Cyclin B1/Cdk1 Phosphorylation of Mitochondrial p53 Induces Anti-Apoptotic Response

The pro-apoptotic function of p53 has been well defined in preventing genomic instability and cell transformation. However, the intriguing fact that p53 contributes to a pro-survival advantage of tumor cells under DNA damage conditions raises a critical question in radiation therapy for the 50% human cancers with intact p53 function. Herein, we reveal an anti-apoptotic role of mitochondrial p53 regulated by the cell cycle complex cyclin B1/Cdk1 in irradiated human colon cancer HCT116 cells with p53^+/+ status. Steady-state levels of p53 and cyclin B1/Cdk1 were identified in the mitochondria of many human and mouse cells, and their mitochondrial influx was significantly enhanced by radiation. The mitochondrial kinase activity of cyclin B1/Cdk1 was found to specifically phosphorylate p53 at Ser-315 residue, leading to enhanced mitochondrial ATP production and reduced mitochondrial apoptosis. The improved mitochondrial function can be blocked by transfection of mutant p53 Ser-315-Ala, or by siRNA knockdown of cyclin B1 and Cdk1 genes. Enforced translocation of cyclin B1 and Cdk1 into mitochondria with a mitochondrial-targeting-peptide increased levels of Ser-315 phosphorylation on mitochondrial p53, improved ATP production and decreased apoptosis by sequestering p53 from binding to Bcl-2 and Bcl-xL. Furthermore, reconstitution of wild-type p53 in p53-deficient HCT116 p53^−/− cells resulted in an increased mitochondrial ATP production and suppression of apoptosis. Such phenomena were absent in the p53-deficient HCT116 p53^−/− cells reconstituted with the mutant p53. These results demonstrate a unique anti-apoptotic function of mitochondrial p53 regulated by cyclin B1/Cdk1-mediated Ser-315 phosphorylation in p53-wild-type tumor cells, which may provide insights for improving the efficacy of anti-cancer therapy, especially for tumors that retain p53.     

Nuclear Accumulation of p21Cip1 at the Onset of Mitosis: a Role at the G2/M-Phase Transition

Cell cycle arrest in G₁ in response to ionizing radiation or senescence is believed to be provoked by inactivation of G₁ cyclin-cyclin-dependent kinases (Cdks) by the Cdk inhibitor p21^{Cip1/Waf1/Sdi1}. We provide evidence that in addition to exerting negative control of the G₁/S phase transition, p21 may play a role at the onset of mitosis. In nontransformed fibroblasts, p21 transiently reaccumulates in the nucleus near the G₂/M-phase boundary, concomitant with cyclin B1 nuclear translocation, and associates with a fraction of cyclin A-Cdk and cyclin B1-Cdk complexes. Premitotic nuclear accumulation of cyclin B1 is not detectable in cells with low p21 levels, such as fibroblasts expressing the viral human papillomavirus type 16 E6 oncoprotein, which functionally inactivates p53, or in tumor-derived cells. Moreover, synchronized E6-expressing fibroblasts show accelerated entry into mitosis compared to wild-type cells and exhibit higher cyclin A- and cyclin B1-associated kinase activities. Finally, primary embryonic fibroblasts derived from p21^−/− mice have significantly reduced numbers of premitotic cells with nuclear cyclin B1. These data suggest that p21 promotes a transient pause late in G₂ that may contribute to the implementation of late cell cycle checkpoint controls.     

