Cyclin-dependent kinase inhibitor p27(Kip1) is required for mouse mammary gland morphogenesis and function

We have studied the role of the cyclin-dependent kinase (Cdk) inhibitor p27(Kip1) in postnatal mammary gland morphogenesis. Based on its ability to negatively regulate cyclin/Cdk function, loss of p27 may result in unrestrained cellular proliferation. However, recent evidence about the stabilizing effect of p27 on cyclin D1-Cdk4 complexes suggests that p27 deficiency might recapitulate the hypoplastic mammary phenotype of cyclin D1-deficient animals. These hypotheses were investigated in postnatal p27-deficient (p27(-/-)), hemizygous (p27(+/)-), or wild-type (p27(+/+)) mammary glands. Mammary glands from p27(+/)- mice displayed increased ductal branching and proliferation with delayed postlactational involution. In contrast, p27(-/-) mammary glands or wild-type mammary fat pads reconstituted with p27(-/-) epithelium produced the opposite phenotype: hypoplasia, low proliferation, decreased ductal branching, impaired lobuloalveolar differentiation, and inability to lactate. The association of cyclin D1 with Cdk4, the kinase activity of Cdk4 against pRb in vitro, the nuclear localization of cyclin D1, and the stability of cyclin D1 were all severely impaired in p27(-/-) mammary epithelial cells compared with p27(+/+) and p27(+/-) mammary epithelial cells. Therefore, p27 is required for mammary gland development in a dose-dependent fashion and positively regulates cyclin D-Cdk4 function in the mammary gland.     

ErbB2 potentiates breast tumor proliferation through modulation of p27(Kip1)-Cdk2 complex formation: receptor overexpression does not determine growth dependency

Overexpression of the ErbB2 receptor, a major component of the ErbB receptor signaling network, contributes to the development of a number of human cancers. ErbB2 presents itself, therefore, as a target for antibody-mediated therapies. In this respect, anti-ErbB2 monoclonal antibody 4D5 specifically inhibits the growth of tumor cells overexpressing ErbB2. We have analyzed the effect of 4D5-mediated ErbB2 inhibition on the cell cycle of the breast tumor cell line BT474. 4D5 treatment of BT474 cells resulted in a G(1) arrest, preceded by rapid dephosphorylation of ErbB2, inhibition of cytoplasmic signal transduction pathways, accumulation of the cyclin-dependent kinase inhibitor p27(Kip1), and inactivation of cyclin-Cdk2 complexes. Time courses demonstrated that 4D5 treatment redirects p27(Kip1) onto Cdk2 complexes, an event preceding increased p27(Kip1) expression; this correlates with the downregulation of c-Myc and D-type cyclins (proteins involved in p27(Kip1) sequestration) and the loss of p27(Kip1) from Cdk4 complexes. Similar events were observed in ErbB2-overexpressing SKBR3 cells, which exhibited reduced proliferation in response to 4D5 treatment. Here, p27(Kip1) redistribution resulted in partial Cdk2 inactivation, consistent with a G1 accumulation. Moreover, p27(Kip1) protein levels remained constant. Antisense-mediated inhibition of p27(Kip1) expression in 4D5-treated BT474 cells further demonstrated that in the absence of p27(Kip1) accumulation, p27(Kip1) redirection onto Cdk2 complexes is sufficient to inactivate Cdk2 and establish the G(1) block. These data suggest that ErbB2 overexpression leads to potentiation of cyclin E-Cdk2 activity through regulation of p27(Kip1) sequestration proteins, thus deregulating the G(1)/S transition. Moreover, through comparison with an ErbB2-overexpressing cell line insensitive to 4D5 treatment, we demonstrate the specificity of these cell cycle events and show that ErbB2 overexpression alone is insufficient to determine the cellular response to receptor inhibition.     

