Regulation of cyclin D1/Cdk4 complexes by calcium/calmodulin-dependent protein kinase I

The selective inhibitor of the multifunctional calcium/calmodulin-dependent kinases (CaMK), KN-93, arrests a variety of cell types in G(1). However, the biochemical nature of this G(1) arrest point and the physiological target of KN-93 in G(1) remain controversial. Here we show that in WI-38 human diploid fibroblasts KN-93 reversibly arrested cells in late G(1) prior to detectable cyclin-dependent kinase 4 (cdk4) activation. At the KN-93 arrest point, we found that cyclin D1/cdk4 complexes had assembled with p21/p27, accumulated in the nucleus, and become phosphorylated on Thr-172, yet were relatively inactive. Additional examination of cdk4 complexes by gel filtration analysis demonstrated that, in late G(1), cyclin D1-containing complexes migrated toward lower molecular weight (M(r)) fractions and this altered migration was accompanied by the appearance of two peaks of cdk4 activity, at 150-200 and 70 kDa, respectively. KN-93 prevented both the activation of cdk4, and this shift in cyclin D1 migration and overexpression of cyclin D1/cdk4 overcame the KN-93 arrest. To determine which multifunctional CaMK acts in G(1), we expressed kinase-deficient forms of CaMKI and CaMKII. Overexpression of kinase-deficient CaMKI, but not CaMKII, prevented cdk4 activation, mimicking the KN-93 arrest point. Therefore, we hypothesize that KN-93 prevents a very late, uncharacterized step in cyclin D/cdk4 activation that involves CaMKI and follows complex assembly, nuclear entry, and phosphorylation.     

Cyclin D3-associated Kinase Activity Is Regulated by p27kip1 in BALB/c 3T3 Cells

We report that cyclin D3/cdk4 kinase activity is regulated by p27^kip1 in BALB/c 3T3 cells. The association of p27^kip1 was found to result in inhibition of cyclin D3 activity as measured by immune complex kinase assays utilizing cyclin D3-specific antibodies. The ternary p27^kip1/cyclin D3/cdk4 complexes do exhibit kinase activity when measured in immune complex kinase assays utilizing p27^kip1-specific antibodies. The association of p27^kip1 with cyclin D3 was highest in quiescent cells and declined upon mitogenic stimulation, concomitantly with declines in the total level of p27^kip1 protein. The decline in this association could be elicited by PDGF treatment alone; this was not sufficient, however, for activation of cyclin D3 activity, which also required the presence of factors in platelet-poor plasma in the culturing medium. Unlike cyclin D3 activity, which was detected only in growing cells, p27^kip1 kinase activity was present throughout the cell cycle. Since we found that the p27^kip1 activity was dependent on cyclin D3 and cdk4, we compared the substrate specificity of the active ternary complex containing p27^kip1 and the active cyclin D3 lacking p27^kip1 by tryptic phosphopeptide mapping of GST-Rb phosphorylated in vitro and also by comparing the relative phosphorylation activity toward a panel of peptide substrates. We found that ternary p27^kip1/cyclin D3/cdk4 complexes exhibited a different specificity than the active binary cyclin D3/cdk4 complexes, suggesting that p27^kip1 has the capacity to both inhibit cyclin D/cdk4 activity as well as to modulate cyclin D3/cdk4 activity by altering its substrate preference.     

Inhibition of G1 phase cyclin dependent kinases by transforming growth factor β1

Transforming growth factor β1 (TGFβ1) inhibits epithelial cell proliferation late in the G1 phase of the cell cycle. We examined the effect of TGFβ1 on known late G1 cell cycle regulators in an attempt to determine the molecular mechanism of growth inhibition by this physiological inhibitor. The results demonstrate the TGFβ1 inhibits the late G1 and S phase specific histone H1 kinase activity of p33^cdk2. This inhibitiion is not dur to TGFβ1's effect on p33^cdk2 synthesis, but rather due to its negative effect on the late G1 phosphorylation of p33^cdk2. It is also shown that TGFβ1 inhibits both late G1 cyclin A and cyclin E associated histon H1 kinase activities. The inhibitor has no effects on the synthesis of cyclin E but to inhibit the synthesis of cyclin A protein in a cell cycle dependent manner. If TGFβ1 is added to cells which have progressed futher than 8 hours into G1, then it is without inhibitory effect on cyclin A synthesis. These effect on TGFβ1 on late G1 cell cycle regulators correlate well with its inhibitory effects on cellular growth and suggest that these G1 cyclin dependent kinases might serve as targets for TGFβ1-mediated growth arrest.     

