Might be fertile? (Ciemerych et al., 2002) Cyclin D2/D3 Megaloblastic anemia E17.5 If viable, might be sterile (Ciemerych et al., similar to D2À/À ? 2002) Cyclin D1/ Proliferative defects in hematopoietic cells E16.5 If viable, might be sterile (Kozar et al., 2004) D2/D3 and cardiac myocytes similar to D2À/À ? Cyclin E1 No abnormalities Viable Fertile Nucleus (Parisi et al., 2003) Cyclin E2 No abnormalities Viable Reduced male fertility Nucleus (Geng et al., 2003) Cyclin E1/E2 Severe defects in extraembryonic tissues E11.5 Not known (Geng et al., 2003) Cyclin F Defects in extraembryonic tissues E10.5 Not known Nucleus (Tetzlaff et al., 2004) is possible that cyclin A2 might not be needed or cyclin Wolgemuth, 1992; Gallant and Nigg, 1992; Lozano A1 could partially compensate for cyclin A2. It was also et al., 2002; Nguyen et al., 2002). Cyclins B1 and B2 are predicted that cyclin B3 might replace cyclin A2, which expressed in the majority of proliferating cells and shares some homology with the A-type cyclins (Winston localize to different compartments of the cell. Cyclin B1 et al., 2000). On the other hand in cultured somatic cells, is associated with microtubules, whereas cyclin B2 cyclin A2 appears to play a nonoverlapping role in S and associates with intracellular membranes (Ookata et al., G2/M phase of cell cycle (Pagano et al., 1992; Resnitzky 1993; Jackman et al., 1995). Cyclin B1 localizes to the et al., 1995; Furuno et al., 1999). nucleus in mitosis whereas cyclin B2 does not change its localization, suggesting that the two proteins play different roles during cell-cycle progression (Pines and B-type cyclins Hunter, 1991; Toyoshima et al., 1998; Yang et al., 1998). B-type cyclins represent essential components of mitosis. Cyclins B1 and B2 interact with Cdk1 (Draetta et al., Three mammalian B-type cyclins have been identified, 1989; Pines and Hunter, 1989) but can also bind to Cdk2 B1, B2 and B3 (Pines and Hunter, 1989; Chapman and (Aleem et al., 2005). It was described that Cdk1/cyclin B1

Oncogene Cyclins and Cdks in mammalian cell-cycle regulation A Satyanarayana and P Kaldis 2930 Table 2 Cdk and cyclin knockin mice Knockin gene Phenotypes rescued (or aggravated) Survival Meiotic Reference phenotype

cyclin D1cyclin E Rescued all the phenotypes associated with loss of cyclin D1 Viable Fertile (Geng et al., 1999) cyclin D1cyclin D2 Mostly rescued the defective retina and mammary gland phenotype, Viable Fertile (Carthon et al., 2005) and partially rescued neurological defects observed in cyclin D1 knockout Cyclin E T74A T393A Disruption of cyclin E regulation by Fbw7, increased cyclin E activity. Viable Fertile (Minella et al., 2008) Proliferative abnormalities including abnormal erythropoiesis, dysplasia and anemia. Delayed mammary gland involution Cdk4R24C Insensitive to INK4 inhibitors. Multiple tumors including endocrine tumors Viable Fertile (Sotillo et al., 2001) and hemangiosarcomas Cdk1Cdk2 Early embryonic lethality. Failed to rescue loss of Cdk1 BE2.5 Both male (Satyanarayana and female et al., 2008a) are sterile

complexes promote nuclear envelope breakdown, chro- temperature-sensitive Cdk1 mutation arrested in G2 at mosome condensation, and mitotic spindle assembly. the restrictive temperature (Th’ng et al., 1990; Yasuda Cytoplasmic Cdk1/cyclin B2 complexes are essential for et al., 1991). Comparable results were obtained by the mitotic reorganization of the Golgi apparatus microinjecting antibodies against Cdks (or their corre- (Draviam et al., 2001). Meanwhile it was described that sponding cyclin partners) or by using chemical Cdk cyclin B3 shares the properties of both A- and B-type inhibitors. These results suggested that Cdks perform cyclins (Gallant and Nigg, 1994; Nieduszynski et al., functions that are specific for one phase of the cell cycle. 2002). Cyclin B3 is mainly localized in the nucleus To study the validity of these conclusions in vivo, mice (Gallant and Nigg, 1994) and associates with Cdk2 lacking the catalytic partners of cyclins, Cdks, were (Nguyen et al., 2002). Cyclin B3 expression is restricted generated with the expectation of severe phenotypes to testes and fetal ovaries (Nguyen et al., 2002). To because Cdks can interact with several cyclins. There- explore the functions of B-type cyclins, B1 and B2 fore, loss of one Cdk would have a similar effect as knockout mice were generated. Deletion of cyclin B1 led knocking out a whole family of cyclins. to embryonic lethality before E10.5 (Brandeis et al., 1998). However, the exact reason for embryonic lethality was not reported. Several gene knockout mice have been Cdk4/Cdk6 reported to die around E10.5 due to severe defects in Mice lacking catalytic partners of the D-type cyclins, extra embryonic tissues (Parekh et al., 2004; Argraves Cdk4 and Cdk6, are viable and displayed defects in the and Drake, 2005). In this regard it would be interesting same tissues and organs as those affected by the lack of to