Overexpression of integrin beta1 inhibits proliferation of hepatocellular carcinoma cell SMMC-7721 through preventing Skp2-dependent degradation of p27 via PI3K pathway

Integrins may play important roles in many cellular events, such as cell proliferation, differentiation, and apoptosis. We showed previously that overexpression of integrin beta1 inhibits cell proliferation in SMMC-7721 cells. Here we reported that one of the cyclin-dependent kinase (CDK) inhibitors, p27(Kip1) was involved in proliferation-inhibition induced by overexpression of integrin beta1. Overexpression of integrin beta1 upregulated p27(Kip1) at the protein level, but not mRNA level. The knock-down of p27(Kip1) expression restored cell growth in integrin beta1-overexpressing cells. Cycloheximide (Chx) treatment and pulse-chase experiments revealed that overexpression of integrin beta1 prolonged the half-life of p27(Kip1) by inhibiting its degradation. Proteasome inhibitor (MG132) treatment of the cells indicated that proteasome mediated degradation of p27, and Skp2-dependent degradation might be prevented. Overexpression of integrin beta1 decreased Skp2 at mRNA level, which was regulated by cell adhesion and the subsequent adhesion-dependent signaling. Overexpression of integrin beta1 reduced cell adhesion, accordingly, inactivated the phosphoinositide 3-kinase (PI3K) signaling. PI3K inhibitor LY294002 upregulated p27(Kip1) at post-translational level and downregulate Skp2 at mRNA level, which could mimic the effects of integrin beta1 overexpression on p27(Kip1) and Skp2. Together, these results suggested that overexpression of integrin beta1 inhibited cell proliferation by preventing the Skp2-dependent degradation of p27(Kip1) via PI3K pathway.     

p27 Kip1 nuclear localization and cyclin-dependent kinase inhibitory activity are regulated by glycogen synthase kinase-3 in human colon cancer cells

The cellular mechanisms regulating intestinal differentiation are poorly understood. Sodium butyrate (NaBT), a short-chain fatty acid, increases p27 Kip1 expression and induces cell cycle arrest associated with intestinal cell differentiation. Here, we show that treatment of intestinal-derived cells with NaBT induced G0/G1 arrest and intestinal alkaline phosphatase, a marker of differentiation, activity and mRNA expression; this induction was attenuated by inhibition of glycogen synthase kinase-3 (GSK-3). Moreover, treatment with NaBT increased the nuclear, but not the cytosolic, expression and activity of GSK-3beta. NaBT decreased cyclin-dependent kinase CDK2 activity and induced p27 Kip1 expression; inhibition of GSK-3 rescued NaBT-inhibited CDK2 activity and blocked NaBT-induced p27 Kip1 expression in the nucleus but not in the cytoplasm. In addition, we demonstrate that NaBT decreased the expression of S-phase kinase-associated protein 2 (Skp2), and this decrease was attenuated by GSK-3 inhibition. Furthermore, NaBT increased p27 Kip1 binding to CDK2, which was completely abolished by GSK-3 inhibition. Overexpression of an active form of GSK-3beta reduced Skp2 expression, increased p27 Kip1 in the nucleus and increased p27 Kip1 binding to CDK2. Our results suggest that GSK-3 not only regulates nuclear p27 Kip1 expression through the downregulation of nuclear Skp2 expression but also functions to regulate p27 Kip1 assembly with CDK2, thereby playing a critical role in the G0/G1 arrest associated with intestinal cell differentiation.     