RyR channel-mediated increase of cytosolic free calcium level signals cyclin B1 degradation during abortive spontaneous egg activation in rat

In few mammalian species including rat, post-ovulatory aging induces abortive spontaneous egg activation (SEA), which is morphologically characterized by exit from metaphase-II (M-II) arrest. A possibility exists that the RyR channel-mediated insufficient increase of cytosolic free Ca²⁺ level could be one of the causes for post-ovulatory aging-induced abortive SEA. To test this possibility, eggs collected after 17 h post-hCG surge were cultured with or without various concentrations of nifedipine (NF), ruthenium red (RR), and KN-93 for 3 h in vitro. Morphological changes characteristic of abortive SEA, cytosolic free Ca²⁺ level, cyclin B1 level, and meiotic status were analyzed. Data of the present study indicate that NF and RR inhibited post-ovulatory aging-induced abortive SEA in a concentration-dependent manner. Further, RR protected against RyR channel as well as caffeine-mediated increase of cytosolic free Ca²⁺ level. In addition, KN-93 inhibited post-ovulatory aging-induced abortive SEA in a concentration-dependent manner. An increase of cytosolic free Ca²⁺ level was associated with a reduction of cyclin B1 level during post-ovulatory aging-induced abortive SEA. These data indirectly suggest the involvement of RyR channels in the increase of cytosolic free Ca²⁺ level. The increased cytosolic free Ca²⁺ level triggers cyclin B1 degradation possibly through CaMK-II activity during post-ovulatory aging-induced abortive SEA in rat eggs cultured in vitro.     

Differential regulation of the cyclin-dependent kinase inhibitors p21(Cip1) and p27(Kip1) by phosphorylation directed by the cyclin encoded by Murine Herpesvirus 68

Members of the gamma2-herpesvirus family encode cyclin-like proteins that have the ability to deregulate mammalian cell cycle control. Here we report the key features of the viral cyclin encoded by Murine Herpesvirus 68, M cyclin. M cyclin preferentially associated with and activated cdk2; the M cyclin/cdk2 holoenzyme displayed a strong reliance on phosphorylation of the cdk T loop for activity. cdk2 associated with M cyclin exhibited substantial resistance to the cdk inhibitor proteins p21(Cip) and p27(Kip). Furthermore, M cyclin directed cdk2 to phosphorylate p27(Kip1) on threonine 187 (T187) and cellular expression of M cyclin led to down-regulation of p27(Kip1) and the partial subversion of the associated G1 arrest. Mutation of T187 to a non-phosphorylatable alanine rendered the p27(Kip1)-imposed G1 arrest resistant to M cyclin expression. Unlike the related K cyclin, M cyclin was unable to circumvent the G1 arrest associated with p21(Cip1) and was unable to direct its associated catalytic subunit to phosphorylate this cdk inhibitor. These results imply that M cyclin has properties that are distinct from other viral cyclins and that M cyclin expression alone is insufficient for S phase entry.     

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.     

Parathyroid hormone-related protein induces G1 phase growth arrest of vascular smooth muscle cells