investigate whether a similar defect exists in the cyclin D-type cyclins. The body size and size of several organs B1-deficient mice. In contrast to cyclin B1, cyclin B2 is decreased in Cdk4À/À mice due to a reduced number of knockout mice are viable and did not display any cells rather than a decrease in cell size. Cdk4À/À MEFs growth abnormalities, suggesting that cyclin B1 fully proliferate normally with a 4 h delay in S phase entry compensates for the loss of B2 (Figure 2). The precise from quiescence. This delay was indirectly linked to functions of cyclin B3 in mouse embryogenesis and inhibition of Cdk2 activity due to redistribution of p27. development is still unclear because cyclin B3 knockout Accordingly, combined deletion of Cdk4 and p27 mice have not yet been generated. Of all the different restored Cdk2 activity and normal S phase entry cyclin members (A, B, D, E), cyclin A2 and B1 appear to (Tsutsui et al., 1999). Although Cdk4 is dispensable be the most nonredundant cyclins, as deletion of either for proliferation of MEFs, it is essential for the A2 (E5.5) or B1 (E10.5) led to embryonic lethality, proliferation of certain endocrine cell types. Cdk4À/À suggesting an important role in development and/or cell- mice displayed similar defects as observed in cyclin cycle regulation (Figure 3). To dissect the specific D2À/À mice such as insulin-deficient diabetes due to a functions of cyclin B1 and A2 it will be essential to severe decrease in pancreatic b cells (Rane et al., 1999; generate conditional knockout mice to investigate their Tsutsui et al., 1999). The requirement of Cdk4 activity role during embryogenesis and in adult organs. for b-cell proliferation could be that Cdk6 might not be expressed in this cell type. Accordingly, mutations in negative regulators of Cdk4 activity lead to abnormal b-cell proliferation indicating a specific role of Cdk4 in Compensatory mechanisms among Cdks this cell type (Franklin et al., 2000; Latres et al., 2000). These defects could be due to a cell autonomous In human cell lines, inactivation of Cdk1, 2 or 3 resulted requirement of Cdk4 in these cell types as reexpression in cell-cycle arrest in one specific phase. For example, of endogenous Cdk4 in b cells and in the pituitary gland D145N À/À expression of Cdk2 caused a G1 arrest, whereas of Cdk4 mice restores cell proliferation and normo- D146N Cdk1 resulted in a G2/M arrest (van den Heuvel glycemic condition. However, reexpression of Cdk4 and Harlow, 1993). Similarly, cell lines carrying a does not rescue the small body size of Cdk4À/À mice,

Oncogene Cyclins and Cdks in mammalian cell-cycle regulation A Satyanarayana and P Kaldis 2931 controlling cell movement (Fahraeus and Lane, 1999). These observed differences in subcellular localization could explain a mechanism for regulating kinase activity, or may be indicative of the presence of a cytoplasmic role of Cdk6. However, Cdk4 and Cdk6 are widely expressed in embryos and their expression is similar in most of the cell types, suggesting that these two kinases might be distinct regarding their substrate specificity. Accordingly it was reported that Cdk4 is more efficient in phosphorylating Rb than Cdk6 and displays different residue selectivity (Takaki et al., 2005). This could explain the divergence in phenotype of corresponding Cdk4- and Cdk6-deficient mice. More- over, recent findings suggest a new role for Cdk6 in the differentiation of a variety of cell types. This function, which affects the transcription of genes involved in terminal differentiation, is not shared with Cdk4 and could be independent of Rb (Grossel and Hinds, 2006). Figure 4 Array of compensations among Cdks. Under normal All these evidences suggest that Cdk4 and Cdk6 may circumstances the functions of Cdks are limited to a particular phase of the cell cycle but in the absence of one or more Cdks, the have independent functions in the maintenance of the remaining Cdks will take over the functions of lost Cdks and delicate balance between cellular division and differ- faithfully control the cell cycle. For example, in the absence of entiation. In contrast to single knockouts, double Cdk2, Cdk4 and Cdk6, Cdk1 alone can drive the cell cycle by deletion of Cdk4 and Cdk6 led to embryonic lethality complexing with all the phase-specific cyclins. and mice die during the late stages of embryonic development due to severe anemia (Table 1). Cdk4À/ÀCdk6À/À double knockout mice survived a few days indicating that this phenotype is not because of longer than triple cyclin DÀ/À mice perhaps due to the endocrine defects. Therefore mammalian Cdk4 is not ability of D-type cyclins to interact with Cdk2 and/or only involved in controlling proliferation of specific cell Cdk1 in the absence of Cdk4 and Cdk6 (Malumbres types but might also play a crucial role in establishing et al., 2004). DKO embryos displayed normal organo- homeostasis. On the other hand mice lacking Cdk6 do genesis and most cell