Skp2 regulates G2/M progression in a p53-dependent manner

Targeted proteasomal degradation mediated by E3 ubiquitin ligases controls cell cycle progression, and alterations in their activities likely contribute to malignant cell proliferation. S phase kinase-associated protein 2 (Skp2) is the F-box component of an E3 ubiquitin ligase complex that targets p27^Kip1 and cyclin E1 to the proteasome. In human melanoma, Skp2 is highly expressed, regulated by mutant B-RAF, and required for cell growth. We show that Skp2 depletion in melanoma cells resulted in a tetraploid cell cycle arrest. Surprisingly, co-knockdown of p27^Kip1 or cyclin E1 failed to prevent the tetraploid arrest induced by Skp2 knockdown. Enhanced Aurora A phosphorylation and repression of G2/M regulators cyclin B1, cyclin-dependent kinase 1, and cyclin A indicated a G2/early M phase arrest in Skp2-depleted cells. Furthermore, expression of nuclear localized cyclin B1 prevented tetraploid accumulation after Skp2 knockdown. The p53 status is most frequently wild type in melanoma, and the tetraploid arrest and down-regulation of G2/M regulatory genes were strongly dependent on wild-type p53 expression. In mutant p53 melanoma lines, Skp2 depletion did not induce cell cycle arrest despite up-regulation of p27^Kip1. These data indicate that elevated Skp2 expression may overcome p53-dependent cell cycle checkpoints in melanoma cells and highlight Skp2 actions that are independent of p27^Kip1 degradation.     

sst2 Somatostatin receptor inhibits cell proliferation through Ras-, Rap1-, and B-Raf-dependent ERK2 activation

The G protein-coupled sst2 somatostatin receptor is a critical negative regulator of cell proliferation. sstII prevents growth factor-induced cell proliferation through activation of the tyrosine phosphatase SHP-1 leading to induction of the cyclin-dependent kinase inhibitor p27Kip1. Here, we investigate the signaling molecules linking sst2 to p27Kip1. In Chinese hamster ovary-DG-44 cells stably expressing sst2 (CHO/sst2), the somatostatin analogue RC-160 transiently stimulates ERK2 activity and potentiates insulin-stimulated ERK2 activity. RC-160 also stimulates ERK2 activity in pancreatic acini isolated from normal mice, which endogenously express sst2, but has no effect in pancreatic acini derived from sst2 knock-out mice. RC-160-induced p27Kip1 up-regulation and inhibition of insulin-dependent cell proliferation are both prevented by pretreatment of CHO/sst2 cells with the MEK1/2 inhibitor PD98059. In addition, using dominant negative mutants, we show that sst2-mediated ERK2 stimulation is dependent on the pertussis toxin-sensitive Gi/o protein, the tyrosine kinase Src, both small G proteins Ras and Rap1, and the MEK kinase B-Raf but is independent of Raf-1. Phosphatidylinositol 3-kinase (PI3K) and both tyrosine phosphatases, SHP-1 and SHP-2, are required upstream of Ras and Rap1. Taken together, our results identify a novel mechanism whereby a Gi/o protein-coupled receptor inhibits cell proliferation by stimulating ERK signaling via a SHP-1-SHP-2-PI3K/Ras-Rap1/B-Raf/MEK pathway.     

The p42/p44 mitogen-activated protein kinase activation triggers p27Kip1 degradation independently of CDK2/cyclin E in NIH 3T3 cells.

The p42/p44 mitogen-activated protein (MAP) kinase is stimulated by various mitogenic stimuli, and its sustained activation is necessary for cell cycle G(1) progression and G(1)/S transition. G(1) progression and G(1)/S transition also depend on sequential cyclin-dependent kinase (CDK) activation. Here, we demonstrate that MAP kinase inhibition leads to accumulation of the CDK inhibitor p27(Kip1) in NIH 3T3 cells. Blocking the proteasome-dependent degradation of p27(Kip1) impaired this accumulation, suggesting that MAP kinase does not act on p27(Kip1) protein synthesis. In the absence of extracellular signals (growth factors or cell adhesion), genetic activation of MAP kinase decreased the expression of p27(Kip1) as assessed by cotransfection experiments and by immunofluorescence detection. Importantly, MAP kinase activation also decreased the expression of a p27(Kip1) mutant, which cannot be phosphorylated by CDK2, suggesting that MAP kinase-dependent p27(Kip1) regulation is CDK2-independent. Accordingly, expression of dominant-negative CDK2 did not impair the down-regulation of p27(Kip1) induced by MAP kinase activation. These data demonstrate that the MAP kinase pathway regulates p27(Kip1) expression in fibroblasts essentially through a degradation mechanism, independently of p27(Kip1) phosphorylation by CDK2. This strengthens the role of this CDK inhibitor as a key effector of G(1) growth arrest, whose expression can be controlled by extracellular stimuli-dependent signaling pathways.     