In this study, we investigated the mechanisms responsible for the growth-inhibitory action of parathyroid hormone-related protein (PTHRP) in A10 vascular smooth muscle cells (VSMC). Fluorescence-activated cell sorting analysis of serum-stimulated VSMC treated with PTHRP or dibutyryl-cAMP (DBcAMP) demonstrated an enrichment of cells in G1 and a reduction in the S phase. Measurement of DNA synthesis in platelet-derived growth factor-stimulated VSMC treated with DBcAMP revealed that cells became refractory to growth inhibition by 12-16 h, consistent with blockade in mid-G1. cAMP treatment blunted the serum-induced rise in cyclin D1 during cell cycle progression without altering levels of the cyclin-dependent kinase cdk4 or cyclin E and its associated kinase, cdk2. Exposure of cells to PTHRP or cAMP resulted in a reduction in retinoblastoma gene product (Rb) phosphorylation. Immunoblotting of extracts from cAMP-treated cells with antibodies to cdk inhibitors revealed a striking increase in p27(kip1) abundance coincident with the G1 block. Immunoprecipitation with an anti-cyclin D1 antibody of cell lysates prepared from cAMP-treated cells followed by immunoblotting with antisera to p27(kip1) disclosed a threefold increase in p27(kip1) associated with cyclin D1 compared with lysates treated with serum alone. We conclude that PTHRP, by increasing intracellular cAMP, induces VSMC cycle arrest in mid-G1. This occurs secondary to a suppression in cyclin D1 and induction of p27(kip1) expression, which in turn inhibits Rb phosphorylation.     

Involvement of p27(kip1) and cyclin D3 in the regulation of cdk2 activity during skeletal muscle differentiation

Terminal myogenic differentiation involves an irreversible transition from a proliferative state to a post-mitotic quiescent state. We showed here that in addition to the previously reported down regulation of G(1)-related cyclin-associated kinase activities, this transition was also accompanied by an extensive reorganization of the cyclin-cdk complexes, including a dramatic shift of cdk2 from cyclin A to cyclin D3. Moreover, the inhibition of cdk activity also correlated with an increase in the expression of the p27(kip1) cdk inhibitor and in its association with the cyclin-cdk2 complexes. Since depletion of p27 substantially reduced the cdk inhibitor activity present in differentiated muscle cells, we believe that the increase in p27 expression along with the reorganization of the cyclin-cdk2 complexes may play an important role in the inhibition of cdk2 activity during the differentiation process.     

Fbxl12 triggers G1 arrest by mediating degradation of calmodulin kinase I

Cell cycle progression through its regulatory control by changes in intracellular Ca^2 + levels at the G1/S transition mediates cellular proliferation and viability. Ca^2 +/CaM-dependent kinase 1 (CaMKI) appears critical in regulating the assembly of the cyclin D1/cdk4 complex essential for G1 progression, but how this occurs is unknown. Cyclin D1/cdk4 assembly in the early G1 phase is also regulated via binding to p27. Here, we show that a ubiquitin E3 ligase component, F-box protein Fbxl12, mediates CaMKI degradation via a proteasome-directed pathway leading to disruption of cyclin D1/cdk4 complex assembly and resultant G1 arrest in lung epithelia. We also demonstrate that i) CaMKI phosphorylates p27 at Thr¹⁵⁷ and Thr¹⁹⁸ in human cells and at Thr¹⁷⁰ and Thr¹⁹⁷ in mouse cells to modulate its subcellular localization; ii) Fbxl12-induced CaMKI degradation attenuates p27 phosphorylation at these sites in early G1 and iii) activation of CaMKI during G1 transition followed by p27 phosphorylation appears to be upstream to other p27 phosphorylation events, an effect abrogated by Fbxl12 overexpression. Lastly, known inducers of G1 arrest significantly increase Fbxl12 levels in cells. Thus, Fbxl12 may be a previously uncharacterized, functional growth inhibitor regulating cell cycle progression that might be used for mechanism-based therapy.     

Integrin Signaling at the M/G1 Transition Induces Expression of Cyclin E

The activities of the mammalian G1 cyclins, cyclin D and cyclin E, during cell cycle progression (G1/S) are believed to be regulated by cell attachment and the presence of growth factors. In order to study the importance of cell attachment and concomitant integrin signaling on the expression of G1 cyclins during the natural adhesion process from mitosis to interphase, protein expression was monitored in cells that were synchronized by mitotic shake off. Here we show that in Chinese hamster ovary (CHO) and neuroblastoma (N2A) cells, expression of cyclin E at the M/G1 transition is regulated by both growth factors and cell attachment, while expression of cyclin D seems to be entirely dependent on the presence of serum. Expression of cyclin E appears to be correlated with the phosphorylation of the retinoblastoma protein, suggesting a link with the activity of the cyclin D/cdk4 complex. Expression of the cdk inhibitors p21^cip1/Waf1 and p27^Kip1 is not changed upon serum depletion or detachment of cells during early G1, suggesting no direct role for these CKIs in the regulation of cyclin activity. Although inhibition of cyclin E/cdk2 kinase activity has been reported previously, this is the first time that cyclin E expression is shown to be dependent on cell attachment.     