types proliferated normally. MEFs not display any defects in cell-cycle progression but lacking Cdk4 and Cdk6 proliferate with a lower female mice were reduced in size. Consistent with a efficiency but became immortal upon serial passage prescribed role for Cdk6 in blood cell differentiation, (Malumbres et al., 2004). Overall, none of the compo- these mice displayed defects in hematopoiesis including nents of the cyclin D-associated complexes are required decreased cellularity of the thymus, red blood cells and in early embryogenesis and in cell-cycle progression, lymphocytes (Malumbres et al., 2004). The mild suggesting the presence of alternative mechanisms to phenotype of this knockout mouse suggests that the initiate the cell cycle. functions of Cdk6 are mainly compensated by Cdk4, or possibly by Cdk2 (Figure 4), which was identified to be interacting with D-type cyclins in Cdk4/Cdk6 double Cdk2 knockout mice (Malumbres et al., 2004). The compensation of loss of Cdk4 by Cdk6 or vice versa is perhaps not very surprising because both proteins were identified to play a role in the early G1 phase by Cdk4À/ÀCdk6À/À double mutant mice binding to the same D-type cyclin partners. In contrast, On the basis of these studies it was hypothesized that it was widely anticipated that deletion of Cdk2 could Cdk4 and Cdk6 could simply compensate for each other lead to severe phenotypes because it was believed that (Figure 4). Cdk4 and Cdk6 share 71% amino-acid Cdk2 would be absolutely essential for the G1/S identity, and both partner with all three D-type cyclins transition of the cell cycle. This hypothesis was mainly to phosphorylate Rb. Nevertheless, a few differences based on in vitro studies where inhibition of Cdk2 led to have been described between Cdk4 and Cdk6 including cell-cycle arrest (van den Heuvel and Harlow, 1993). differences in timing or pattern of expression. Similarly, Surprisingly, Cdk2 knockout mice are viable and Cdk4 and Cdk6 also display distinct patterns of develop normally with a reduced body size (Berthet subcellular localization. For example in mouse astro- et al., 2003; Ortega et al., 2003). Cell proliferation is only cytes, Cdk6 was localized predominately to the cyto- slightly affected with a 4 h delay in S phase entry in plasm whereas Cdk4 localized almost exclusively to the Cdk2-deficient MEFs released from quiescence. Later it nucleus (Ericson et al., 2003). In T cells, Cdk6 was was demonstrated that Cdk1 binds to cyclin E in the detected in both the nucleus and cytoplasm with only absence of Cdk2 and promotes the G1/S transition the nuclear fraction active as Rb kinase (Mahony et al., (Aleem et al., 2005). It appears that Cdk2 is not 1998). Cdk6 was also localized to the ruffling edge of necessary for cell proliferation but it is not known spreading fibroblasts, suggesting a role for Cdk6 in whether Cdk2 is rate-limiting in certain cell types or at

Oncogene Cyclins and Cdks in mammalian cell-cycle regulation A Satyanarayana and P Kaldis 2932 certain stages of development. Recently it has been Cdk2 might be essential to keep Cdk1 in the cytoplasm demonstrated that neural progenitor cells of the until mitosis, and in the absence of Cdk2, Cdk1 is free to subventricular zone displayed impaired proliferation in localize to the nucleus prematurely. Although this is the absence of Cdk2 in the adult animals although they unlikely due to the fact that to date no NLS was do not require Cdk2 perinatally. Interestingly, in this identified in Cdk1 and it appears that Cdk1 localizes to cell type, Cdk4, but not Cdk1, compensates for Cdk2. the nucleus only after complexing with its cyclin This compensatory mechanism in the absence of Cdk2 partners. Moreover due to earlier localization of Cdk1 persisted only until postnatal day 15 involving increased to the nucleus, cells could face catastrophic conse- Cdk4 expression with concomitant Rb inactivation quences because this could lead to premature initiation (Jablonska et al., 2007). It appears that the concept of mitosis. Interestingly, premature initiation of mitosis derived from MEFs, Cdk1 compensates for Cdk2, might was not observed although Cdk1 is mainly present in the not be universal and there are certain cell types, which nucleus in the absence of Cdk2 (Satyanarayana et al., still require Cdk2 and have not developed a full 2008b). The molecular mechanism behind prevention of spectrum of compensatory mechanisms in response to premature initiation of mitosis is still unknown. One loss of Cdk2. Cdk2 was also proposed to function as a possibility is that the Cdk1/cyclin E complexes are master regulator of centrosome duplication (Hinchcliffe perhaps unable to phosphorylate the substrates that are et al., 1999; Meraldi et al., 1999; Matsumoto and essential for initiation of mitosis. Alternatively even Maller, 2004). Nevertheless, it was described recently though Cdk1 is physically present in the nucleus, that Cdk2 is not required for normal