Blockade of the extracellular signal-regulated kinase pathway induces marked G1 cell cycle arrest and apoptosis in tumor cells in which the pathway is constitutively activated: up-regulation of p27(Kip1)

Constitutive activation of the ERK pathway is associated with the neoplastic phenotype of a relatively large number of human tumor cells. Blockade of the ERK pathway by treatment with PD98059, a specific inhibitor of mitogen-activated protein (MAP) kinase/ERK kinase (MEK), completely suppressed the growth of tumor cells in which the pathway is constitutively activated (RPMI-SE and HT1080 cells). Consistent with its prominent antiproliferative effect, PD98059 induced a remarkable G(1) cell cycle arrest, followed by a modest apoptotic response, in these tumor cells. Selective up-regulation of p27(Kip1) was observed after PD98059 treatment of RPMI-SE and HT1080 cells. Overexpression in RPMI-SE cells of either a kinase-negative form of MEK1 or wild-type MAP kinase phosphatase-3 also induced up-regulation of p27(Kip1). The up-regulation of p27(Kip1) correlated with increased association of p27(Kip1) with cyclin E-cyclin-dependent kinase (CDK) 2 complexes, a concomitant inhibition of cyclin E-CDK2 kinase activity, and a consequent decrease in the phosphorylation state of retinoblastoma protein, which would culminate in the marked G(1) cell cycle arrest observed in these tumor cells. These results suggest that the complete growth suppression that follows specific blockade of the ERK pathway in tumor cells in which the pathway is constitutively activated is mediated by up-regulation of p27(Kip1).     

p27(Kip1) stabilization and G(1) arrest by 1,25-dihydroxyvitamin D(3) in ovarian cancer cells mediated through down-regulation of cyclin E/cyclin-dependent kinase 2 and Skp1-Cullin-F-box protein/Skp2 ubiquitin ligase

p27(Kip1) (p27) is a tumor suppressor whose stability is controlled by proteasome-mediated degradation, a process directed in part by cyclin-dependent kinase 2 (CDK2)-mediated phosphorylation of p27 at Thr(187) and its subsequent interaction with the Skp1-Cullin-F-box protein/Skp2 (Skp2) ubiquitin ligase. The present study shows that 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) arrests ovarian cancer cells in G(1) by stabilizing the p27 protein. 1,25(OH)(2)D(3) initiates a chain of events by decreasing the amounts of cyclin E and cyclin E-associated CDK2 activity. As a result, p27 phosphorylation at Thr(187) and consequently the interaction with Skp2 are decreased. 1,25(OH)(2)D(3) also increases p27 stability by decreasing the abundance of Skp2. It is the combined effect of 1,25(OH)(2)D(3) on both the CDK2-dependent phosphorylation of p27, and thus its affinity for Skp2, and Skp2 expression that dramatically increases the stability of the p27 protein. Similar to its effects in ovarian cancer cells, 1,25(OH)(2)D(3) induces p27 accumulation in wild type mouse embryo fibroblasts and arrests wild type but not p27-null mouse embryo fibroblasts in G(1). Stable expression of Skp2 in OVCAR3 cells diminishes the G(1) arrest and decreases the growth response to 1,25(OH)(2)D(3). Taken together, the results of this study identify p27 as the key mediator of 1,25(OH)(2)D(3)-induced growth suppression in G(1) and show that the hormone achieves this by decreasing the activity of CDK2 and reducing the abundance of Skp2, which act together to degrade p27.     

p27Kip1 serine 10 phosphorylation determines its metabolism and interaction with cyclin-dependent kinases