The proto-oncogene c-myc acts through the cyclin-dependent kinase (Cdk) inhibitor p27(Kip1) to facilitate the activation of Cdk4/6 and early G(1) phase progression

Progression through the early G(1) phase of the cell cycle requires mitogenic stimulation, which ultimately leads to the activation of cyclin-dependent kinases 4 and 6 (Cdk4/6). Cdk4/6 activity is promoted by D-type cyclins and opposed by Cdk inhibitor proteins. Loss of c-myc proto-oncogene function results in a defect in the activation of Cdk4/6. c-myc(-/-) cells express elevated levels of the Cdk inhibitor p27(Kip1) and reduced levels of Cdk7, the catalytic subunit of Cdk-activating kinase. We show here that in normal (c-myc(+/+)) cells, the majority of cyclin D-Cdk4/6 complexes are assembled with p27 and remain inactive during cell cycle progression; their function is presumably to sequester p27 from Cdk2 complexes. A small fraction of Cdk4/6 protein was found in lower molecular mass catalytically active complexes. Conditional overexpression of p27 in c-myc(+/+) cells caused inhibition of Cdk4/6 activity and elicited defects in G(0)-to-S phase progression very similar to those seen in c-myc(-/-) cells. Overexpression of cyclin D1 in c-myc(-/-) cells rescued the defect in Cdk4/6 activity, indicating that the limiting factor is the number of cyclin D-Cdk4/6 complexes. Cdk-activating kinase did not rescue Cdk4/6 activity. We propose that the defect in Cdk4/6 activity in c-myc(-/-) cells is caused by the elevated levels of p27, which convert the low abundance activable cyclin D-Cdk4/6 complexes into unactivable complexes containing higher stoichiometries of p27. These observations establish p27 as a physiologically relevant regulator of cyclin D-Cdk4/6 activity as well as mechanistically a target of c-Myc action and provide a model by which c-Myc influences the early-to-mid G(1) phase transition.     

The cyclin-dependent kinase inhibitor p27 (Kip1) regulates both DNA synthesis and apoptosis in mammary epithelium but is not required for its functional development during pregnancy

Decreased expression of the cyclin-dependent kinase (CDK) inhibitor p27(Kip1) is common in breast cancer and is associated with poor prognosis. p27 is also an important mediator of steroidal regulation of cell cycle progression. We have therefore investigated the role of p27 in mammary epithelial cell proliferation. Examination of the two major functions of p27, assembly of cyclin D1-Cdk4 complexes and inhibition of Cdk2 activity, revealed that cyclin D1-Cdk4 complex formation was not impaired in p27-/- mammary epithelial cells in primary culture. However, cyclin E-Cdk2 activity was increased approximately 3-fold, indicating that the CDK inhibitory function of p27 is important in mammary epithelial cells. Increased epithelial DNA synthesis was observed during pregnancy in p27-/- mammary gland transplants, but this was paralleled by increased apoptosis. During pregnancy and at parturition, development and differentiation of p27+/+ and p27-/- mammary tissue were indistinguishable. These results demonstrate a role for p27 in both the proliferation and survival of mammary epithelial cells. However, the absence of morphological and cellular defects in p27-/- mammary tissue during pregnancy raises the possibility that loss of p27 in breast cancer may not confer an overall growth advantage unless apoptosis is also impaired.     

Cooperation of p27(Kip1) and p18(INK4c) in progestin-mediated cell cycle arrest in T-47D breast cancer cells

The steroid hormone progesterone regulates proliferation and differentiation in the mammary gland and uterus by cell cycle phase-specific actions. The long-term effect of progestins on T-47D breast cancer cells is inhibition of cellular proliferation. This is accompanied by decreased G(1) cyclin-dependent kinase (CDK) activities, redistribution of the CDK inhibitor p27(Kip1) among these CDK complexes, and alterations in the elution profile of cyclin E-Cdk2 upon gel filtration chromatography, such that high-molecular-weight complexes predominate. This study aimed to determine the relative contribution of CDK inhibitors to these events. Following progestin treatment, the majority of cyclin E- and D-CDK complexes were bound to p27(Kip1) and few were bound to p21(Cip1). In vitro, recombinant His(6)-p27 could quantitatively reproduce the effects on cyclin E-Cdk2 kinase activity and the shift in molecular weight observed following progestin treatment. In contrast, cyclin D-Cdk4 was not inhibited by His(6)-p27 in vitro or p27(Kip1) in vivo. However, an increase in the expression of the Cdk4/6 inhibitor p18(INK4c) and its extensive association with Cdk4 and Cdk6 were apparent following progestin treatment. Recombinant p18(INK4c) led to the reassortment of cyclin-CDK-CDK inhibitor complexes in vitro, with consequent decrease in cyclin E-Cdk2 activity. These results suggest a concerted model of progestin action whereby p27(Kip1) and p18(INK4c) cooperate to inhibit cyclin E-Cdk2 and Cdk4. Since similar models have been developed for growth inhibition by transforming growth factor beta and during adipogenesis, interaction between the Cip/Kip and INK4 families of inhibitors may be a common theme in physiological growth arrest and differentiation.     