centrosome initiation components for mitosis may not yet be duplication, maturation and bipolar mitotic spindle available to be phosphoylated by Cdk1. formation. In contrast, Cdk2 deficiency completely abrogated aberrant centrosome duplication induced by a viral oncogene. This indicates that normal and Cdk2À/ÀCdk4À/À double knockout mice aberrant centrosome duplication have significantly In contrast to Cdk2 deletion, simultaneous deletion of different requirements for Cdk2, and it appears that in Cdk2/Cdk4 leads to embryonic lethality and embryos the absence of Cdk2 other kinases including cyclin die between E14.5 and E16.5 due to heart defects A-associated Cdk activity may compensate for the loss (Figure 3). Compound mutants of Cdk4 and Cdk2 leads of Cdk2 to allow normal centrosome duplication to an impairment of cell-cycle progression due to a (Duensing et al., 2006). progressive decline of Rb phosphorylation (Berthet et al., 2006). This suggests that Cdk6 and Cdk1 can regulate cell proliferation, but these two kinases might Compensation of Cdk2 functions by Cdk1 not phosphorylate Rb to full extent, leading to repres- Although the compensation of Cdk2 by Cdk1 in the sion of E2Fs and resulting eventually in decreased Cdk1 mitotic cell-cycle regulation appears to be a simple expression. The declining Cdk1 expression might act as mechanism, it left us with additional questions mainly a negative loop leading to proliferation defects. The fact due to the discrepancies in the properties of the two that more Cdk6 is bound to cyclin D1, in the absence of kinases. Cdk2 translocates to the nucleus as soon as it is Cdk4, is apparently not sufficient to compensate for the translated in complex with cyclin E and is present lack of Cdk2 and Cdk4. In contrast, when Cdk6 is mainly in the nucleus irrespective of the cell-cycle stage. deleted together with Cdk2, Cdk2À/ÀCdk6À/À mice are In contrast, Cdk1 localizes mainly to the cytoplasm but viable and exhibit similar phenotypes that were observed is found in the nucleus during mitosis after the break- in respective single knockouts (Malumbres et al., 2004), down of nuclear lamina (Moore et al., 1999; Satyanar- and have no impact on cell proliferation. On the basis of ayana et al., 2008b). If Cdk1 has to perform the mitotic these studies, the idea of a minimal-essential Cdk in cell-cycle functions of Cdk2, Cdk1 needs to be in the mammalian cells, like in yeast, started to emerge. nucleus at the G1/S transition. Another important Recently, it was described that MEFs lacking all three question arising in this context is whether Cdk1 will interphase Cdks (Cdk2, Cdk4 and Cdk6) can progress localize exclusively in the nucleus similar to Cdk2 to through different phases of cell-cycle with a delay perform both G1/S and G2/M transitions, or will still but complete cell division (Santamaria et al., 2007) shuttle between cytoplasm and nucleus? A recent study (Figure 5). No apparent changes in the expression of described that Cdk1 localizes to the nucleus earlier in the Cdk1, Cdk7, Cdk9 as well as cyclins D1, D2, E1, A2 and absence of Cdk2 and mainly resides in the nucleus B1 were detected in the triple knockout cells. In (Satyanarayana et al., 2008b). The molecular mechan- contrast, a reduction in the p27 levels was detected ism behind Cdk1’s premature localization is puzzling (Santamaria et al., 2007). In these cells, Cdk1 binds to especially due to the fact that Cdk1 not only binds to all cyclins resulting in phosphorylation of the retino- cyclin B and cyclin A but also to cyclin E. One blastoma protein Rb. However, they become immortal possibility could be that a subpopulation of Cdk1 on continuous passage when grown at low oxygen. The localizes to the nucleus by complexing with cyclin E and triple knockout embryos survived up to E13.5 and died this complex functions in the G1/S transition. Later, due to heart and severe hematopoietic defects (Santa- during mitosis another subset of Cdk1 binds to cyclin B maria et al., 2007). Even though it appears that Cdk1 and localizes to the nucleus to execute mitosis. At this can drive cell-cycle progression alone by binding to point, it can also be speculated that the kinase activity of all phase-specific cyclins, it cannot be excluded the