Repression of p27kip1 synthesis by platelet-derived growth factor in BALB/c 3T3 cells.

We have investigated the regulation of p27kip1, a cyclin-dependent kinase inhibitor, in BALB/c 3T3 cells during growth factor-stimulated transition from quiescence (G0) to a proliferative (G1) state. The level of p27kip1 protein falls dramatically after mitogenic stimulation and is accompanied by a decrease in cyclin E associated p27kip1, as well as a transient increase in cyclin D1-associated p27kip1 that later declines concomitantly with the loss of total p27kip1. Analysis of metabolically labelled cells revealed that cyclin D2, cyclin D3, and cdk4 were also partnered with p27kip1 in quiescent BALB/c 3T3 cells and that this association decreased after platelet-derived growth factor (PDGF) treatment. Furthermore, the decline in p27kip1 and reduced association with cyclin D3, initiated by the addition of PDGF but not plasma-derived factors, suggested that these changes are involved in competence, the first step in the exit from G0. Synthesis of p27kip1 as determined by incorporation of [35S]methionine was repressed upon mitogenic stimulation, and PDGF was sufficient to elicit this repression within 2 to 3 h. Pulse-chase experiments demonstrated the reduced rate of synthesis was not the result of an increased rate of degradation. Full repression of p27kip1 synthesis required the continued presence of PDGF and failed to occur in the presence of the RNA polymerase inhibitor 5,6-dichlorobenzimidazole riboside. These characteristics demonstrate that repression was a late effect of PDGF and was consistent with our finding that conditional expression of activated H-ras did not affect synthesis of p27kip1. Northern (RNA) analysis of p27kip1 mRNA revealed that the repression was not accompanied by a corresponding decrease in p27kip1 mRNA, suggesting that the PDGF-regulated decrease in p27kip1 expression occurred through a translational mechanism.     

Transforming growth factor beta(1) selectively inhibits the cyclic AMP-dependent proliferation of primary thyroid epithelial cells by preventing the association of cyclin D3-cdk4 with nuclear p27(kip1)

Dog thyroid epithelial cells in primary culture constitute a physiologically relevant model of positive control of DNA synthesis initiation and G0-S prereplicative phase progression by cAMP as a second messenger for thyrotropin (thyroid-stimulating hormone [TSH]). As previously shown in this system, the cAMP-dependent mitogenic pathway differs from growth factor cascades as it stimulates the accumulation of p27(kip1) but not cyclins D. Nevertheless, TSH induces the nuclear translocations and assembly of cyclin D3 and cdk4, which are essential in cAMP-dependent mitogenesis. Here we demonstrate that transforming growth factor beta(1) (TGFbeta(1)) selectively inhibits the cAMP-dependent cell cycle in mid-G1 and various cell cycle regulatory events, but it weakly affects the stimulation of DNA synthesis by epidermal growth factor (EGF), hepatocyte growth factor, serum, and phorbol esters. EGF+serum and TSH did not interfere importantly with TGFbeta receptor signaling, because they did not affect the TGFbeta-induced nuclear translocation of Smad 2 and 3. TGFbeta inhibited the phosphorylation of Rb, p107, and p130 induced by TSH, but it weakly affected the phosphorylation state of Rb-related proteins in EGF+serum-treated cells. TGFbeta did not inhibit c-myc expression. In TSH-stimulated cells, TGFbeta did not affect the expression of cyclin D3, cdk4, and p27(kip1), nor the induced formation of cyclin D3-cdk4 complexes, but it prevented the TSH-induced relocalization of p27(kip1) from cdk2 to cyclin D3-cdk4. It prevented the nuclear translocations of cdk4 and cyclin D3 without altering the assembly of cyclin D3-cdk4 complexes probably formed in the cytoplasm, where they were prevented from sequestering nuclear p27(kip1) away from cdk2. This study dissociates the assembly of cyclin D3-cdk4 complexes from their nuclear localization and association with p27(kip1). It provides a new mechanism of regulation of proliferation by TGFbeta, which points out the subcellular location of cyclin D-cdk4 complexes as a crucial factor integrating mitogenic and antimitogenic regulations in an epithelial cell in primary culture.     