Retinoic acid-mediated G1 arrest is associated with induction of p27(Kip1) 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 beta (RARbeta) 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.     

Vitamin D inhibits G1 to S progression in LNCaP prostate cancer cells through p27Kip1 stabilization and Cdk2 mislocalization to the cytoplasm

1,25-(OH)2 vitamin D3 (1,25-(OH)2D3) exerts antiproliferative effects via cell cycle regulation in a variety of tumor cells, including prostate. We have previously shown that in the human prostate cancer cell line LN-CaP, 1,25-(OH)2D3 mediates an increase in cyclin-dependent kinase inhibitor p27Kip1 levels, inhibition of cyclin-dependent kinase 2 (Cdk2) activity, hypophosphorylation of retinoblastoma protein, and accumulation of cells in G1. In this study, we investigated the mechanism whereby 1,25-(OH)2D3 increases p27 levels. 1,25-(OH)2D3 had no effect on p27 mRNA levels or on the regulation of a 3.5-kb fragment of the p27 promoter. The rate of p27 protein synthesis was not affected by 1,25-(OH)2D3 as measured by luciferase activity driven by the 5'- and 3'-untranslated regions of p27 that regulate p27 protein synthesis. Pulse-chase analysis of 35S-labeled p27 revealed an increased p27 protein half-life with 1,25-(OH)2D3 treatment. Because Cdk2-mediated phosphorylation of p27 at Thr187 targets p27 for Skp2-mediated degradation, we examined the phosphorylation status of p27 in 1,25-(OH)2D3-treated cells. 1,25-(OH)2D3 decreased levels of Thr187 phosphorylated p27, consistent with inhibition of Thr187 phosphorylation-dependent p27 degradation. In addition, 1,25-(OH)2D3 reduced Skp2 protein levels in LNCaP cells. Cdk2 is activated in the nucleus by Cdk-activating kinase through Thr160 phosphorylation and by cdc25A phosphatase via Thr14 and Tyr15 dephosphorylation. Interestingly, 1,25-(OH)2D3 decreased nuclear Cdk2 levels as assessed by subcellular fractionation and confocal microscopy. Inhibition of Cdk2 by 1,25-(OH)2D3 may thus involve two mechanisms: 1) reduced nuclear Cdk2 available for cyclin binding and activation and 2) impairment of cyclin E-Cdk2-dependent p27 degradation through cytoplasmic mislocalization of Cdk2. These data suggest that Cdk2 mislocalization is central to the antiproliferative effects of 1,25-(OH)2D3.     