Characterization of the p53-Dependent Postmitotic Checkpoint following Spindle Disruption

The p53 tumor suppressor gene product is known to act as part of a cell cycle checkpoint in G₁ following DNA damage. In order to investigate a proposed novel role for p53 as a checkpoint at mitosis following disruption of the mitotic spindle, we have used time-lapse videomicroscopy to show that both p53^+/+ and p53^−/− murine fibroblasts treated with the spindle drug nocodazole undergo transient arrest at mitosis for the same length of time. Thus, p53 does not participate in checkpoint function at mitosis. However, p53 does play a critical role in nocodazole-treated cells which have exited mitotic arrest without undergoing cytokinesis and have thereby adapted. We have determined that in nocodazole-treated, adapted cells, p53 is required during a specific time window to prevent cells from reentering the cell cycle and initiating another round of DNA synthesis. Despite having 4N DNA content, adapted cells are similar to G₁ cells in that they have upregulated cyclin E expression and hypophosphorylated Rb protein. The mechanism of the p53-dependent arrest in nocodazole-treated adapted cells requires the cyclin-dependent kinase inhibitor p21, as p21^−/− fibroblasts fail to arrest in response to nocodazole treatment and become polyploid. Moreover, p21 is required to a similar extent to maintain cell cycle arrest after either nocodazole treatment or irradiation. Thus, the p53-dependent checkpoint following spindle disruption functionally overlaps with the p53-dependent checkpoint following DNA damage.     

The p53-p21WAF1 checkpoint pathway plays a protective role in preventing DNA rereplication induced by abrogation of FOXF1 function

We previously identified FOXF1 as a potential tumor suppressor gene with an essential role in preventing DNA rereplication to maintain genomic stability, which is frequently inactivated in breast cancer through the epigenetic mechanism. Here we further addressed the role of the p53-p21^WAF1 checkpoint pathway in DNA rereplication induced by silencing of FOXF1. Knockdown of FOXF1 by small interference RNA (siRNA) rendered colorectal p53-null and p21^WAF1-null HCT116 cancer cells more susceptible to rereplication and apoptosis than the wild-type parental cells. In parental HCT116 cells with a functional p53 checkpoint, the p53-p21^WAF1 checkpoint pathway was activated upon FOXF1 knockdown, which was concurrent with suppression of the CDK2-Rb cascade and induction of G₁ arrest. In contrast, these events were not observed in FOXF1-depleted HCT116-p53−/− and HCT116-p21−/− cells, indicating that the p53-dependent checkpoint function is vital for inhibiting CDK2 to induce G₁ arrest and protect cells from rereplication. The pharmacologic inhibitor (caffeine) of ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3 related (ATR) protein kinases abolished activation of the p53-p21^WAF1 pathway upon FOXF1 knockdown, suggesting that suppression of FOXF1 function triggered the ATM/ATR-mediated DNA damage response. Cosilencing of p53 by siRNA synergistically enhanced the effect of FOXF1 depletion on the stimulation of DNA rereplication and apoptosis in wild-type HCT116. Finally, we show that FOXF1 expression is predominantly silenced in breast and colorectal cancer cell lines with inactive p53. Our study demonstrated that the p53-p21^WAF1 checkpoint pathway is an intrinsically protective mechanism to prevent DNA rereplication induced by silencing of FOXF1.     

The Role of MMP7 and Its Cross-Talk with the FAS/FASL System during the Acquisition of Chemoresistance to Oxaliplatin

Background

The efficacy of oxaliplatin in cancer chemotherapy is limited by the development of drug resistance. MMP7 has been related to the loss of tumor cell response to cytotoxic agents although the exact mechanism is not fully understood. Moreover, MMP7 is an independent prognosis factor for survival in patients with colorectal cancer. The aim of the present study was to analyze the role of MMP7 and its cross-talk with the Fas/FasL system during the acquisition of oxaliplatin resistance in colon cancer cells.