p27Kip1 is a critical modulator of cell proliferation by controlling assembly, localization and activity of cyclin-dependent kinase (CDK). p27Kip1 also plays important roles in malignant transformation, modulating cell movement and interaction with the extracellular matrix. A critical p27Kip1 feature is the lack of a stable tertiary structure that enhances its “adaptability” to different interactors and explains the heterogeneity of its function. The absence of a well-defined folding underlines the importance of p27Kip1 post-translational modifications that might highly impact the protein functions. Here, we characterize the metabolism and CDK interaction of phosphoserine10-p27Kip1 (pS10- p27Kip1), the major phosphoisoform of p27Kip1. By an experimental strategy based on specific immunoprecipitation and bidimensional electrophoresis, we established that pS10-p27Kip1 is mainly bound to cyclin E/CDK2 rather than to cyclin A/CDK2. pS10- p27Kip1 is more stable than non-modified p27Kip1, since it is not (or scarcely) phosphorylated on T187, the post-translational modification required for p27Kip1 removal in the nucleus. pS10-p27Kip1 does not bind CDK1. The lack of this interaction might represent a mechanism for facilitating CDK1 activation and allowing mitosis completion. In conclusion, we suggest that nuclear p27Kip1 follows 2 almost independent pathways operating at different rates. One pathway involves threonine-187 and tyrosine phosphorylations and drives the protein toward its Skp2-dependent removal. The other involves serine-10 phosphorylation and results in the elongation of p27Kip1 half-life and specific CDK interactions. Thus, pS10-p27Kip1, due to its stability, might be thought as a major responsible for the p27Kip1-dependent arrest of cells in G1/G0 phase.     

Degradation of p27(Kip1) at the G(0)-G(1) transition mediated by a Skp2-independent ubiquitination pathway.

Targeting of the cyclin-dependent kinase inhibitor p27(Kip1) for proteolysis has been thought to be mediated by Skp2, the F-box protein component of an SCF ubiquitin ligase complex. Degradation of p27(Kip1) at the G(0)-G(1) transition of the cell cycle has now been shown to proceed normally in Skp2(-/-) lymphocytes, whereas p27(Kip1) proteolysis during S-G(2) phases is impaired in these Skp2-deficient cells. Degradation of p27(Kip1) at the G(0)-G(1) transition was blocked by lactacystin, a specific proteasome inhibitor, suggesting that it is mediated by the ubiquitin-proteasome pathway. The first cell cycle of stimulated Skp2(-/-) lymphocytes appeared normal, but the second cycle was markedly inhibited, presumably as a result of p27(Kip1) accumulation during S-G(2) phases of the first cell cycle. Polyubiquitination of p27(Kip1) in the nucleus is dependent on Skp2 and phosphorylation of p27(Kip1) on threonine 187. However, polyubiquitination activity was also detected in the cytoplasm of Skp2(-/-) cells, even with a threonine 187 --> alanine mutant of p27(Kip1) as substrate. These results suggest that a polyubiquitination activity in the cytoplasm contributes to the early phase of p27(Kip1) degradation in a Skp2-independent manner, thereby promoting cell cycle progression from G(0) to G(1).     

Phosphorylation of p27Kip1 on serine 10 is required for its binding to CRM1 and nuclear export

Phosphorylation of the cyclin-dependent kinase inhibitor p27(Kip1) has been thought to regulate its stability. Ser(10) is the major phosphorylation site of p27(Kip1), and phosphorylation of this residue affects protein stability. Phosphorylation of p27(Kip1) on Ser(10) has now been shown to be required for the binding of CRM1, a carrier protein for nuclear export. The p27(Kip1) protein was translocated from the nucleus to the cytoplasm at the G(0)-G(1) transition of the cell cycle, and this export was inhibited by leptomycin B, a specific inhibitor of CRM1-dependent nuclear export. The nuclear export and subsequent degradation of p27(Kip1) at the G(0)-G(1) transition were observed in cells lacking Skp2, the F-box protein component of an SCF ubiquitin ligase complex, indicating that these early events are independent of Skp2-mediated proteolysis. Substitution of Ser(10) with Ala (S10A) markedly reduced the extent of p27(Kip1) export, whereas substitution of Ser(10) with Asp (S10D) or Glu (S10E) promoted export. Co-immunoprecipitation analysis showed that CRM1 preferentially interacted with S10D and S10E but not with S10A, suggesting that the phosphorylation of p27(Kip1) on Ser(10) is required for its binding to CRM1 and for its subsequent nuclear export.     