Genetic evidence for the interactions of cyclin D1 and p27(Kip1) in mice

The cell cycle of cultured cells appears to be regulated by opposing actions of the cyclins together with their partners, the cyclin-dependent kinases (Cdk), and their inhibitors (Cki). Consistent with this situation null mutations in the genes for cyclin D1 and Cki p27(Kip1) in mice give opposite phenotypes of dwarfism and gigantism. To test their genetic interactions, we generated mice nullizygous for both genes. Correction of cyclin D1 or p27 null to wild-type phenotypes was observed for many but not all traits. These included, for cyclin D1(-/-) mice, body weight, early lethality, retinal hypoplasia, and male aggressiveness and, for p27(-/-) mice, body weight, retinal hyperplasia, and embryo implantation. p27(-/-) traits that were not corrected were the aberrant estrus cycles, luteal cell proliferation, and susceptibility to pituitary tumors. This mutual correction of these phenotypes is the first genetic demonstration of the interaction of these inhibitory and stimulatory cell cycle-regulatory molecules in vivo. The molecular basis for the correction was analyzed in the neonatal retina. Retinal cellularity was rescued in the cyclin D1 null mouse by loss of p27 with only a partial restoration of phosphorylation of retinoblastoma protein (Rb) and Cdk4 activity but with a dramatic elevation of Cdk2 activity. Our data provide in vivo genetic validation of cell culture experiments that indicated that p27 acts as a negative regulator of cyclin E-Cdk2 activity and that it can be titrated away by cyclin D-Cdk4 complexes. It also supports the suggestion that the cyclin E/Cdk2 pathway can largely bypass Rb in regulating the cell cycle in vivo.     

Redistribution of the CDK inhibitor p27 between different cyclin.CDK complexes in the mouse fibroblast cell cycle and in cells arrested with lovastatin or ultraviolet irradiation.

The cyclin-dependent kinase (CDK) inhibitor p27 binds and inhibits the kinase activity of several CDKs. Here we report an analysis of the behavior and partners of p27 in Swiss 3T3 mouse fibroblasts during normal mitotic cell cycle progression, as well as in cells arrested at different stages in the cycle by growth factor deprivation, lovastatin treatment, or ultraviolet (UV) irradiation. We found that the level of p27 is elevated in cells arrested in G0 by growth factor deprivation or contact inhibition. In G0, p27 was predominantly monomeric, although some portion was associated with residual cyclin A.Cdk2. During G1, all of p27 was associated with cyclin D1.Cdk4 and was then redistributed to cyclin A.Cdk2 as cells entered S phase. The loss of the monomeric p27 pool as cyclins accumulate in G1 is consistent with the in vivo and in vitro data showing that p27 binds better to cyclin.CDK complexes than to monomeric CDKs. In growing cells, the majority of p27 was associated with cyclin D1 and the level of p27 was significantly lower than the level of cyclin D1. In cells arrested in G1 with lovastatin, cyclin D1 was degraded and p27 was redistributed to cyclin A.Cdk2. In contrast to p21 (which is a p27-related CDK inhibitor and is induced by UV irradiation), the level of p27 was reduced after UV irradiation, but because cyclin D1 was degraded more rapidly than p27, there was a transient increase in binding of p27 to cyclin A.Cdk2. These data suggest that cyclin D1.Cdk4 acts as a reservoir for p27, and p27 is redistributed from cyclin D1.Cdk4 to cyclin A.Cdk2 complexes during S phase, or when cells are arrested by growth factor deprivation, lovastatin treatment, or UV irradiation. It is likely that a similar principle of redistribution of p27 is used by the cell in other instances of cell cycle arrest.     

Non-malignant and tumor-derived cells differ in their requirement for p27Kip1 in transforming growth factor-beta-mediated G1 arrest

Transforming growth factor beta (TGF-beta) induces G(1) arrest in susceptible cells by multiple mechanisms that inhibit the G(1) cyclin-dependent kinases (Cdks), including Cdk2, Cdk4, and Cdk6. TGF-beta treatment of early passage finite lifespan human mammary epithelial cells (HMECs) led to an accumulation of p27(Kip1) in cyclin E1-Cdk2 complexes and kinase inhibition. The requirement for p27 in the G(1) arrest by TGF-beta was assessed by transfection of antisense p27 (ASp27) oligonucleotides into TGF-beta-treated HMECs. Despite a reduction in total and cyclin E-Cdk2 bound p27 after ASp27 transfection, HMECs remained arrested in the G(1) phase. Maintenance of the G(1) arrest was accompanied by increased association of the Cdk inhibitor p21(WAF-1/Cip-1) and the retinoblastoma family member p130(Rb2) in cyclin E1-Cdk2 complexes along with kinase inhibition. In contrast to the findings in HMECs, p27 was essential for G(1) arrest by TGF-beta in two tumor-derived lines. ASp27 transfection into two TGF-beta-responsive, cancer-derived lines was not associated with increased compensatory binding of p21 and p130 to cyclin E1-Cdk2, and these cell lines failed to maintain G(1) arrest despite the continued presence of TGF-beta. Progressive cell cycle deregulation leading to impaired checkpoint controls during malignant tumor progression may alter the role of p27 from a redundant to an essential inhibitor of G(1)-to-S phase progression.     