Principal Findings

For this purpose we have developed three different oxaliplatin-resistant cell lines (RHT29, RHCT116 p53^+/+, RHCT116 p53^−/−) from the parental HT29, HCT116 p53^+/+ and HCT116 p53^−/− colon cancer cells. MMP7 basal expression was higher in the resistant compared to the parental cell lines. MMP7 was also upregulated by oxaliplatin in both HT29 (p53 mutant) and RHCT116 p53^−/− but not in the RHCT116 p53^+/+. Inhibition of MMP by 1,10-phenantroline monohydrate or siRNA of MMP7 restores cell sensitivity to oxaliplatin-induced apoptosis in both HT29 and RHCT116 p53^−/− but not in the RHCT116 p53^+/+. Some of these effects are caused by alterations in Fas receptor. Fas is upregulated by oxaliplatin in colon cancer cells, however the RHT29 cells treated with oxaliplatin showed a 3.8-fold lower Fas expression at the cell surface than the HT29 cells. Decrease of Fas at the plasma membrane seems to be caused by MMP7 since its inhibition restores Fas levels. Moreover, functional analysis of Fas demonstrates that this receptor was less potent in inducing apoptosis in RHT29 cells and that its activation induces MAPK signaling in resistant cells.

Conclusions

Taking together, these results suggest that MMP7 is related to the acquisition of oxaliplatin-resistance and that its inhibition restores drug sensitivity by increasing Fas receptor. Furthermore, Fas undergoes a change in its functionality in oxaliplatin-resistant cells inducing survival pathways instead of apoptotic signals.     

Hyperoxia Induces S-Phase Cell-Cycle Arrest and p21-Independent Cdk2 Inhibition in Human Carcinoma T47D-H3 Cells

Little is known about cell-cycle checkpoint activation by oxidative stress in mammalian cells. The effects of hyperoxia on cell-cycle progression were investigated in asynchronous human T47D-H3 cells, which contain mutated p53 and fail to arrest at G1/S in response to DNA damage. Hyperoxic exposure (95% O₂, 40–64 h) induced an S-phase arrest associated with acute inhibition of Cdk2 activity and DNA synthesis. In contrast, exit from G2/M was not inhibited in these cells. After 40 h of hyperoxia, these effects were partially reversible during recovery under normoxic conditions. The inhibition of Cdk2 activity was not due to degradation of Cdk2, cyclin E or A, nor impairment of Cdk2 complex formation with cyclin A or E and p21^Cip1. The loss of Cdk2 activity occurred in the absence of induction and recruitment of cdk inhibitor p21^Cip1 or p27^Kip1 in cyclin A/Cdk2 or cyclin E/Cdk2 complexes. In contrast, Cdk2 inhibition was associated with increased Cdk2-Tyr15 phosphorylation, increased E2F-1 recruitment, and decreased PCNA contents in Cdk2 complexes. The latter results indicate a p21^Cip1/p27^Kip1-independent mechanism of S-phase checkpoint activation in the hyperoxic T47D cell model investigated.     

Haploinsufficiency of the Mus81-Eme1 endonuclease activates the intra-S-phase and G2/M checkpoints and promotes rereplication in human cells

The Mus81–Eme1 complex is a structure-specific endonuclease that preferentially cleaves nicked Holliday junctions, 3′-flap structures and aberrant replication fork structures. Mus81^−/− mice have been shown to exhibit spontaneous chromosomal aberrations and, in one of two models, a predisposition to cancers. The molecular mechanisms underlying its role in chromosome integrity, however, are largely unknown. To clarify the role of Mus81 in human cells, we deleted the gene in the human colon cancer cell line HCT116 by gene targeting. Here we demonstrate that Mus81 confers resistance to DNA crosslinking agents and slight resistance to other DNA-damaging agents. Mus81 deficiency spontaneously promotes chromosome damage such as breaks and activates the intra-S-phase checkpoint through the ATM-Chk1/Chk2 pathways. Furthermore, Mus81 deficiency activates the G₂/M checkpoint through the ATM-Chk2 pathway and promotes DNA rereplication. Increased rereplication is reversed by the ectopic expression of Cdk1. Haploinsufficiency of Mus81 or Eme1 also causes similar phenotypes. These findings suggest that a complex network of the checkpoint pathways that respond to DNA double-strand breaks may participate in some of the phenotypes associated with Mus81 or Eme1 deficiency.     

