p107 inhibits G1 to S phase progression by down-regulating expression of the F-box protein Skp2

Cell cycle progression is negatively regulated by the pocket proteins pRb, p107, and p130. However, the mechanisms responsible for this inhibition are not fully understood. Here, we show that overexpression of p107 in fibroblasts inhibits Cdk2 activation and delays S phase entry. The inhibition of Cdk2 activity is correlated with the accumulation of p27, consequent to a decreased degradation of the protein, with no change of Thr187 phosphorylation. Instead, we observed a marked decrease in the abundance of the F-box receptor Skp2 in p107-overexpressing cells. Reciprocally, Skp2 accumulates to higher levels in p107-/- embryonic fibroblasts. Ectopic expression of Skp2 restores p27 down-regulation and DNA synthesis to the levels observed in parental cells, whereas inactivation of Skp2 abrogates the inhibitory effect of p107 on S phase entry. We further show that the serum-dependent increase in Skp2 half-life observed during G1 progression is impaired in cells overexpressing p107. We propose that p107, in addition to its interaction with E2F, inhibits cell proliferation through the control of Skp2 expression and the resulting stabilization of p27.     

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.     

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.     

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.     

Role of the SCFSkp2 ubiquitin ligase in the degradation of p21Cip1 in S phase

The cyclin-dependent kinase inhibitor p21Cip1 has important roles in the control of cell proliferation, differentiation, senescence, and apoptosis. It has been observed that p21 is a highly unstable protein, but the mechanisms of its degradation remained unknown. We show here that p21 is a good substrate for an SCF (Skp1-Cullin1-F-box protein) ubiquitin ligase complex, which contains the F-box protein Skp2 (S phase kinase-associated protein 2) and the accessory protein Cks1 (cyclin kinase subunit 1). A similar ubiquitin ligase complex has been previously shown to be involved in the degradation of a related cyclin-dependent kinase inhibitor, p27Kip1. The levels of Skp2 oscillate in the cell cycle, reaching a maximum in S phase. The ubiquitylation of p21 in vitro required the supplementation of all components of the SCF complex as well as of Cks1 and Cdk2-cyclin E. The protein kinase Cdk2-cyclin E acts both by the phosphorylation of p21 on Ser-130 and by the formation of a complex with p21, which is required for its presentation to the ubiquitin ligase. As opposed to the case of p27, the phosphorylation of p21 stimulates its ubiquitylation but is not absolutely required for this process. Levels of p21 are higher in Skp2-/- mouse embryo fibroblasts than in wild-type fibroblasts in the S phase, and the rates of the degradation of p21 are slower in cells that lack Skp2. It is suggested that SCFSkp2 participates in the degradation of p21 in the S phase.     

Ubiquitination of p27Kip1 requires physical interaction with cyclin E and probable phosphate recognition by SKP2

p27Kip1 is an essential cell cycle inhibitor of Cyclin-dependent kinases. Ubiquitin-mediated proteolysis of p27Kip1 is an important mechanism for activation of Cyclin E-Cdk2 and facilitates G1/S transition. Ubiquitination of p27 is primarily catalyzed by a multisubunit E3 ubiquitin ligase, SCF(Skp2), and requires an adapter protein Cks1. In addition, phosphorylation of p27 at Thr187 by Cyclin E and Cdk2 is also essential for triggering substrate ubiquitination. Here we investigate the molecular mechanism of p27 ubiquitination. We show that Cyclin E-Cdk2 is essential for targeting the p27 substrate to SCF(Skp2). Direct physical contact between Cyclin E but not Cdk2 and p27 is required for p27 recruitment to SCF(Skp2). In a search for positively charged amino acid residues that may be involved in recognition of the Thr187 phosphate group, we found that Arg306 of Skp2 is required for association and ubiquitination of phosphorylated p27 but dispensable for ubiquitination of unphosphorylated p21. Thus, our data unravel the molecular organization of the ubiquitination complex that catalyzes p27 ubiquitination and provide unique insights into the specificity of substrate recognition by SCF(Skp2).     

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).     

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.     

Role of the F-box protein Skp2 in adhesion-dependent cell cycle progression.

