Combined loss of Cdk2 and Cdk4 results in embryonic lethality and Rb hypophosphorylation

Mouse knockouts of Cdk2 and Cdk4 have demonstrated that, individually, these genes are not essential for viability. To investigate whether there is functional redundancy, we have generated double knockout (DKO) mice. Cdk2-/- Cdk4-/- DKOs die during embryogenesis around E15 as a result of heart defects. We observed a gradual decrease of Retinoblastoma protein (Rb) phosphorylation and reduced expression of E2F-target genes, like Cdc2 and cyclin A2, during embryogenesis and in embryonic fibroblasts (MEFs). DKO MEFs are characterized by a decreased proliferation rate, impaired S phase entry, and premature senescence. HPV-E7-mediated inactivation of Rb restored normal expression of E2F-inducible genes, senescence, and proliferation in DKO MEFs. In contrast, loss of p27 did not rescue Cdk2-/- Cdk4-/- phenotypes. Our results demonstrate that Cdk2 and Cdk4 cooperate to phosphorylate Rb in vivo and to couple the G1/S phase transition to mitosis via E2F-dependent regulation of gene expression.     

Cdc2-cyclin E complexes regulate the G1/S phase transition

The cyclin-dependent kinase inhibitor p27(Kip1) is known as a negative regulator of cell-cycle progression and as a tumour suppressor. Cdk2 is the main target of p27 (refs 2, 3) and therefore we hypothesized that loss of Cdk2 activity should modify the p27(-/-) mouse phenotype. Here, we show that although p27(-/-) Cdk2(-/-) mice developed ovary tumours and tumours in the anterior lobe of the pituitary, we failed to detect any functional complementation in p27(-/-) Cdk2(-/-) double-knockout mice, indicating a parallel pathway regulated by p27. We observed elevated levels of S phase and mitosis in tissues of p27(-/-) Cdk2(-/-) mice concomitantly with elevated Cdc2 activity in p27(-/-) Cdk2(-/-) extracts. p27 binds to Cdc2, cyclin B1, cyclin A2, or suc1 complexes in wild-type and Cdk2(-/-) extracts. In addition, cyclin E binds to and activates Cdc2. Our in vivo results provide strong evidence that Cdc2 may compensate the loss of Cdk2 function.     

CIB1 is essential for mouse spermatogenesis

CIB1 is a 22-kDa calcium binding, regulatory protein with approximately 50% homology to calmodulin and calcineurin B. CIB1 is widely expressed and binds to a number of effectors, such as integrin alphaIIb, PAK1, and polo-like kinases, in different tissues. However, the in vivo functions of CIB1 are not well understood. To elucidate the function of CIB1 in whole animals, we used homologous recombination in embryonic stem cells to generate Cib1(-/-) mice. Although Cib1(-/-) mice grow normally, the males are sterile due to disruption of the haploid phase of spermatogenesis. This is associated with reduced testis size and numbers of germ cells in seminiferous tubules, increased germ cell apoptosis, and the loss of elongated spermatids and sperm. Cib1(-/-) testes also show increased mRNA and protein expression of the cell cycle regulator Cdc2/Cdk1. In addition, mouse embryonic fibroblasts (MEFs) derived from Cib1(-/-) mice exhibit a much slower growth rate compared to Cib1(+/+) MEFs, suggesting that CIB1 regulates the cell cycle, differentiation of spermatogenic germ cells, and/or differentiation of supporting Sertoli cells.     

PDK1 regulates cell proliferation and cell cycle progression through control of cyclin D1 and p27Kip1 expression

PDK1 (3-phosphoinositide-dependent protein kinase 1) is a key mediator of signaling by phosphoinositide 3-kinase. To gain insight into the physiological importance of PDK1 in cell proliferation and cell cycle control, we established immortalized mouse embryonic fibroblasts (MEFs) from mice homozygous for a "floxed" allele of Pdk1 and from wild-type mice. Introduction of Cre recombinase by retrovirus-mediated gene transfer resulted in the depletion of PDK1 in Pdk1(lox/lox) MEFs but not in Pdk1(+/+) MEFs. The insulin-like growth factor-1-induced phosphorylation of various downstream effectors of PDK1, including Akt, glycogen synthase kinase 3, ribosomal protein S6, and p70 S6 kinase, was markedly inhibited in the PDK1-depleted (Pdk1-KO) MEFs. The rate of serum-induced cell proliferation was reduced; progression of the cell cycle from the G(0)-G(1) phase to the S phase was delayed, and cell cycle progression at G(2)-M phase was impaired in Pdk1-KO MEFs. These cells also manifested an increased level of p27(Kip1) expression and a reduced level of cyclin D1 expression during cell cycle progression. The defect in cell cycle progression from the G(0)-G(1) to the S phase in Pdk1-KO MEFs was rescued by forced expression of cyclin D1, whereas rescue of the defect in G(2)-M progression in these cells required both overexpression of cyclin D1 and depletion of p27(Kip1) by RNA interference. These data indicate that PDK1 plays an important role in cell proliferation and cell cycle progression by controlling the expression of both cyclin D1 and p27(Kip1).     