Oncogene Cyclins and Cdks in mammalian cell-cycle regulation A Satyanarayana and P Kaldis 2933 copies of Cdk1 by Cdk2 leads to early embryonic lethality similar to deletion of Cdk1 (Satyanarayana et al., 2008a; Table 2). It indicates that Cdk2 cannot substitute for Cdk1 functions even when it is expressed directly from the Cdk1 locus. This result suggests that there are intrinsic differences between Cdk1 and Cdk2 despite the fact that they bind to the same cyclins and inhibitors. Although differences in substrate specificity cannot be excluded, most likely Cdk2 cannot compensate because of its nuclear localiza- tion compared to the cytoplasmic localization of Cdk1. Figure 5 Mammalian cell-cycle regulation Now and Then. According to classical model of cell-cycle regulation Cdk4, Cdk6, One could speculate that Cdk1 fulfills essential functions Cdk2 and Cdk1 complex with phase-specific cyclins at different in the cytoplasm, which would be quite contrary to the stages of cell cycle to coordinate initiation and progression of cell current view. Future experiments will have to explore cycle. Recent evidences suggest that most of the Cdks and cyclins this possibility. are dispensable for mammalian cell-cycle regulation and Cdk1 At the same time, we also analysed how efficiently alone by complexing with minimum number of cyclins can drive cells through different phases of cell cycle. Although it appears that Cdk2 expressed from the Cdk1 locus could perform its Cdk1 alone can drive the cell-cycle regulation it cannot be excluded meiotic function in germ cells. Analysis of Cdk2À/À that other Cdks such as Cdk3, Cdk7 and Cdk11 might participate Cdk1 þ /Cdk2KI mice where one copy of Cdk2 and one copy more actively in cell-cycle regulation in the absence of Cdk2, Cdk4 of Cdk1 are expressed from the Cdk1 locus and the and Cdk6. Cdk2 gene was deleted in the endogenous Cdk2 locus, revealed that both Cdk2À/ÀCdk1 þ /Cdk2KI male and female mice are sterile, similar to Cdk2À/À mice, although they express functional Cdk2 protein from participation of other Cdk/cyclin complexes such Cdk1 locus. This indicates that the genetic relocation as Cdk3/cyclin C, Cdk7/cyclin H or Cdk11/cyclin L. of Cdk2 from the Cdk2 to the Cdk1 locus abolished In the absence of Cdk2, Cdk4 and Cdk6 there is a Cdk2’s own meiotic function (Satyanarayana et al., possibility that these Cdk/cyclin complexes might 2008a). It is still puzzling why Cdk2 is unable to perform play more active roles (Figure 5). On the basis of the its meiotic function when expressed from Cdk1 locus. above result, one could expect that disruption of the One possibility could be that the timing of Cdk2 Cdk1 gene could lead to early embryonic lethality. expression might be crucial to participate fully in In accordance with this hypothesis, it was observed meiosis. that deletion of Cdk1 (our unpublished results) or a The studies in the knockout mouse models of cell- gene trap mutation in the Cdk1 gene (Santamaria et al., cycle regulators clearly produced contrasting results 2007) led to early embryonic lethality and the embryos compared to in vitro studies, where inhibition of Cdks fail to reach even the blastocyst stage in the absence of by drugs, antibodies and RNAi led to dramatic effects Cdk1. These results indicate that Cdk1 can drive the on cell-cycle progression. Several possible reasons could mammalian cell cycle alone similar to yeast Cdk1 explain why the wide range of compensatory mechan- (Figure 4). isms observed in vivo was not evident in vitro. One reason could be that when Cdks are inhibited in vitro, they do not make their cyclin subunits available for Replacement of Cdk2 by Cdk1 other Cdks to compensate. In contrast, in in vivo models, So far all the studies were focused on identifying the complete absence of specific Cdks, cyclin subunits compensatory mechanisms for the loss of Cdk2, Cdk4 become available to form novel, compensatory Cdk and Cdk6. It was not explored if any of the Cdks can complexes. In line with this hypothesis a recent study compensate for the loss of Cdk1. However, it was illustrates that acute elimination of a protein’s function described that deletion of Cdk1 leads to early embryonic in somatic cells may have consequences different than in lethality (Santamaria et al., 2007; our unpublished the mouse knockout setting, where there may be results), and no other Cdk, including Cdk2, can compensation by related proteins during development compensate for the loss of Cdk1 under normal (Sage et al., 2003). On the basis of these results it conditions. However, the inability of Cdk2 to take appears that both in vitro and in vivo systems have their over the functions of Cdk1 could be due to (1) the own advantages. The in vivo systems help us to differences in the timing of expression of Cdk1 and understand the effect of complete loss of a protein and Cdk2 during different phases of cell cycle, (2) substrate the concomitant compensatory mechanisms during specificity or (3) subcellular localization. It is interesting development and disease. On the other hand the to explore whether Cdk2 acquires some of the properties in vitro systems, where the acute inhibition of proteins of Cdk1, if Cdk2 is expressed directly from the and the resulting effects on the function of the Cdk1 locus. Therefore, we generated a knockin proteins, help us to develop drugs for different diseases mouse in which Cdk2 was knocked into the Cdk1 because some of the drugs are designed to inhibit a locus (Cdk1Cdk2KI) and studied whether Cdk2 can rescue particular protein or a group of proteins for a limited the loss of Cdk1 in vivo. Substitution of both the amount of time.