Cell adhesion to the extracellular matrix (ECM) is a requirement for proliferation that is typically lost in malignant cells. In the absence of adhesion, nontransformed cells arrest in G1 with increased levels of the cyclin-dependent kinase inhibitor p27. We have reported previously that the degradation of p27 requires its phosphorylation on Thr-187 and is mediated by Skp2, an F-box protein that associates with Skp1, Cul1, and Roc1/Rbx1 to form the SCF(Skp2) ubiquitin ligase complex. Here, we show that the accumulation of Skp2 protein is dependent on both cell adhesion and growth factors but that the induction of Skp2 mRNA is exclusively dependent on cell adhesion to the ECM. Conversely, the expression of the other three subunits of the SCF(Skp2) complex is independent of cell anchorage. Phosphorylation of p27 on Thr-187 is also not affected significantly by the loss of cell adhesion, demonstrating that increased p27 stability is not dependent on p27 dephosphorylation. Significantly, ectopic expression of Skp2 in nonadherent G1 cells resulted in p27 downregulation, entry into S phase, and cell division. The ability to induce adhesion-independent cell cycle progression was potentiated by coexpressing Skp2 with cyclin D1 but not with cyclin E, indicating that Skp2 and cyclin D1 cooperate to rescue proliferation in suspension cells. Our study shows that Skp2 is a key target of ECM signaling that controls cell proliferation.     

Cks1 promotion of S phase entry and proliferation is independent of p27Kip1 suppression

Cks1 is an activator of the SCF(Skp2) ubiquitin ligase complex that targets the cell cycle inhibitor p27(Kip1) for degradation. The loss of Cks1 results in p27(Kip1) accumulation and decreased proliferation and inhibits tumorigenesis. We identify here a function of Cks1 in mammalian cell cycle regulation that is independent of p27(Kip1). Specifically, Cks1(-/-); p27(Kip1-/-) mouse embryonic fibroblasts retain defects in the G(1)-S phase transition that are coupled with decreased Cdk2-associated kinase activity and defects in proliferation that are associated with Cks1 loss. Furthermore, concomitant loss of Cks1 does not rescue the tumor suppressor function of p27(Kip1) that is manifest in various organs of p27(Kip1-/-) mice. In contrast, defects in mitotic entry and premature senescence manifest in Cks1(-/-) cells are p27(Kip1) dependent. Collectively, these findings establish p27(Kip1)-independent functions of Cks1 in regulating the G(1)-S transition.     

Estrogens down-regulate p27Kip1 in breast cancer cells through Skp2 and through nuclear export mediated by the ERK pathway

The cyclin-dependent kinase (CDK) inhibitor p27Kip1 plays a key role in growth and development of the mammary epithelium and in breast cancer. p27Kip1 levels are regulated through ubiquitin/proteasome-mediated proteolysis, promoted by CDK2 and the F box protein Skp2 at the G1/S transition, and independent of Skp2 in mid-G1. We investigated the respective roles of Skp2 and subcellular localization of p27Kip1 in down-regulation of p27Kip1 induced in MCF-7 cells by estrogens. 17beta-Estradiol treatment increased Skp2 expression in MCF-7 cells; however, this increase was prevented by G1 blockade mediated by p16Ink4a or the CDK inhibitor roscovitine, whereas down-regulation of p27Kip1 was maintained. Exogenous Skp2 prevented growth arrest of MCF-7 cells by antiestrogen, coinciding with decreased p27Kip1 expression. Under conditions of G1 blockade, p27Kip1 was stabilized by inhibition of CRM1-dependent nuclear export with leptomycin B or by mutation of p27Kip1 (Ser10 --> Ala; S10A) interfering with CRM1/p27Kip1 interaction. Antisense Skp2 oligonucleotides and a dominant-interfering Cul-1(1-452) mutant prevented down-regulation of p27Kip1S10A, whereas Skp2 overexpression elicited its destruction in mitogen-deprived cells. Active mediators of the extracellular signal-regulated kinase (ERK) pathway including Raf-1caax induced cytoplasmic localization of p27Kip1 in antiestrogen-treated cells and prevented accumulation of p27Kip1 in these cells independent of Skp2 expression and coinciding with ERK activation. Genetic or chemical blockade of the ERK pathway prevented down-regulation and cytoplasmic localization of p27Kip1 in response to estrogen. Our studies indicate that estrogens elicit down-regulation of p27Kip1 in MCF-7 cells through Skp2-dependent and -independent mechanisms that depend upon subcellular localization of p27Kip1 and require the participation of mediators of the Ras/Raf-1/ERK signaling pathway.     

P27 expression is regulated by separate signaling pathways, downstream of Ras, in each cell cycle phase

The cyclin inhibitory protein p27Kip1 (p27) plays a vital role in regulating cell proliferation in response to the extracellular growth environment. Active proliferation requires the suppression of p27 levels throughout the cell cycle. Late in the cell cycle, p27 degradation requires phosphorylation of Thr 187 by cyclin dependent kinase 2, leading to recognition by the SCF ubiquitin ligase containing the Skp2 F-box protein. Suppression of p27 is also essential for cell proliferation early in the cell cycle, but this occurs independently of Skp2, whose expression is suppressed during G1 phase. In this study, we use a time lapse and quantitative imaging approach to study the connection between proliferative signaling and the degradation of p27 during each cell cycle period in actively cycling cells. Ras activity was required for the suppression of p27 levels throughout the cell cycle, but separate pathways downstream of Ras signaling were required in different cell cycle periods. For example, inhibitors of MEK and phosphatidylinositol-3-kinase induced p27 expression primarily in G1 phase, while inhibitors of AKT activity stimulated these levels primarily in S phase. Skp2 was expressed in a Ras-dependent manner at higher levels late in the cell cycle. Its ablation resulted in higher p27 levels primarily in G2 phase as expected. The fact that separate signaling pathways downstream of Ras function in each cell cycle phase to suppress p27 levels helps explain the vital connection between proliferative signaling, cell cycle control, and p27 expression.     