Two Distinct Controls of Mitotic Cdk1/Cyclin B1 Activity Requisite for Cell Growth Prior to Cell Division

Cell growth prior to cell division is restricted by the activity of cyclin-dependent kinase 1 (Cdk1)/cyclin B1 complexes. Recently, we identified that the death-effector domain (DED) containing protein, DEDD, acts as a novel inhibitor of mitotic Cdk1/cyclin B1, influencing cell size. Like cyclin B1, DEDD protein levels specifically peak during the G2/M phase. In the nucleus, DEDD associates with Cdk1/cyclin B1 complexes, via direct binding to cyclin B1, and reduces their function. In agreement, kinase activity of nuclear Cdk1/cyclin B1 in DEDD-null (DEDD-/-) embryonic fibroblasts is increased compared to that in DEDD+/+ cells. This accelerates mitotic progression in DEDD-/- cells, with a shortened G2/M phase, reduced rRNA, and diminished cell volume. Likewise, DEDD-/- mice show decreased body and organ weights relative to DEDD+/+ mice. Interestingly, the DED domain is not involved in the association of DEDD with Cdk1/cyclin B1, but is indispensable for the cell sizing function of DEDD. Together, in addition to the well-established machinery for activation of Cdk1 through dephosphorylation of its inhibitory-residues, we propose a novel mechanism for impeditive regulation of mitotic Cdk1/cyclin B1 mediated by DEDD within the nucleus, which allows sufficient cell growth prior to cell division.     

Unmasking the Redundancy Between Cdk1 and Cdk2 at G2 Phase in Human Cancer Cell Lines

A series of studies published in 2003 has challenged the essentiality of Cdk2. A recently published work indicates that cyclin E-Cdk1 compensates for Cdk2’s function at G1/S transition in Cdk2-/- Mefs. In this study, we uncovered a redundant mechanism between Cdk1 and Cdk2 at G2 in multiple cancer cell lines. When either Cdk2 or Cdk1 is ablated using RNAi, there were complex shifts of cyclin A towards its reciprocal partner, i.e., when Cdk2 is ablated, cyclin A redistributes to Cdk1; when Cdk1 is ablated, cyclin A forms more abundant complexes with Cdk2. Further, cyclin B redistributes to Cdk2 upon Cdk1 knockdown. These redistributions bring about increased kinase activities of corresponding complexes. Elimination of the compensatory mechanism by knockdown of both Cdk1 and Cdk2 using RNAi reveals phenotypes at G2 phase. The results suggest that the redistributed complexes contribute to the cyclin B-Cdk1 activation when either Cdk1 or Cdk2 alone is ablated and this redundancy masks Cdk2’s role when Cdk2 is singly ablated. It is also worth noting that the predominant G2 arrest described here, unlike those Cdk1-Cdk2 double ablated Mefs, raises a question of whether different Cdk activities are required for G1/S or G2/M progression in normal vs. cancer cells.     