Involvement of p53 and p21 in Cellular Defects and Tumorigenesis in Atm−/− Mice

Disruption of the mouse Atm gene, whose human counterpart is consistently mutated in ataxia-telangiectasia (A-T) patients, creates an A-T mouse model exhibiting most of the A-T-related systematic and cellular defects. While ATM plays a major role in signaling the p53 response to DNA strand break damage, Atm^−/− p53^−/− mice develop lymphomas earlier than Atm^−/− or p53^−/− mice, indicating that mutations in these two genes lead to synergy in tumorigenesis. The cell cycle G₁/S checkpoint is abolished in Atm^−/− p53^−/− mouse embryonic fibroblasts (MEFs) following γ-irradiation, suggesting that the partial G₁ cell cycle arrest in Atm^−/− cells following γ-irradiation is due to the residual p53 response in these cells. In addition, the Atm^−/− p21^−/− MEFs are more severely defective in their cell cycle G₁ arrest following γ-irradiation than Atm^−/− and p21^−/− MEFs. The Atm^−/− MEFs exhibit multiple cellular proliferative defects in culture, and an increased constitutive level of p21 in these cells might account for these cellular proliferation defects. Consistent with this notion, Atm^−/− p21^−/− MEFs proliferate similarly to wild-type MEFs and exhibit no premature senescence. These cellular proliferative defects are also rescued in Atm^−/− p53^−/− MEFs and little p21 can be detected in these cells, indicating that the abnormal p21 protein level in Atm^−/− cells is also p53 dependent and leads to the cellular proliferative defects in these cells. However, the p21 mRNA level in Atm^−/− MEFs is lower than that in Atm^+/+ MEFs, suggesting that the higher level of constitutive p21 protein in Atm^−/− MEFs is likely due to increased stability of the p21 protein.     

Murine coronavirus replication induces cell cycle arrest in G0/G1 phase

Mouse hepatitis virus (MHV) replication in actively growing DBT and 17Cl-1 cells resulted in the inhibition of host cellular DNA synthesis and the accumulation of infected cells in the G₀/G₁ phase of the cell cycle. UV-irradiated MHV failed to inhibit host cellular DNA synthesis. MHV infection in quiescent 17Cl-1 cells that had been synchronized in the G₀ phase by serum deprivation prevented infected cells from entering the S phase after serum stimulation. MHV replication inhibited hyperphosphorylation of the retinoblastoma protein (pRb), the event that is necessary for cell cycle progression through late G₁ and into the S phase. While the amounts of the cellular cyclin-dependent kinase (Cdk) inhibitors p21^Cip1, p27^Kip1, and p16^INK4a did not change in infected cells, MHV infection in asynchronous cultures induced a clear reduction in the amounts of Cdk4 and G₁ cyclins (cyclins D1, D2, D3, and E) in both DBT and 17Cl-1 cells and a reduction in Cdk6 levels in 17Cl-1 cells. Infection also resulted in a decrease in Cdk2 activity in both cell lines. MHV infection in quiescent 17Cl-1 cells prevented normal increases in Cdk4, Cdk6, cyclin D1, and cyclin D3 levels after serum stimulation. The amounts of cyclin D2 and cyclin E were not increased significantly after serum stimulation in mock-infected cells, whereas they were decreased in MHV-infected cells, suggesting the possibility that MHV infection may induce cyclin D2 and cyclin E degradation. Our data suggested that a reduction in the amounts of G₁ cyclin-Cdk complexes in MHV-infected cells led to a reduction in Cdk activities and insufficient hyperphosphorylation of pRb, resulting in inhibition of the cell cycle in the G₀/G₁ phase.     