Incomplete folding upon binding mediates Cdk4/cyclin D complex activation by tyrosine phosphorylation of inhibitor p27 protein

p27(Kip1) (p27), an intrinsically disordered protein, regulates the various Cdk/cyclin complexes that control cell cycle progression. The kinase inhibitory domain of p27 contains a cyclin-binding subdomain (D1), a Cdk-binding subdomain (D2), and a linker helix subdomain that connects D1 and D2. Here, we report that, despite extensive sequence conservation between Cdk4/cyclin D1 (hereafter Cdk4/cyclin D) and Cdk2/cyclin A, the thermodynamic details describing how the individual p27 subdomains contribute to equally high affinity binding to these two Cdk/cyclin complexes are strikingly different. Differences in enthalpy/entropy compensation revealed that the D2 subdomain of p27 folds incompletely when binding Cdk4/cyclin D versus Cdk2/cyclin A. Incomplete binding-induced folding exposes tyrosine 88 of p27 for phosphorylation by the nonreceptor tyrosine kinase Abl. Importantly, tyrosine phosphorylation (of p27) relieves Cdk inhibition by p27, enabling cell cycle entry. Furthermore, the interaction between a conserved hydrophobic patch on cyclin D and subdomain D1 is much weaker than that with cyclin A; consequently, a construct containing subdomains D1 and LH (p27-D1LH) does not inhibit substrate binding to Cdk4/cyclin D as it does to Cdk2/cyclin A. Our results provide a mechanism by which Cdk4 (within the p27/Cdk4/cyclin D complex) is poised to be activated by extrinsic mitogenic signals that impinge upon p27 at the earliest stage of cell division. More broadly, our results further illustrate the regulatory versatility of intrinsically disordered proteins.     

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.     

Increased expression of MDM2, cyclin D1, and p27Kip1 in carcinogen-induced rat mammary tumors

It is thought that environmental pollutants, such as polycyclic aromatic hydrocarbons (PAH), contribute to human breast tumorigenesis, yet their roles remain incompletely elucidated. The prototypical PAH 7,12-dimethylbenz(alpha)anthracene (DMBA) specifically and effectively induces mammary tumor formation in rodent models. In an attempt to explore the molecular mechanisms by which PAH initiates and promotes mammary tumorigenesis, we examined the expression of several cell cycle regulators in rat mammary tumors induced by DMBA. Expression of cyclin D1, murine double minute-2 (MDM2), and Akt was up-regulated in tumors in comparison to normal mammary glands, as indicated by RT-PCR, Western blot analysis, and immunohistochemical staining. Expression of p27Kip1 protein was also elevated in the tumors with increased cytoplasmic localization. However, RB protein remained hyperphosphorylated. To directly test the effects of DMBA, the MCF-7 human breast cancer cells were treated. DMBA induced MDM2 expression in a dose- and time-dependent fashion in the MCF-7 cells, and this activation appeared to be p53 dependent. These data suggest that activation of cyclin D1, MDM2, and AKT as well as increased expression and cytoplasmic localization of p27Kip1 may play a role in this model of environmental pollutant-induced mammary tumorigenesis.     

Mechanism of cell cycle entry mediated by the intrinsically disordered protein p27(Kip1)

p27(Kip1) (p27), a prototypical intrinsically disordered protein (IDP), regulates eukaryotic cell division through interactions with cyclin-dependent kinase (Cdk)/cyclin complexes. The activity, stability, and subcellular localization of p27 are regulated by phosphorylation. We illustrate how p27 integrates regulatory signals from several non-receptor tyrosine kinases (NRTKs) to activate Cdk4 and initiate cell cycle entry. Unmodified p27 potently inhibits Cdk/cyclin complexes, including Cdk4/cyclin D (IC(50), 1 nM). Some NRTKs (e.g., Abl) phosphorylate p27 on Tyr 88, which facilitates a second modification on Tyr 74 by another NRTK (e.g., Src). Importantly, this second modification causes partial reactivation of Cdk4 within ternary complexes containing doubly Tyr phosphorylated p27. Partial activation of Cdk4 initiates entry into the cell division cycle. Therefore, p27's disordered features enable NRTKs to sequentially promote a phosphorylation cascade that controls cell fate. Beyond cell cycle control, these results illustrate general concepts regarding why IDPs are well-suited for roles in signaling and regulation in biological systems.     