Genetic reevaluation of the role of F-box proteins in cyclin D1 degradation

D-type cyclins play a pivotal role in G(1)-S progression of the cell cycle, and their expression is frequently deregulated in cancer. Cyclin D1 has a half-life of only ~30 min as a result of its ubiquitylation and proteasomal degradation, with various F-box proteins, including Fbxo4, Fbxw8, Skp2, and Fbxo31, having been found to contribute to its ubiquitylation. We have now generated Fbxo4-deficient mice and found no abnormalities in these animals. Cyclin D1 accumulation was thus not observed in Fbxo4(-/-) mouse tissues. The half-life of cyclin D1 in mouse embryonic fibroblasts (MEFs) prepared from Fbxo4(-/-), Fbxw8(-/-), and Fbxo4(-/-); Fbxw8(-/-) mice also did not differ from that in wild-type MEFs. Additional depletion of Skp2 and Fbxo31 in Fbxo4(-/-); Fbxw8(-/-) MEFs by RNA interference did not affect cyclin D1 stability. Although Fbxo31 depletion in MEFs increased cyclin D1 abundance, this effect appeared attributable to upregulation of cyclin D1 mRNA. Furthermore, abrogation of the function of the Skp1-Cul1-F-box protein (SCF) complex or the anaphase-promoting complex/cyclosome (APC/C) complexes did not alter the half-life of cyclin D1, whereas cyclin D1 degradation was dependent largely on proteasome activity. Our genetic analyses thus do not support a role for any of the four F-box proteins examined in cyclin D1 degradation during normal cell cycle progression. They suggest the existence of other ubiquitin ligases that target cyclin D1 for proteolysis.     

Cyclin A2/cyclin-dependent kinase 1-dependent phosphorylation of Top2a is required for S phase entry during retinal development in zebrafish

Cyclin-dependent kinase 1 (CDK1) plays an essential role in cell cycle regulation.However,as mouse Cdk1embryos die early,the role of CDK1 in regulating the cell cycle and embryo development remains unclear.Here,we showed that zebrafish cdk1~(-/-)embryos exhibit severe microphthalmia accompanied by multiple defects in S phase entry,M phase progression,and cell differentiation but not in interkinetic nuclear migration.We identified Top2a as a potential downstream target and cyclin A2 and cyclin B1 as partners of Cdk1 in cell cycle regulation via an in silico analysis.While depletion of either cyclin A2 or Top2a led to the decreased S phase entry in zebrafish retinal cells,the depletion of cyclin B1 led to M phase arrest.Moreover,phosphorylation of Top2a at serine 1213 (S1213) was nearly abolished in both cdk1 and ccna2mutants,but not in ccnb1 mutants.Furthermore,overexpression of TOP2A~(S1213D),the phosphomimetic form of human TOP2A,rescued S phase entry and alleviated the microphthalmia defects in both cdk1~(-/-)and ccna2~(-/-)embryos.Taken together,our data suggest that Cdk1 interacts with cyclin A2 to regulate S phase entry partially through Top2a phosphorylation and interacts with cyclin B1 to regulate M phase progression.     

Initiation of human DNA replication in vitro using nuclei from cells arrested at an initiation-competent state

Initiation of human DNA replication is investigated in vitro, using initiation-competent nuclei isolated from cells arrested in late G(1) phase by a 24-h treatment with 0.5 mm mimosine (Krude, T. (1999) Exp. Cell Res. 247, 148-159). Nuclei isolated from mimosine-arrested HeLa cells initiate semiconservative DNA replication upon incubation in cytosolic extracts from proliferating human cells. Initiation occurs in the absence and presence of a nuclear membrane. The cyclin-dependent kinase (Cdk) inhibitors roscovitine and olomoucine inhibit initiation of DNA replication, indicating a dependence of initiation on Cdk activity. Cell fractionation shows that cyclins A, E, and Cdk2 are bound to nuclei from mimosine-arrested cells. Exogenously added human cyclin A.Cdk2 and cyclin E.Cdk2 complexes, but not cyclin B1/Cdk1 or cyclin D2/Cdk6, can overcome inhibition of initiation by roscovitine in vitro. Depleting Cdk2 from cytosolic extract does not prevent initiation, demonstrating that cyclin.Cdk2 complexes are not required in the soluble extract, but are provided by the nuclei. Initiation depends further on an essential and soluble activity present in cytosolic extracts from proliferating cells, but not from mimosine-arrested cells, acting together with nuclear cyclin/Cdk2 activity.     

Is It Easy to Stop RNA Polymerase?

Cdk2 has been viewed as a key cell cycle regulator that is essential for S phase progression. The recent discovery that Cdk2 is not required for cell proliferation in mice now shows that other factors must be able to replace Cdk2 in stimulating DNA replication. Experiments performed in Xenopus egg extracts identify the mitotic protein kinases Cdk1/Cyclin B and Cdk1/Cyclin A as likely candidates. These observations raise the intriguing possibility that Cdk1 normally participates in genome duplication in wild type cells.     

Cdk1: unsung hero of S phase?