Analysis of cyclin D3-cdk4 complexes in fibroblasts expressing and lacking p27(kip1) and p21(cip1)

Our studies examined the effects of p27(kip1) and p21(cip1) on the assembly and activity of cyclin D3-cdk4 complexes and determined the composition of the cyclin D3 pool in cells containing and lacking these cyclin-dependent kinase inhibitors. We found that catalytically active cyclin D3-cdk4 complexes were present in fibroblasts derived from p27(kip1)-p21(cip1)-null mice and that immunodepletion of extracts of wild-type cells with antibody to p27(kip1) and/or p21(cip1) removed cyclin D3 protein but not cyclin D3-associated activity. Similar results were observed in experiments assaying cyclin D1-cdk4 activity. Data obtained using mixed cell extracts demonstrated that p27(kip1) interacted with cyclin D3-cdk4 complexes in vitro and that this interaction was paralleled by a loss of cyclin D3-cdk4 activity. In p27(kip1)-p21(cip1)-deficient cells, the cyclin D3 pool consisted primarily of cyclin D3 monomers, whereas in wild-type cells, the majority of cyclin D3 molecules were complexed to cdk4 and either p27(kip1) or p21(cip1) or were monomeric. We conclude that neither p27(kip1) nor p21(cip1) is required for the formation of cyclin D3-cdk4 complexes and that cyclin D3-cdk4 complexes containing p27(kip1) or p21(cip1) are inactive. We suggest that only a minor portion of the total cyclin D3 pool accounts for all of the cyclin D3-cdk4 activity in the cell regardless of whether the cell contains p27(kip1) and p21(cip1).     

p27Kip1, a double-edged sword in Shh-mediated medulloblastoma

Medulloblastoma, a brain tumor arising in the cerebellum, is the most common solid childhood malignancy. The current standard of care for medulloblastoma leaves survivors with life-long side effects. Gaining insight into mechanisms regulating transformation of medulloblastoma cells-of-origin may lead to development of better treatments for these tumors. Cerebellar granule neuron precursors (CGNPs) are proposed cells-of-origin for certain classes of medulloblastoma, specifically those marked by aberrant Sonic hedgehog (Shh) signaling pathway activation. CGNPs require signaling by Shh for proliferation during brain development. In mitogen-stimulated cells, nuclear localized cyclin dependent kinase (cdk) inhibitor p27 (Kip1) functions as a checkpoint control at the G1- to S-phase transition by inhibiting cdk2. Recent studies have suggested cytoplasmically localized p27^kip1 acquires oncogenic functions. Here, we show that p27^Kip1 is cytoplasmically localized in CGNPs and mouse Shh-mediated medulloblastomas. Tranasgenic mice bearing an activating mutation in the Shh pathway and lacking one or both p27^Kip1 alleles have accelerated tumor incidence compared to mice bearing both p27^Kip1 alleles. Interestingly, mice heterozygous for p27^Kip1 have decreased survival latency compared to p27^Kip1-null animals. Our data indicate that this may reflect the requirement for at least one copy of p27^Kip1 for recruiting cyclin D/cdk4/6 to promote cell cycle progression yet insufficient expression in the heterozygous or null state to inhibit cyclin E/cdk2. Finally, we find that mis-localized p27^Kip1 may play a positive role in motility in medulloblastoma cells. Together, our data indicate that the dosage of p27^Kip1 plays a role in cell cycle progression and tumor suppression in Shh-mediated medulloblastoma expansion.     