Phosphorylation of p27Kip1 regulates assembly and activation of cyclin D1-Cdk4

p27 mediates Cdk2 inhibition and is also found in cyclin D1-Cdk4 complexes. The present data support a role for p27 in the assembly of D-type cyclin-Cdk complexes and indicate that both cyclin D1-Cdk4-p27 assembly and kinase activation are regulated by p27 phosphorylation. Prior work showed that p27 can be phosphorylated by protein kinase B/Akt (PKB/Akt) at T157 and T198. Here we show that PKB activation and the appearance of p27pT157 and p27pT198 precede p27-cyclin D1-Cdk4 assembly in early G₁. PI3K/PKB inhibition rapidly reduced p27pT157 and p27pT198 and dissociated cellular p27-cyclin D1-Cdk4. Mutant p27 allele products lacking phosphorylation at T157 and T198 bound poorly to cellular cyclin D1 and Cdk4. Cellular p27pT157 and p27pT198 coprecipitated with Cdk4 but were not detected in Cdk2 complexes. The addition of p27 to recombinant cyclin D1 and Cdk4 led to cyclin D1-Cdk4-p27 complex formation in vitro. p27 phosphorylation by PKB increased p27-cyclin D1-Cdk4 assembly in vitro but yielded inactive Cdk4. In contrast, Src pretreatment of p27 did not affect p27-cyclin D1-Cdk4 complex formation. However, Src treatment led to tyrosine phosphorylation of p27 and catalytic activation of assembled cyclin D1-Cdk4-p27 complexes. Thus, while PKB-dependent p27 phosphorylation appears to increase cyclin D1-Cdk4-p27 assembly or stabilize these complexes in vitro, cyclin D1-Cdk4-p27 activation requires the tyrosine phosphorylation of p27. Constitutive activation of PKB and Abl or Src family kinases in cancers would drive p27 phosphorylation, increase cyclin D1-Cdk4 assembly and activation, and reduce the cyclin E-Cdk2 inhibitory function of p27. Combined therapy with both Src and PI3K/PKB inhibitors may reverse this process.     

Cyclin D2 translocates p27 out of the nucleus and promotes its degradation at the G0-G1 transition

The nuclear export and cytoplasmic degradation of the cyclin-dependent kinase inhibitor p27 are required for effective progression of the cell cycle through the G(0)-G(1) transition. The mechanism responsible for this translocation of p27 has remained unclear, however. We now show that cyclin D2 directly links growth signaling with the nuclear export of p27 at the G(0)-G(1) transition in some cell types. The up-regulation of cyclin D2 in response to mitogenic stimulation was found to occur earlier than that of other D-type cyclins and in parallel with down-regulation of p27 at the G(0)-G(1) transition. RNA interference-mediated depletion of cyclin D2 inhibited the nuclear export of p27 and delayed its degradation at the G(0)-G(1) transition. In contrast, overexpression of cyclin D2 in G(0) phase shifted the localization of p27 from the nucleus to the cytoplasm and reduced the stability of p27. Overexpression of the cyclin D2(T280A) mutant, whose export from the nucleus is impaired, prevented the translocation and degradation of p27. These results indicate that cyclin D2 translocates p27 from the nucleus into the cytoplasm for its KPC-dependent degradation at the G(0)-G(1) transition.     