Cdk2 has been viewed as a key cell cycle regulator that is essential for S phase progression. The recent discovery that Cdk2 is not required for cell proliferation in mice now shows that other factors must be able to replace Cdk2 in stimulating DNA replication. Experiments performed in Xenopus egg extracts identify the mitotic protein kinases Cdk1/Cyclin B and Cdk1/Cyclin A as likely candidates. These observations raise the intriguing possibility that Cdk1 normally participates in genome duplication in wild type cells.     

Functional Interaction between the Bovine Papillomavirus Virus Type 1 Replicative Helicase E1 and Cyclin E-Cdk2

We have found that the replicative helicase E1 of bovine papillomavirus type 1 (BPV-1) interacts with a key cell cycle regulator of S phase, the cyclin E-Cdk2 kinase. The E1 helicase, which interacts with cyclin E and not with Cdk2, presents the highest affinity for catalytically active kinase complexes. In addition, E1, cyclin E, and Cdk2 expressed in Xenopus egg extracts are quantitatively coimmunoprecipitated from crude extracts by either anti-Cdk2 or anti-E1 antibodies. E1 protein is also a substrate of the cyclin E-Cdk2 kinase in vitro. Using the viral components required for in vitro BPV-1 replication and free-membrane cytosol from Xenopus eggs, we show that efficient replication of BPV plasmids is dependent on the addition of E1-cyclin E-Cdk2 complexes. Thus, the BPV initiator of replication and cyclin E-Cdk2 are likely to function together as a protein complex which may be the key to the cell cycle regulation of papillomavirus replication.     

Cyclin F disruption compromises placental development and affects normal cell cycle execution

Human cyclin F was originally isolated as a cDNA capable of suppressing the temperature sensitivity of a Saccharomyces cerevisiae cdc4-1 mutant. Its tightly regulated expression and high conservation in the evolutionary progression from amphibians to mammals suggest that it coordinates the timing of a critical cell cycle event. The fact that it contains an F box and can form an SCF (Skp1-Cul1/Cdc53-F-box) complex in vivo further suggests that it may also function in proteolysis. To investigate the role of cyclin F in vivo, we generated mice deficient for cyclin F and conditionally deficient mice as well as mouse embryonic fibroblasts (MEFs) conditionally deficient for cyclin F. Heterozygous animals are normal and fertile, but CycF(-/-) animals, with a myriad of developmental anomalies due in large part to failures in yolk sac and chorioallantoic placentation, die around embryonic day 10.5. Tissue-specific deletion of cyclin F revealed that it was not required for the development and function of a number of different embryonic and adult tissues. In contrast, MEFs lacking cyclin F, while viable, do exhibit cell cycle defects, including reduced population-doubling time and a delay in cell cycle reentry from quiescence, indicating that cyclin F plays a role in cell cycle regulation.     

F-box proteins: more than baits for the SCF?

Progression through the mammalian cell cycle is associated with the activity of four cyclin dependent kinases (Cdc2/Cdk1, Cdk2, Cdk4, and Cdk6). Knockout mouse models have provided insight into the interplay of these Cdks. Most of these models do not exhibit major cell cycle defects revealing redundancies, and suggesting that a single Cdk might be sufficient to drive the cell cycle, similar as in yeast. Recent work on Cdk2/Cdk4 double knockouts has indicated that these two Cdks are required to phosphorylate Rb during late embryogenesis. The lack of Rb phosphorylation is progressive and associated with reduced E2F-inducible gene expression. Cdk2 and Cdk4 share the essential function of coupling the G1/S transition with mitosis. However, proliferation in early embryogenesis appears to be independent of Cdk2 and Cdk4. We discuss these observations and propose molecular mechanisms that establish the requirement for Cdk2 and Cdk4 at the G1/S transition. We are considering that the balance between proliferation and differentiation is disturbed, which affects especially heart development and leads to embryonic lethality in Cdk2 ^-/- Cdk4 ^-/- mutants. We also discuss the specific functions of Cdk4 and Cdk6, which ironically do not compensate for each other.     