Phosphorylation of the cyclin-dependent kinase inhibitor p21Cip1 on serine 130 is essential for viral cyclin-mediated bypass of a p21Cip1-imposed G1 arrest

K cyclin encoded by Kaposi's sarcoma-associated herpesvirus confers resistance to the cyclin-dependent kinase (cdk) inhibitors p16Ink4A, p21Cip1, and p27Kip1 on the associated cdk6. We have previously shown that K cyclin expression enforces S-phase entry on cells overexpressing p27Kip1 by promoting phosphorylation of p27Kip1 on threonine 187, triggering p27Kip1 down-regulation. Since p21Cip1 acts in a manner similar to that of p27Kip1, we have investigated the subversion of a p21Cip1-induced G1 arrest by K cyclin. Here, we show that p21Cip1 is associated with K cyclin both in overexpression models and in primary effusion lymphoma cells and is a substrate of the K cyclin/cdk6 complex, resulting in phosphorylation of p21Cip1 on serine 130. This phosphoform of p21Cip1 appeared unable to associate with cdk2 in vivo. We further demonstrate that phosphorylation on serine 130 is essential for K cyclin-mediated release of a p21Cip1-imposed G1 arrest. Moreover, we show that under physiological conditions of cell cycle arrest due to elevated levels of p21Cip1 resulting from oxidative stress, K cyclin expression enabled S-phase entry and was associated with p21Cip1 phosphorylation and partial restoration of cdk2 kinase activity. Thus, expression of the viral cyclin enables cells to subvert the cell cycle inhibitory function of p21Cip1 by promoting cdk6-dependent phosphorylation of this antiproliferative protein.     

Human ribosomal protein S13 promotes gastric cancer growth through down‐regulating p27Kip1

Our previous works revealed that human ribosomal protein S13 (RPS13) was up‐regulated in multidrug‐resistant gastric cancer cells and overexpression of RPS13 could protect gastric cancer cells from drug‐induced apoptosis. The present study was designed to explore the role of RPS13 in tumorigenesis and development of gastric cancer. The expression of RPS13 in gastric cancer tissues and normal gastric mucosa was evaluated by immunohistochemical staining and Western blot analysis. It was found RPS13 was expressed at a higher level in gastric cancer tissues than that in normal gastric mucosa. RPS13 was then genetically overexpressed in gastric cancer cells or knocked down by RNA interference. It was demonstrated that up‐regulation of RPS13 accelerated the growth, enhanced in vitro colony forming and soft agar cologenic ability and promoted in vivo tumour formation potential of gastric cancer cells. Meanwhile, down‐regulation of RPS13 in gastric cancer cells resulted in complete opposite effects. Moreover, overexpression of RPS13 could promote G1 to S phase transition whereas knocking down of RPS13 led to G1 arrest of gastric cancer cells. It was further demonstrated that RPS13 down‐regulated p27^kip1 expression and CDK2 kinase activity but did not change the expression of cyclin D, cyclin E, CDK2, CDK4 and p16^INK4A. Taken together, these data indicate that RPS13 could promote the growth and cell cycle progression of gastric cancer cells at least through inhibiting p27^kip1 expression.     

Low levels of cyclin D and nonfunctional Rb protein affect cdk6 association with cyclin-dependent kinase inhibitor p27(Kip1)

p27(Kip1) associates with cyclin/cdk complexes and inhibiting cdk activity, and overexpression of p27(Kip1) induces G1 arrest. We found that p27(Kip1) overexpression inhibits cdk2 kinase activity, but not cdk6 kinase activity in HeLa cells. The amount of p27(Kip1) associated with cdk2 was significantly higher than that associated with cdk6. cdk6 complexes contained detectable amounts of p27(Kip1) in all human cell lines examined, except in HeLa cells where p27(Kip1) preferentially associated with cdk2. It appears that in HeLa cells overexpressed p27(Kip1) fails to inhibit cdk6 kinase activity because of low binding affinity of cdk6 to p27(Kip1). The low binding affinity is due to a low level of the cdk6/cyclin D complexes. Functional inactivation of pRb has an effect on p27(Kip1) association with cdk6/cyclin D complexes.