Cul4A and DDB1 associate with Skp2 to target p27Kip1 for proteolysis involving the COP9 signalosome

DDB1, a subunit of the damaged-DNA binding protein DDB, has been shown to function also as an adaptor for Cul4A, a member of the cullin family of E3 ubiquitin ligase. The Cul4A-DDB1 complex remains associated with the COP9 signalosome, and that interaction is conserved from fission yeast to human. Studies with fission yeast suggested a role of the Pcu4-Ddb1-signalosome complex in the proteolysis of the replication inhibitor Spd1. Here we provide evidence that the function of replication inhibitor proteolysis is conserved in the mammalian DDB1-Cul4A-signalosome complex. We show that small interfering RNA-mediated knockdown of DDB1, CSN1 (a subunit of the signalosome), and Cul4A in mammalian cells causes an accumulation of p27Kip1. Moreover, expression of DDB1 reduces the level of p27Kip1 by increasing its decay rate. The DDB1-induced proteolysis of p27Kip1 requires signalosome and Cul4A, because DDB1 failed to increase the decay rate of p27Kip1 in cells deficient in CSN1 or Cul4A. Surprisingly, the DDB1-induced proteolysis of p27Kip1 also involves Skp2, an F-box protein that allows targeting of p27Kip1 for ubiquitination by the Skp1-Cul1-F-box complex. Moreover, we provide evidence for a physical association between Cul4A, DDB1, and Skp2. We speculate that the F-box protein Skp2, in addition to utilizing Cul1-Skp1, utilizes Cul4A-DDB1 to induce proteolysis of p27Kip1.     

Structural basis of the Cks1-dependent recognition of p27(Kip1) by the SCF(Skp2) ubiquitin ligase

The ubiquitin-mediated proteolysis of the Cdk2 inhibitor p27(Kip1) plays a central role in cell cycle progression, and enhanced degradation of p27(Kip1) is associated with many common cancers. Proteolysis of p27(Kip1) is triggered by Thr187 phosphorylation, which leads to the binding of the SCF(Skp2) (Skp1-Cul1-Rbx1-Skp2) ubiquitin ligase complex. Unlike other known SCF substrates, p27(Kip1) ubiquitination also requires the accessory protein Cks1. The crystal structure of the Skp1-Skp2-Cks1 complex bound to a p27(Kip1) phosphopeptide shows that Cks1 binds to the leucine-rich repeat (LRR) domain and C-terminal tail of Skp2, whereas p27(Kip1) binds to both Cks1 and Skp2. The phosphorylated Thr187 side chain of p27(Kip1) is recognized by a Cks1 phosphate binding site, whereas the side chain of an invariant Glu185 inserts into the interface between Skp2 and Cks1, interacting with both. The structure and biochemical data support the proposed model that Cdk2-cyclin A contributes to the recruitment of p27(Kip1) to the SCF(Skp2)-Cks1 complex.     

Targeted disruption of Skp2 results in accumulation of cyclin E and p27(Kip1), polyploidy and centrosome overduplication

The ubiquitin-proteasome pathway plays an important role in control of the abundance of cell cycle regulators. Mice lacking Skp2, an F-box protein and substrate recognition component of an Skp1-Cullin-F-box protein (SCF) ubiquitin ligase, were generated. Although Skp2(-/-) animals are viable, cells in the mutant mice contain markedly enlarged nuclei with polyploidy and multiple centrosomes, and show a reduced growth rate and increased apoptosis. Skp2(-/-) cells also exhibit increased accumulation of both cyclin E and p27(Kip1). The elimination of cyclin E during S and G(2) phases is impaired in Skp2(-/-) cells, resulting in loss of cyclin E periodicity. Biochemical studies showed that Skp2 interacts specifically with cyclin E and thereby promotes its ubiquitylation and degradation both in vivo and in vitro. These results suggest that specific degradation of cyclin E and p27(Kip1) is mediated by the SCF(Skp2) ubiquitin ligase complex, and that Skp2 may control chromosome replication and centrosome duplication by determining the abundance of cell cycle regulators.     

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.