PAM14, a novel MRG- and Rb-associated protein, is not required for development and T-cell function in mice

PAM14 has been found to associate in complexes with the MORF4/MRG family of proteins as well as Rb, the tumor suppressor protein. This suggested that it might be involved in cell growth, immortalization, and/or senescence. To elucidate the in vivo function of PAM14, we characterized the expression pattern of mouse Pam14 and generated PAM14-deficient (Pam14(-/-)) mice. Pam14 was widely expressed in all mouse tissues and as early as 7 days during embryonic development. Despite this ubiquitous expression in wild-type mice, Pam14(-/-) mice were healthy and fertile. Response to mitogenic stimulation and production of interleukin-2 were the same in stimulated splenic T cells from Pam14(-/-) mice as in control littermates. Cell growth rates of mouse embryonic fibroblasts (MEFs) from all three genotypes were the same, and immortalized cells were obtained from all cell cultures during continuous culture. There was also no difference in expression of growth-related genes in response to serum stimulation in the null versus control MEFs. These data demonstrate that PAM14 is not essential for normal mouse development and cell cycle control. PAM14 likely acts as an adaptor protein in nucleoprotein complexes and is probably compensated for by another functionally redundant protein(s).     

The Fbx4 tumor suppressor regulates cyclin D1 accumulation and prevents neoplastic transformation

Skp1-Cul1-F-box (SCF) E3 ubiquitin ligase complexes modulate the accumulation of key cell cycle regulatory proteins. Following the G(1)/S transition, SCF(Fbx4) targets cyclin D1 for proteasomal degradation, a critical event necessary for DNA replication fidelity. Deregulated cyclin D1 drives tumorigenesis, and inactivating mutations in Fbx4 have been identified in human cancer, suggesting that Fbx4 may function as a tumor suppressor. Fbx4(+/-) and Fbx4(-/-) mice succumb to multiple tumor phenotypes, including lymphomas, histiocytic sarcomas and, less frequently, mammary and hepatocellular carcinomas. Tumors and premalignant tissue from Fbx4(+/-) and Fbx4(-/-) mice exhibit elevated cyclin D1, an observation consistent with cyclin D1 as a target of Fbx4. Molecular dissection of the Fbx4 regulatory network in murine embryonic fibroblasts (MEFs) revealed that loss of Fbx4 results in cyclin D1 stabilization and nuclear accumulation throughout cell division. Increased proliferation in early passage primary MEFs is antagonized by DNA damage checkpoint activation, consistent with nuclear cyclin D1-driven genomic instability. Furthermore, Fbx4(-/-) MEFs exhibited increased susceptibility to Ras-dependent transformation in vitro, analogous to tumorigenesis observed in mice. Collectively, these data reveal a requisite role for the SCF(Fbx4) E3 ubiquitin ligase in regulating cyclin D1 accumulation, consistent with tumor suppressive function in vivo.     

Cell Cycle Sibling Rivalry: Cdc2 Versus Cdk2

It has been long believed that the cyclin-dependent kinase 2 [Cdk2] binds to cyclin E or cyclin Aand exclusively promotes the G1/S phase transition and that Cdc2/cyclin B complexes play a majorrole in mitosis. We now provide evidence that Cdc2 binds to cyclin E [in addition to cyclin A & B]and is able to promote the G1/S transition. This new concept indicates that both Cdk2 and/or Cdc2can drive cells through G1/S phase in parallel. In this review we discuss the classic cell cycle modeland how results from knockout mice provide new evidence that refute this model. We focus on newroles of Cdc2 and p27 in regulating the mammalian cell cycle and propose a new model for cellcycle regulation that accommodates these novel findings.     

Cell cycle-dependent dynamic association of cyclin/Cdk complexes with human DNA replication proteins

We have previously described the isolation of a replication competent (RC) complex from calf thymus, containing DNA polymerase alpha, DNA polymerase delta and replication factor C. Here, we describe the isolation of the RC complex from nuclear extracts of synchronized HeLa cells, which contains DNA replication proteins associated with cell-cycle regulation factors like cyclin A, cyclin B1, Cdk2 and Cdk1. In addition, it contains a kinase activity and DNA polymerase activities able to switch from a distributive to a processive mode of DNA synthesis, which is dependent on proliferating cell nuclear antigen. In vivo cross-linking of proteins to DNA in synchronized HeLa cells demonstrates the association of this complex to chromatin. We show a dynamic association of cyclins/Cdks with the RC complex during the cell cycle. Indeed, cyclin A and Cdk2 associated with the complex in S phase, and cyclin B1 and Cdk1 were present exclusively in G(2)/M phase, suggesting that the activity, as well the localization, of the RC complex might be regulated by specific cyclin/Cdk complexes.