Cyclin E2, a Novel G1 Cyclin That Binds Cdk2 and Is Aberrantly Expressed in Human Cancers

A novel cyclin gene was discovered by searching an expressed sequence tag database with a cyclin box profile. The human cyclin E2 gene encodes a 404-amino-acid protein that is most closely related to cyclin E. Cyclin E2 associates with Cdk2 in a functional kinase complex that is inhibited by both p27^Kip1 and p21^Cip1. The catalytic activity associated with cyclin E2 complexes is cell cycle regulated and peaks at the G₁/S transition. Overexpression of cyclin E2 in mammalian cells accelerates G₁, demonstrating that cyclin E2 may be rate limiting for G₁ progression. Unlike cyclin E1, which is expressed in most proliferating normal and tumor cells, cyclin E2 levels were low to undetectable in nontransformed cells and increased significantly in tumor-derived cells. The discovery of a novel second cyclin E family member suggests that multiple unique cyclin E-CDK complexes regulate cell cycle progression.     

Cell Cycle-Dependent Regulation of Human DNA Polymerase α-Primase Activity by Phosphorylation

DNA polymerase α-primase is known to be phosphorylated in human and yeast cells in a cell cycle-dependent manner on the p180 and p68 subunits. Here we show that phosphorylation of purified human DNA polymerase α-primase by purified cyclin A/cdk2 in vitro reduced its ability to initiate simian virus 40 (SV40) DNA replication in vitro, while phosphorylation by cyclin E/cdk2 stimulated its initiation activity. Tryptic phosphopeptide mapping revealed a family of p68 peptides that was modified well by cyclin A/cdk2 and poorly by cyclin E/cdk2. The p180 phosphopeptides were identical with both kinases. By mass spectrometry, the p68 peptide family was identified as residues 141 to 160. Cyclin A/cdk2- and cyclin A/cdc2-modified p68 also displayed a phosphorylation-dependent shift to slower electrophoretic mobility. Mutation of the four putative phosphorylation sites within p68 peptide residues 141 to 160 prevented its phosphorylation by cyclin A/cdk2 and the inhibition of replication activity. Phosphopeptide maps of the p68 subunit of DNA polymerase α-primase from human cells, synchronized and labeled in G₁/S and in G₂, revealed a cyclin E/cdk2-like pattern in G₁/S and a cyclin A/cdk2-like pattern in G₂. The slower-electrophoretic-mobility form of p68 was absent in human cells in G₁/S and appeared as the cells entered G₂/M. Consistent with this, the ability of DNA polymerase α-primase isolated from synchronized human cells to initiate SV40 replication was maximal in G₁/S, decreased as the cells completed S phase, and reached a minimum in G₂/M. These results suggest that the replication activity of DNA polymerase α-primase in human cells is regulated by phosphorylation in a cell cycle-dependent manner.     

Targeted Inactivation of p12Cdk2ap1, CDK2 Associating Protein 1, Leads to Early Embryonic Lethality

Targeted disruption of murine Cdk2ap1, an inhibitor of CDK2 function and hence G1/S transition, results in the embryonic lethality with a high penetration rate. Detailed timed pregnancy analysis of embryos showed that the lethality occurred between embryonic day 3.5 pc and 5.5 pc, a period of implantation and early development of implanted embryos. Two homozygous knockout mice that survived to term showed identical craniofacial defect, including a short snout and a round forehead. Examination of craniofacial morphology by measuring Snout Length (SL) vs. Face Width (FW) showed that the Cdk2ap1^+/− mice were born with a reduced SL/FW ratio compared to the Cdk2ap1^+/+ and the reduction was more pronounced in Cdk2ap1^−/− mice. A transgenic rescue of the lethality was attempted by crossing Cdk2ap1^+/− animals with K14-Cdk2ap1 transgenic mice. Resulting Cdk2ap1^{+/−:K14-Cdk2ap1} transgenic mice showed an improved incidence of full term animals (16.7% from 0.5%) on a Cdk2ap1^−/− background. Transgenic expression of Cdk2ap1 in Cdk2ap1^{−/−:K14-Cdk2ap1} animals restored SL/FW ratio to the level of Cdk2ap1^{+/−:K14-Cdk2ap1} mice, but not to that of the Cdk2ap1^{+/+:K14-Cdk2ap1} mice. Teratoma formation analysis using mESCs showed an abrogated in vivo pluripotency of Cdk2ap1^−/− mESCs towards a restricted mesoderm lineage specification. This study demonstrates that Cdk2ap1 plays an essential role in the early stage of embryogenesis and has a potential role during craniofacial morphogenesis.     