Cytoplasmic ubiquitin ligase KPC regulates proteolysis of p27(Kip1) at G1 phase

The cyclin-dependent kinase inhibitor p27(Kip1) is degraded at the G0-G1 transition of the cell cycle by the ubiquitin-proteasome pathway. Although the nuclear ubiquitin ligase (E3) SCF(Skp2) is implicated in p27(Kip1) degradation, proteolysis of p27(Kip1) at the G0-G1 transition proceeds normally in Skp2(-/-) cells. Moreover, p27(Kip1) is exported from the nucleus to the cytoplasm at G0-G1 (refs 9-11). These data suggest the existence of a Skp2-independent pathway for the degradation of p27(Kip1) at G1 phase. We now describe a previously unidentified E3 complex: KPC (Kip1 ubiquitination-promoting complex), consisting of KPC1 and KPC2. KPC1 contains a RING-finger domain, and KPC2 contains a ubiquitin-like domain and two ubiquitin-associated domains. KPC interacts with and ubiquitinates p27(Kip1) and is localized to the cytoplasm. Overexpression of KPC promoted the degradation of p27(Kip1), whereas a dominant-negative mutant of KPC1 delayed p27(Kip1) degradation. The nuclear export of p27(Kip1) by CRM1 seems to be necessary for KPC-mediated proteolysis. Depletion of KPC1 by RNA interference also inhibited p27(Kip1) degradation. KPC thus probably controls degradation of p27(Kip1) in G1 phase after export of the latter from the nucleus.     

Novel PKCs activate ERK through PKD1 in MCF-7 cells

PKCs can have opposite effects on ERK phosphorylation. Novel (n)PKCs can inhibit ERK by phosphorylation of Raf-1, classical and atypical PKCs can activate ERK by removing an inhibitory protein from Raf-1. The aim of this work was to clarify how PMA-activated PKCs lead to ERK activation in MCF-7 cells expressing mainly nPKCs. Using chemical inhibitors and antibodies against PKCs, delivered into cells by the Chariot transfection system, we found that nPKCs activate ERK through transphosphorylation of PKD1, the blockage of which prevented PMA-stimulated ERK activation. We conclude that the nPKCs/PKD1 cascade is determinant for ERK activation by PMA in MCF-7 cells.     

High expression of Cks1 in human non-small cell lung carcinomas

Enhanced degradation of cyclin-dependent kinase (CDK) inhibitor p27(Kip1) is known to be a powerful prognostic marker in many types of human cancers. Human CDK subunit 1 (Cks1) and S-phase kinase associated protein 2 (Skp2) are components of the SCF(Skp2) complex, which acts as a ubiquitin ligase for p27(Kip1). There are no reports about the involvement of Cks1 in the pathogenesis of human cancer. Here we show high expression of Cks1 in non-small cell lung cancers (NSCLCs) using Western blotting and quantitative real-time RT-PCR. The Skp2 mRNA expression level was high in squamous cell carcinomas and was inversely related with the p27(Kip1) protein level in individual clinical samples. In contrast, Cks1 mRNA expression had no such relationship with p27(Kip1), although Cks1 mRNA was significantly elevated in adenocarcinomas. These results suggest that high expression of Skp2 and Cks1 may be involved in the pathogenesis of NSCLCs via different mechanisms.     

Antimitogenesis linked to regulation of Skp2 gene expression

Prostacyclin has many effects in the vasculature; one of the less well understood is the ability to block cell cycle progression through G(1) phase. We previously reported that the prostacyclin mimetic, cicaprost, selectively inhibits cyclin E-cyclin-dependent kinase-2 (Cdk2), and now we show that it acts by regulating the expression of Skp2, the F-box protein that targets p27(Kip1) for ubiquitin-mediated proteolysis. First, we show that cicaprost prevents the late G(1) phase down-regulation of p27(Kip1) and that the inhibitory effect of cicaprost on cyclin E-Cdk2 activity and S phase entry is eliminated by deleting p27(Kip1). Levels of the closely related Cdk2 inhibitor, p21(Cip1), are unaffected by cicaprost. Moreover, we show that cicaprost blocks the induction of Skp2 mRNA and that ectopic expression of a Skp2 cDNA overrides the effect of cicaprost on p27(Kip1) levels and S phase entry. Our data show that inhibition of F-box protein gene expression can underlie the effect of a potent antimitogen.     

A CDK-independent function of mammalian Cks1: targeting of SCF(Skp2) to the CDK inhibitor p27Kip1.