Oncogene Cyclins and Cdks in mammalian cell-cycle regulation A Satyanarayana and P Kaldis 2934 Cdks and cyclins in meiosis because of a cell autonomous requirement of Cdk4 in these cell types as reexpression of Cdk4 restores cell The specific functions of cyclins and Cdks in meiosis proliferation and results in fertile animals. In contrast to have become more apparent with the generation of Cdk4À/À mice, Cdk6 male and female mice are fertile and knockout mouse models. Although Cdk and cyclin no meiotic defects were reported (Malumbres et al., knockout mouse models were extensively used to study 2004). Although Cdk2 was identified to be dispensable embryogenesis, development and mitotic cell-cycle for mitotic cell cycle, it was discovered that Cdk2 is regulation, much less work was done in these mouse essential for meiosis and both male and female Cdk2À/À models to understand the role of individual cyclins and mice are sterile with 100% penetrance (Berthet et al., Cdks in meiosis. These studies revealed that in contrast 2003; Ortega et al., 2003). The germ cells of males do not to the effect of loss of individual cyclins and Cdks on progress beyond the pachytene stage of prophase I. At mitotic cell cycle, meiosis was much more affected. this stage, primary spermatocytes undergo apoptosis Cyclin D1 and D3 single knockout mice are fertile and due to a consequence of defective synaptonemal no detectable meiotic defects were identified in both complex formation (Ortega et al., 2003). However, males and females. In contrast, cyclin D2-deficient oocytes progress normally through pachytene, engage females are sterile (Kozar et al., 2004). When subjected in normal synapsis, progress further to the late diplotene to superovulation, D2À/À females failed to release or dictyate stage and die by apoptosis. The meiotic oocytes, suggesting ovarian failure as the cause of phenotype of Cdk2À/À mice suggests that Cdk2 is infertility. Although the number of ovarian follicles and essential for germ cell development beyond meiotic oocytes was normal in D2À/À females, the number of prophase I in both males and females and Cdk1 cannot somatic granulosa cells surrounding each oocyte was compensate for Cdk2 in germ cells. The assumption that reduced, indicating defective proliferation of ovarian Cdk1 cannot compensate for Cdk2’s meiotic function is granulosa cells. Why do D1 and D3 fail to compensate further strengthened by a recent study, where genetic for D2 function in these cell types is not clearly substitution of Cdk1 by Cdk2 leads to the loss of Cdk2’s understood. In contrast, the D2À/À males are fertile own meiotic function (Satyanarayana et al., 2008a). though they showed hypoplastic testes (Sicinski et al., Both male and female Cdk2À/ÀCdk1 þ /Cdk2KI mice are 1996). Cyclin E1-deficient male and female mice are sterile, similar to Cdk2À/À mice, even though there is fertile but E2 knockout males displayed testicular abundant expression of knockin Cdk2 from the Cdk1 abnormalities, testicular atrophy and reduced fertility locus in the testes. In adult Cdk2À/ÀCdk1 þ /Cdk2KI mice, whereas cyclin E2À/À females are normal and fertile testes and ovaries are atrophic. The seminiferous tubules (Geng et al., 2003). Testes of E2À/À males displayed are smaller than normal with a substantial depletion of thinned walls of the seminiferous tubules with a germ cells, similar to Cdk2À/À mice. The atrophic testes reduction in spermatogenetic cells. Abnormal meiotic lacked round spermatids and displayed extensive apop- figures in the spermatocyte layers and multinuclear giant tosis of germ cells, indicating that although knockin cells in seminiferous epithelium were also observed. This Cdk2 was expressed, it was unable to complete the clearly shows that E1 and E2 have nonredundant pachytene stage and instead the cells underwent functions in meiosis unlike mitosis. Similar to the apoptosis (Satyanarayana et al., 2008a). It appears that E2-deficient females, cyclin A1 females are fertile but the timing of expression of Cdk2 is crucial for its meiotic the males are completely sterile with spermatogenesis function(s). These results suggest the existence of a arresting before the first meiotic division in males. certain time window for the requirement of Cdk2. When Spermatocytes progress normally to the transition from Cdk2 is not expressed at that particular time point, the prophase to metaphase but stop just before metaphase I. cells fail to complete meiosis even though Cdk2 is This arrest leads to extensive germ cell apoptosis and expressed thereafter. This indicates that the genetic locus sterility. Cyclin A1-deficient spermatocytes displayed and timing of Cdk2 expression determine the meiotic impaired Cdk1 activation at the end of the meiotic functions of Cdk2. Whether