Retinoic Acid-Mediated G1 Arrest Is Associated with Induction of p27 and Inhibition of Cyclin-Dependent Kinase 3 in Human Lung Squamous Carcinoma CH27 Cells

Retinoids are promising agents for the prevention and treatment of several human malignancies including lung cancer. In this study, the effect of retinoic acid (RA) on cell growth and the mechanism of growth modulation were examined in human lung squamous carcinoma CH27 cells. Here we report that RA mediated the dose- and time-dependent growth arrest in G1 phase, accompanied by the up-regulation of p27^Kip1 and the down-regulation of the cyclin-dependent kinase 3 (Cdk3) and p21^CIP1/Waf1 proteins. Furthermore, RA-induced growth arrest of CH27 cells was also associated with increased retinoic acid receptor β (RARβ) and reduced c-Myc expression. However, RA had no effect on the levels of cyclins A, D1, D3, E, or H, or on Cdk2, Cdk4, Cdk5, CDk6, Cdk7, p16^Ink4A, p15^Ink4B, p53, or pRb proteins in CH27 cells. Evaluation of the kinase activity of cyclin–Cdk complexes showed that RA increases p27^Kip1 expression in CH27 cells leading to markedly reduced cyclin A/Cdk2 kinase activity and slightly reduced cyclin E/Cdk2 kinase activity, with no effect on cyclin D/Cdk4 and cyclin D/Cdk6 activities. Moreover, coincident with the decrease in kinase activity was a drastic increase in cyclin A-bound p27^Kip1. These results suggest that increases in the levels of p27^Kip1 and its binding to cyclin A, as well as reduction of Cdk3 protein expression, are strong candidates for the cell cycle regulator that prevents the entry into the S phase in RA-treated CH27 cells, with prolongation of G1 phase and inhibition of DNA synthesis.     

Chemical DNA damage activates p21 WAF1/CIP1-dependent intra-S checkpoint

When cells progressing in G₁ phase are irradiated with UV light, two damage checkpoint pathways are activated: CHK1-Cdc25A and p53-p21^WAF1/CIP1, both targeting Cdk2 but the latter inducing long lasting inactivation. In similarly irradiated S phase cells, however, p21^WAF1/CIP1-dependent checkpoint is largely inactive. We report here that p21-dependent checkpoint can effectively be activated and induce a prolonged S phase arrest with similarly extended inactivation of Cdk2 by association of p21 if mid-S phase cells are damaged with a base-modifying agent instead of UV light, indicating that the poor utilization of p21-dependent checkpoint is not an innate property of S phase cells.     

Requirement of Cyclin E-Cdk2 Inhibition in p16INK4a-Mediated Growth Suppression

Loss-of-function mutations of p16^INK4a have been identified in a large number of human tumors. An established biochemical function of p16 is its ability to specifically inhibit cyclin D-dependent kinases in vitro, and this inhibition is believed to be the cause of the p16-mediated G₁ cell cycle arrest after reintroduction of p16 into p16-deficient tumor cells. However, a mutant of Cdk4, Cdk4^N158, designed to specifically inhibit cyclin D-dependent kinases through dominant negative interference, was unable to arrest the cell cycle of the same cells (S. van den Heuvel and E. Harlow, Science 262:2050–2054, 1993). In this study, we determined functional differences between p16 and Cdk4^N158. We show that p16 and Cdk4^N158 inhibit the kinase activity of cellular cyclin D1 complexes through different mechanisms. p16 dissociated cyclin D1-Cdk4 complexes with the release of bound p27^KIP1, while Cdk4^N158 formed complexes with cyclin D1 and p27. In cells induced to overexpress p16, a higher portion of cellular p27 formed complexes with cyclin E-Cdk2, and Cdk2-associated kinase activities were correspondingly inhibited. Cells engineered to express moderately elevated levels of cyclin E became resistant to p16-mediated growth suppression. These results demonstrate that inhibition of cyclin D-dependent kinase activity may not be sufficient to cause G₁ arrest in actively proliferating tumor cells. Inhibition of cyclin E-dependent kinases is required in p16-mediated growth suppression.     