The Cks/Suc1 proteins associate with CDK/cyclin complexes, but their precise function(s) is not well defined. Here we demonstrate that Cks1 directs the ubiquitin-mediated proteolysis of the CDK-bound substrate p27Kip1 by the protein ubiquitin ligase (E3) SCF(Skp2). Cks1 associates with the F box protein Skp2 and is essential for recognition of the p27Kip1 substrate for ubiquitination in vivo and in vitro. Using purified recombinant proteins, we reconstituted p27Kip1 ubiquitination activity and show that it is dependent on Cks1. CKS1-/- mice are abnormally small, and cells derived from them proliferate poorly, particularly under limiting mitogen conditions, possibly due to elevated levels of p27Kip1.     

Regulation of IGF-1-dependent cyclin D1 and E expression by hEag1 channels in MCF-7 cells: The critical role of hEag1 channels in G1 phase progression

Insulin-like Growth Factor-1 (IGF-1) plays a key role in breast cancer development and cell cycle regulation. It has been demonstrated that IGF-1 stimulates cyclin expression, thus regulating the G1 to S phase transition of the cell cycle. Potassium (K⁺) channels are involved in the G1 phase progression of the cell cycle induced by growth factors. However, mechanisms that allow growth factors to cooperate with K⁺ channels in order to modulate the G1 phase progression and cyclin expression remain unknown. Here, we focused on hEag1 K⁺ channels which are over-expressed in breast cancer and are involved in the G1 phase progression of breast cancer cells (MCF-7). As expected, IGF-1 increased cyclin D1 and E expression of MCF-7 cells in a cyclic manner, whereas the increase of CDK4 and 2 levels was sustained. IGF-1 stimulated p21^WAF1/Cip1 expression with a kinetic similar to that of cyclin D1, however p27^Kip1 expression was insensitive to IGF-1. Interestingly, astemizole, a blocker of hEag1 channels, but not E4031, a blocker of HERG channels, inhibited the expression of both cyclins after 6-8 h of co-stimulation with IGF-1. However, astemizole failed to modulate CDK4, CDK2, p21^WAF1/Cip1 and p27^Kip1 expression. The down-regulation of hEag1 by siRNA provoked a decrease in cyclin expression. This study is the first to demonstrate that K⁺ channels such as hEag1 are directly involved in the IGF-1-induced up-regulation of cyclin D1 and E expression in MCF-7 cells. By identifying more specifically the temporal position of the arrest site induced by the inhibition of hEag1 channels, we confirmed that hEag1 activity is predominantly upstream of the arrest site induced by serum-deprivation, prior to the up-regulation of both cyclins D1 and E.     

The role of cyclin-dependent kinase inhibitor p27Kip1 in anti-HER2 antibody-induced G1 cell cycle arrest and tumor growth inhibition

Cyclin-dependent kinase (CDK) inhibitor p27Kip1 binds to the cyclin E.CDK2 complex and plays a major role in controlling cell cycle and cell growth. Our group and others have reported that anti-HER2 monoclonal antibodies exert inhibitory effects on HER2-overexpressing breast cancers through G1 cell cycle arrest associated with induction of p27Kip1 and reduction of CDK2. The role of p27Kip1 in anti-HER2 antibody-induced cell cycle arrest and growth inhibition is, however, still uncertain. Here we have provided several lines of evidence supporting a critical role for p27Kip1 in the anti-HER2 antibody-induced G1 cell cycle arrest and tumor growth inhibition. Induction of p27Kip1 and G1 growth arrest by anti-HER2 antibody, murine 4D5, or humanized trastuzumab (Herceptin) are concentration-dependent, time-dependent, irreversible, and long-lasting. The magnitude of G1 cell cycle arrest induced by trastuzumab or 4D5 is well correlated with the level of p27Kip1 protein induced. Up-regulation of p27Kip1 and G1 growth arrest could no longer be removed with as little as 14 h of treatment with trastuzumab. Anti-HER2 antibody-induced p27Kip1 protein, G1 arrest, and growth inhibition persist at least 5 days after a single treatment. The magnitude of growth inhibition of breast cancer cells induced by anti-HER2 antibody closely parallels the level of p27Kip1 induced. Induced expression of exogenous p27Kip1 results in a p27Kip1 level-dependent G1 cell cycle arrest and growth inhibition similar to that obtained with anti-HER2 antibodies. Reducing p27Kip1 expression using p27Kip1 small interfering RNA blocks anti-HER2 antibody-induced p27Kip1 up-regulation and G1 arrest. Treatment with anti-HER2 antibody significantly increases the half-life of p27Kip1 protein. Inhibition of ubiquitin-proteasome pathway, but not inhibition of calpain and caspase activities, up-regulates p27Kip1 protein to a degree comparable with that obtained with anti-HER2 antibodies. We have further demonstrated that anti-HER2 antibody significantly decreases threonine phosphorylation of p27Kip1 protein at position 187 (Thr-187) and increases serine phosphorylation of p27Kip1 protein at position 10 (Ser-10). Expression of S10A and T187A mutant p27Kip1 protein increases the fraction of cells in G1 and reduces a further antibody-induced G1 arrest. Consequently, p27Kip1 plays an important role in the anti-HER2 antibody-induced G1 cell cycle arrest and tumor growth inhibition through post-translational regulation. Regulation of the phosphorylation of p27Kip1 protein is one of the post-translational mechanisms by which anti-HER2 antibody upregulates the protein.     