Cdk1 has any specific role prophase. As a result, the first meiotic division failed to in meiosis is still not known due to early embryonic be induced (Liu et al., 1998). However, cyclin B2 lethality of Cdk1 knockout mouse. Specific deletion of knockout males and females are fertile, suggesting that Cdk1 in germ cells of mice will disclose the functions of there is no obvious meiotic defect (Brandeis et al., 1998). Cdk1 in meiosis in future studies. These studies disclosed that cyclin D2, E2 and A1 have specific functions in meiosis. Whether cyclin A2 and B1 have any function in meiosis is still unclear because deletion of either of the cyclins leads to early embryonic Other Cdks and cyclins lethality (Table 1). The specific role of cyclin partners, Cdks, was also Although the Cdks and cyclins discussed above studied in meiosis in the Cdk-deficient mice. Cdk4À/À attracted major attention due to their crucial roles in females are sterile due to a decrease in prolactin cell-cycle regulation, several other Cdks and cyclins have producing pituitary lactotrophs (Rane et al., 1999; also been discovered. These include Cdk3, Cdk5, Cdk7, Tsutsui et al., 1999). These mice also display decreased Cdk8, Cdk9, Cdk10, Cdk11 and cyclins C, F, G, H, K, male fertility due to defective spermatogenesis and lower L, T1, T2a, T2b. These Cdk/cyclin complexes have numbers of Leydig cells. These defects are mainly diverse cellular functions, including cell-cycle regulation,

Oncogene Cyclins and Cdks in mammalian cell-cycle regulation A Satyanarayana and P Kaldis 2935 neuronal development and regulation of transcription. (CAK) complex, responsible for the activating phos- The functions of Cdk8/cyclin E and Cdk9/cyclin T, phorylation of Cdk1, Cdk2, Cdk4 and Cdk6, which is Cdk9/cyclin K and Cdk10 in transcriptional regulation essential for cell-cycle progression (Fisher and Morgan, were extensively reviewed (Akoulitchev et al., 2000; 1994; Fisher, 2005). CAK is also part of the general Sano and Schneider, 2003; Fisher, 2005; Wang and transcription factor TFIIH (Feaver et al., 1994; Fisher Fischer, 2008). Cdk5 was identified to have minor roles and Morgan, 1996), and CAK phosphorylates the RNA in cell-cycle regulation and functions mostly in neuronal polymerase II large subunit C-terminal domain (CTD) development (Dhavan and Tsai, 2001). Cdk5 is ubiqui- (Lu et al., 1992). CTD phosphorylation by TFIIH is tously expressed in neurons and several lines of evidence involved in promoter clearance and in progression of indicate that Cdk5 contributes to normal brain devel- transcription from the preinitiation to the initiation opment where it plays a role in axon guidance and stage (Goodrich and Tjian, 1994). Genetic studies in neuronal migration (Dhavan et al., 2002). Cdk5 was Drosophila, where inactivation of a temperature sensi- identified to play an important role in neurotransmis- tive Cdk7 mutant (cdk7P140S) prevented activation of sion and synaptic plasticity and thus functions in Cdk1 by T-loop phosphorylation and caused embryonic cognitive functions such as learning and memory and larval lethality by blocking mitosis. In contrast, (Fischer et al., 2005; Cheung et al., 2006). Aberrant adult flies bearing the temperature-sensitive allele are Cdk5 signaling has been proposed to be a common viable and are phenotypically nearly normal at the contributor to a variety of central nervous system restrictive temperature, indicating that no impairment of disorders (Cruz and Tsai, 2004; Qu et al., 2007). As a transcription has occurred (Larochelle et al., 1998). This result of Cdk5’s crucial role in neuronal development, suggests that Cdk7 may not be crucial for the TFIIH Cdk5À/À mice exhibited severe defects in the central function. Whether deletion of mammalian Cdk7 can nervous system and 60% of the mice died in utero and have similar effects or any of the other Cdks substitute the remaining 40% died immediately after birth for Cdk7 in its absence is not known because Cdk7 (Ohshima et al., 1996). The brain defects in Cdk5À/À knockout mice have not been generated. Nevertheless, mice include lack of cortical laminar structure and disruption of the murine Mat1 gene leads to peri- cerebellar foliation. Moreover, the large neurons in the implantation lethality (Rossi et al., 2001). Mat1À/À brain stem and in the spinal cord displayed chromato- trophoblast giant cells exhibited a rapid arrest in lytic changes, indicating that Cdk5 is important for endoreduplication, which was characterized by an brain development and neuronal differentiation and inability to enter S phase (Rossi et al., 2001). Whether might also play critical roles in neuronal cytoskeleton deletion of Cdk7 leads to a similar phenotype needs to structure and organization (Ohshima et al., 1996). be resolved.