Cyclin D1 Promotes Cell Cycle Progression through Enhancing NDR1/2 Kinase Activity Independent of Cyclin-dependent Kinase 4

Cyclin/cyclin-dependent kinases (Cdks) are critical protein kinases in regulating cell cycle progression. Among them, cyclin D1/Cdk4 exerts its function mainly in the G₁ phase. By using the tandem affinity purification tag approach, we identified a set of proteins interacting with Cdk4, including NDR1/2. Interestingly, confirming the interactions between NDR1/2 and cyclin D1/Cdk4, we observed that NDR1/2 interacted with cyclin D1 independent of Cdk4, but NDR1/2 and cyclin D1/Cdk4 did not phosphorylate each other. In addition, we found that NDR1/2 did not affect the kinase activity of cyclin D1/Cdk4 upon phosphorylation of GST-Rb. However, cyclin D1 but not Cdk4 promoted the kinase activity of NDR1/2. We also demonstrated that cyclin D1 K112E, which could not bind Cdk4, enhanced the kinase activity of NDR1/2. To test whether cyclin D1 promotes G₁/S transition though enhancing NDR1/2 kinase activity, we performed flow cytometry analysis using cyclin D1 and cyclin D1 K112E Tet-On inducible cell lines. The data show that both cyclin D1 and cyclin D1 K112E promoted G₁/S transition. Importantly, knockdown of NDR1/2 almost completely abolished the function of cyclin D1 K112E in promoting G₁/S transition. Consistently, we found that the protein level of p21 was reduced in cells overexpressing cyclin D1 K112E but not when NDR1/2 was knocked down. Taken together, these results reveal a novel function of cyclin D1 in promoting cell cycle progression by enhancing NDR kinase activity independent of Cdk4.     

Genetic Characterization of the Role of the Cip/Kip Family of Proteins as Cyclin-Dependent Kinase Inhibitors and Assembly Factors

The Cip/Kip family, namely, p21^Cip1, p27^Kip1, and p57^Kip2, are stoichiometric cyclin-dependent kinase inhibitors (CKIs). Paradoxically, they have been proposed to also act as positive regulators of Cdk4/6-cyclin D by stabilizing these heterodimers. Loss of p21^Cip1 and p27^Kip1 reduces Cdk4/6-cyclin D complexes, although with limited phenotypic consequences compared to the embryonic lethality of Cdk4/6 or triple cyclin D deficiency. This milder phenotype was attributed to Cdk2 compensatory mechanisms. To address this controversy using a genetic approach, we generated Cdk2^−/− p21^−/− p27^−/− mice. Triple-knockout mouse embryonic fibroblasts (MEFs) displayed minimal levels of D-type cyclins and Cdk4/6-cyclin D complexes. p57^Kip2 downregulation in the absence of p21^Cip1 and p27^Kip1 aggravated this phenotype, yet MEFs lacking all Cip/Kip proteins exhibited increased retinoblastoma phosphorylation, together with enhanced proliferation and transformation capacity. In vivo, Cdk2 ablation induced partial perinatal lethality in p21^−/− p27^−/− mice, suggesting partial Cdk2-dependent compensation. However, Cdk2^−/− p21^−/− p27^−/− survivors displayed all phenotypes described for p27^−/− mice, including organomegalia and pituitary tumors. Thus, Cip/Kip deficiency does not impair interphasic Cdk activity even in the absence of Cdk2, suggesting that their Cdk-cyclin assembly function is dispensable for homeostatic control in most cell types.