Skp2-mediated p27(Kip1) degradation during S/G2 phase progression of adipocyte hyperplasia

p27(Kip1), an important regulator of Cdk2 activity and G1/S transition, is tightly regulated in a cell-type and condition-specific manner to integrate mitogenic and differentiation signals governing cell cycle progression. We show that p27 protein levels progressively declined from mid-G1 through late-G2 phase as density-arrested 3T3-L1 preadipocytes synchronously reentered the cell cycle during early stages of adipocyte differentiation. This dramatic fall in p27 protein accumulation was due, at least in part, to a decrease in protein stability. Specific inhibitors of the 26S proteasome were shown to completely block the decrease in p27 protein levels throughout G1, increase the abundance of ubiquitylated p27 protein, and inhibit G1/S transition resulting in G1 arrest. It is further demonstrated that p27 was phosphorylated on threonine 187 during S phase progression by Cdk2 and that phosphorylated p27 was polyubiquitylated and degraded. Furthermore, we demonstrate that Skp2 and Cks1 dramatically increased during S/G2 phase progression concomitantly with the maximal fall in p27 protein. Complete knockdown of Skp2 with RNA interference partially prevented p27 degradation equivalent to that observed with Cdk2 blockade suggesting that the SCF(Skp2) E3 ligase and other proteasome-dependent mechanisms contribute to p27 degradation during preadipocyte replication. Interestingly, Skp2-mediated p27 degradation was not essential for G1/S or S/G2 transition as preadipocytes shifted from quiescence to proliferation during adipocyte hyperplasia. Finally, evidence is presented suggesting that elevated p27 protein in the absence of Skp2 was neutralized by sequestration of p27 protein into Cyclin D1/Cdk4 complexes.     

Regulation of p27Kip1 accumulation in murine B-lymphoma cells: role of c-Myc and calcium.

IgM cross-linking induces G1 arrest and apoptosis in murine B-lymphoma cells. It prevents pRb phosphorylation by decreasing cyclin-dependent kinase 2 activity via the up-regulation of cyclin kinase inhibitor p27Kip1. Anti-IgM also causes an increase in cytosolic free calcium and a loss of c-myc mRNA and protein. This down-regulation of c-Myc is prevented by CD40L, which rescues cells from anti-IgM-induced apoptosis. In this study, we addressed the mechanism(s) of anti-IgM-induced p27Kip1 accumulation. We examined effects of early events in B-cell receptor-mediated signaling, c-Myc down-regulation, and an increase in free calcium on p27Kip1. Down-regulation of c-myc alone had no effect on p27Kip1; neither did an increase in free calcium alone. Together, these two events led to p27Kip1 induction, growth arrest, and apoptosis. CD40L, the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester, and cyclosporin A all prevented anti-IgM-induced p27Kip1 accumulation, suggesting that both the decrease in c-Myc expression and an increase in free calcium are necessary for p27Kip1 up-regulation.