Cdk3 Cdk11 Cdk3/cyclin C, Cdk7/cyclin H and Cdk11/cyclin L Another Cdk which has a dual role in cell proliferation appear to function in cell-cycle regulation. In G0 cells, and transcriptional control is Cdk11. The mouse Cdk11 in response to growth factors, Cdk3/cyclin C complexes is present in two different isoforms, Cdk11p110 and become activated and phosphorylate Rb and also the Cdk11p58, expressed from a single gene locus (cdc2l). other Rb family members p107 and p130. Cdk3/cyclin Cdk11p110 associates with RNAP II, cyclin L, pre- C-dependent phosphorylation of Rb family proteins is mRNA splicing proteins RNPS1 and 9G8, several completed in early and late G1 by Cdk4/cyclin D and transcriptional elongation factors and is mainly involved Cdk2/cyclin E complexes (Ren and Rollins, 2004). In in regulation of RNA transcription and processing support to the above studies it was observed that (Loyer et al., 1998; Hu et al., 2003). On the other hand, dominant-negative forms of human Cdk3 block cells in Cdk11p58 is mitosis specific and expressed only during À/À G1 phase, and this arrest is not overcome by over- mitosis (Sachs, 2000). Cdk11p110/p58 mice died early expression of Cdk2 (van den Heuvel and Harlow, 1993). during embryogenesis (E3.5). The blastocysts die Furthermore, Cdk3/cyclin E complexes can promote S between E3.5 and E4.0 due to apoptosis as a result of phase entry in quiescent cells as efficiently as Cdk2/ proliferative defects and mitotic arrest. It appears that cyclin E complexes (Sage, 2004). However, most mouse Cdk11 is essential for proper cellular proliferation and laboratory strains harbor a point mutation in the Cdk3 cell-cycle progression during early embryonic develop- gene that converts a conserved tryptophan (Trp187) to a ment (Li et al., 2004). It can though not be excluded that premature termination codon but did not display any in addition to cell-cycle defects, Cdk11p110/p58À/À mice defects during development or cell-cycle kinetics indi- also have extensive defects in transcriptional regulation. cating a redundancy with other Cdk family members such as Cdk2 and Cdk4 (Ye et al., 2001). Cyclin F In addition to the above discussed Cdks and cyclins, Cdk7 another cyclin which also function in cell-cycle regula- Cdk7 was identified to have a dual role and functions in tion is cyclin F. Cyclin F very closely resembles cyclin A cell-cycle regulation as well as transcriptional control. in amino-acid sequence. Similar to other cyclins, the Cdk7 together with cyclin H and the assembly factor expression of cyclin F strictly occurs in a cell-cycle- MAT1 (me´nage a trios) forms the Cdk activating kinase regulated fashion. Its expression begins in S phase,

Oncogene Cyclins and Cdks in mammalian cell-cycle regulation A Satyanarayana and P Kaldis 2936 peaks in G2 and disappears as cells enter mitosis (Bai cycle regulation and development were mainly revealed et al., 1994). The fact that cyclin F also contains an F from other Cdk knockout mouse models. Cdk1 deletion box and can form an SCF (Skp1-Cul1/Cdc53-F-box) leads to early embryonic lethality (BE2); as a result, the complex in vivo further suggests that it may also specific functions of Cdk1 in mouse development have function in proteolysis (Bai et al., 1996). Cyclin FÀ/À not been fully studied. Generation of Cdk1 conditional mice displayed several developmental abnormalities due knockout mice will allow us to explore several important to defects in yolk sac and chorioallantoic placentation functions of Cdk1. Could the deletion of Cdk1 in and die around E10.5. Conditional deletion of cyclin F conditional Cdk1 MEFs lead to S phase, G2/M, general in several specific tissues revealed that it was not cell-cycle arrest (a senescence-like phenotype) and/or required for development and function of a number apoptosis? So far it appears that Cdk2 is essential in of different embryonic and adult tissues. In contrast, meiosis. However, by specifically deleting Cdk1 in germ cyclin FÀ/À MEFs exhibited cell-cycle defects, including cells, the functions of Cdk1 in meiosis could be reduced population doubling time and a delay in cell- identified. By specifically deleting Cdk1 in certain cell cycle reentry from quiescence, indicating that cyclin F types by specific Cre crossings, it is possible to identify plays a role in cell-cycle regulation (Tetzlaff et al., 2004). cell types, if any, that operate Cdk1-independent cell- cycle regulation. Similarly, by crossing Cdk1 conditional knockout mice with transgenic mouse tumor models and Concluding remarks inducible Cre lines, it is possible to study the effect of Cdk1 deletion on tumor initiation and progression. This Through the characterization of knockout mouse would provide valuable information for the develop- models of cell-cycle regulators, our knowledge of cell- ment and testing of Cdk1 inhibitors in human cancers. cycle regulation has greatly improved. Extensive ana- The most important question is whether the information lyses of these mouse models revealed that most of the derived from these studies is sufficient to specifically Cdks and cyclins, originally thought essential for cell- target cancer therapy. Would inhibition of Cdk1 alone cycle regulation, are in fact largely dispensable. Cyclin be sufficient to prevent cancerous growth? A2 and B1 emerged as the most nonredundant cyclins whereas Cdk1 emerged as the master regulator of mammalian cell-cycle regulation and Cdk1 alone by complexing with all the cyclins can drive the cell cycle Conflict of interest similar than in yeast. Other cyclins and Cdks have specific functions in certain cell types and these cells The authors declare no conflict of interest. have not developed a full spectrum of compensatory mechanisms. Although the finding that Cdk2 is dis- Acknowledgements pensable for cell-cycle progression is astonishing, the finding that Cdk2 loses its own meiotic function when We thank Kaldis lab members for discussions and support. expressed from Cdk1 locus is even more puzzling. It This research was supported by A*STAR of Singapore (PK) appears that Cdk2 and its regulation is vital for meiosis. and the Intramural Research Program of the NIH, National However, the functions of Cdk1 in the mammalian cell- Cancer Institute, Center for Cancer Research (AS).

References

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