Cyclin D1 regulates p27 stability in B cells

p27^Kip1 is a cyclin-dependent kinase inhibitor that plays a critical role in regulating G₁/S transition, and whose activity is, in part, regulated through interactions with D-type cyclins. We have generated the BD1-9 cell line, a BaF3 pro-B cells derivative in which cyclin D1 can be induced rapidly and reversibly by ponasterone A. The induction of cyclin D1 expression leads to a targeted p27^Kip1 accumulation in both cytoplasmic and nuclear compartments. But, only the p27^Kip1 form phosphorylated on serine 10 (pSer10-p27^Kip1) accumulates in BD1-9 cells. We found that the binding of cyclin D1 and pSer10-p27^Kip1 prevents p27^Kip1 degradation by the cytoplasmic Kip1 ubiquitylation-promoting complex (KPC) proteosomic pathway. Importantly, the nuclear CDK2 activity which is crucial for G₁/S transition is not altered by p27^Kip1 increase. Using siRNA techniques, we revealed that p27^Kip1 inhibition does not affect the distribution of BD1-9 cells in the different phases of the cell cycle. Our study demonstrates that aberrant cyclin D1 expression acts as a p27^Kip1 trap in B lymphocytes but does not induce p27^Kip1 relocation from the nucleus to the cytoplasm and does not modulate the G₁/S transition. Since our cellular model mimics what observed in aggressive lymphomas, our data bring new insights into the understanding of their physiopathology.     

Cyclin D1 overexpression induces progestin resistance in T-47D breast cancer cells despite p27(Kip1) association with cyclin E-Cdk2

Long-term growth inhibition, arrest in G(1) phase and reduced activity of both cyclin D1-Cdk4 and cyclin E-Cdk2 are elicited by progestin treatment of breast cancer cells in culture. Decreased cyclin expression, induction of p18(INK4c) and increased association of the CDK inhibitors p21(WAF1/Cip1) and p27(Kip1) with cyclin E-Cdk2 have been implicated in these responses. To determine the role of decreased cyclin expression, T-47D human breast cancer cells constitutively expressing cyclin D1 or cyclin E were treated with the progestin ORG 2058. Overexpression of cyclin E had only a modest effect on growth inhibition. Although cyclin E expression was maintained during progestin treatment, cyclin E-Cdk2 activity decreased by approximately 60%. This was accompanied by p27(Kip1) association with cyclin E-Cdk2, indicating that both cyclin E down-regulation and p27(Kip1) recruitment contribute to the decrease in activity. In contrast, overexpression of cyclin D1 induced progestin resistance and cell proliferation continued despite decreased cyclin E-Cdk2 activity. Progestin treatment of cyclin D1-overexpressing cells was associated with increased p27(Kip1) association with cyclin E-Cdk2. Thus the ability of cyclin D1 to confer progestin resistance does not depend on sequestration of p27(Kip1) away from cyclin E-Cdk2, providing evidence for a critical function of cyclin D1 other than as a high-capacity "sink" for p27(Kip1). These data indicate that regulation of cyclin D1 is a critical element of progestin inhibition in breast cancer cells and suggest that breast cancers overexpressing cyclin D1 may respond poorly to progestin therapy.     

Cdc6 protein activates p27KIP1-bound Cdk2 protein only after the bound p27 protein undergoes C-terminal phosphorylation

In mammalian cells Cdk2 activity during the G(1)-S transition is mainly controlled by p27(KIP1). Although the amount and subcellular localization of p27 influence Cdk2 activity, how Cdk2 activity is regulated during this phase transition still remains virtually unknown. Here we report an entirely new mechanism for this regulation. Cdc6 the AAA+ ATPase, known to assemble prereplicative complexes on chromosomal replication origins and activate p21(CIP1)-bound Cdk2, also activated p27-bound Cdk2 in its ATPase and cyclin binding motif-dependent manner but only after the p27 bound to the Cdk2 was phosphorylated at the C terminus. ROCK, which mediates a signal for cell anchorage to the extracellular matrix and activates the mTORC1 cascade as well as controls cytoskeleton assembly, was partly responsible for C-terminal phosphorylation of the p27. In vitro reconstitution demonstrated ROCK (Rho-associated kinase)-mediated phosphorylation of Cdk2-bound p27 at the C terminus and subsequent activation of the Cdk2 by Cdc6.