Substrate recognition and ubiquitination of SCFSkp2/Cks1 ubiquitin-protein isopeptide ligase

p27, an important cell cycle regulator, blocks the G(1)/S transition in cells by binding and inhibiting Cdk2/cyclin A and Cdk2/cyclin E complexes (Cdk2/E). Ubiquitination and subsequent degradation play a critical role in regulating the levels of p27 during cell cycle progression. Here we provide evidence suggesting that both Cdk2/E and phosphorylation of Thr(187) on p27 are essential for the recognition of p27 by the SCF(Skp2/Cks1) complex, the ubiquitin-protein isopeptide ligase (E3). Cdk2/E provides a high affinity binding site, whereas the phosphorylated Thr(187) provides a low affinity binding site for the Skp2/Cks1 complex. Furthermore, binding of phosphorylated p27/Cdk2/E to the E3 complex showed positive cooperativity. Consistently, p27 is also ubiquitinated in a similarly cooperative manner. In the absence of p27, Cdk2/E and Cks1 increase Skp2 phosphorylation. This phosphorylation enhances Skp2 auto-ubiquitination, whereas p27 inhibits both phosphorylation and auto-ubiquitination of Skp2.     

Evidence for cytosolic p27(Kip1) ubiquitylation and degradation during adipocyte hyperplasia

Objective: Subcellular localization has been shown to play an important role in determining activity and accumulation of p27 protein during cell cycle progression. The purpose of this study was to examine p27 localization and ubiquitylation in relation to E3 ligase expression during adipocyte hyperplasia. Research Methods and Procedures: This study used the murine 3T3‐L1 preadipocyte model to examine p27 regulation during synchronous cell cycle progression. Cell lysates were isolated over time after hormonal stimulation, fractionated to cytosolic and nuclear compartments, and immunoblotted for relative protein determinations. Results: Data presented in this study show that p27 was present in the cytosol and nucleus in density‐arrested preadipocytes and that abundance in both compartments decreased in a phase‐specific manner as preadipocytes synchronously re‐entered the cell cycle during early phases of adipocyte differentiation. Blocking CRM1‐mediated nuclear export did not prevent degradation, nor did it cause nuclear accumulation of p27, suggesting that distinct mechanisms mediating cytosolic and nuclear p27 degradation were involved. Treating preadipocytes with a potent and specific proteasome inhibitor during hormonal stimulation prevented Skp2 accumulation and p27¹⁸⁷ phosphorylation, which are essential events for SCF^Skp2 E3 ligase activity and nuclear p27 ubiquitylation during S/G₂ phase progression. Proteasome blockade also resulted in the first evidence of cytosolic p27 ubiquitylation during late G₁ phase as preadipocytes undergo the transition from quiescence to proliferation. Discussion: These data are consistent with the postulate that p27 is ubiquitylated and targeted for degradation by the 26S proteasome in a phase‐specific manner by distinct ubiquitin E3 ligases localized to the cytosol and nucleus during adipocyte hyperplasia.     

Skp2 induction and phosphorylation is associated with the late G1 phase of proliferating rat hepatocytes.

The changes in phosphoproteins purified with the affinity peptide p9CKShs1 were analyzed from extracts of regenerating rat livers in order to define some G1 and G1/S regulations characteristic of mature hepatocytes stimulated to proliferate. We observed a 47 kDa phosphoprotein that occurred first at the end of G1 before peaking in the S phase. P47 was also found to be phosphorylated in late G1 in primary hepatocyte cultures stimulated with mitogens. P47 was still phosphorylated in extracts depleted of Cdc2, but to a lesser extent after Cdk2 depletion. This phosphoprotein was identified as Skp2. (i) P47 shared the same electrophoretic mobility than Skp2, a cell cycle protein essential for S phase entry in human fibroblasts; (ii) Skp2, like P47, started to be expressed and was highly phosphorylated during the G1/S transition of hepatocytes stimulated to proliferate in vivo and in vitro; (iii) P47 was specifically immunoprecipitated by an antibody directed against Skp2. In addition, cyclin A/Cdk2 complexes from regenerating liver clearly interacted with Skp2. This is the first demonstration that Skp2 is induced and phosphorylated in the late G1 and S phase of hepatocytes in vivo in regenerating liver as well as in vitro in mitogen-stimulated hepatocytes.     

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 degradation of p27 regulates progression into mitosis

Although Skp2 has been thought to mediate the degradation of p27 at the G(1)-S transition, Skp2(-/-) cells exhibit accumulation of p27 in S-G(2) phase with overreplication. We demonstrate that Skp2(-/-)p27(-/-) mice do not exhibit the overreplication phenotype, suggesting that p27 accumulation is required for its development. Hepatocytes of Skp2(-/-) mice entered the endoduplication cycle after mitogenic stimulation, whereas this phenotype was not apparent in Skp2(-/-)p27(-/-) mice. Cdc2-associated kinase activity was lower in Skp2(-/-) cells than in wild-type cells, and a reduction in Cdc2 activity was sufficient to induce overreplication. The lack of p27 degradation in G(2) phase in Skp2(-/-) cells may thus result in suppression of Cdc2 activity and consequent inhibition of entry into M phase. These data suggest that p27 proteolysis is necessary for the activation of not only Cdk2 but also Cdc2, and that Skp2 contributes to regulation of G(2)-M progression by mediating the degradation of p27.     

A Skp2 autoinduction loop and restriction point control

We describe a self-amplifying feedback loop that autoinduces Skp2 during G1 phase progression. This loop, which contains Skp2 itself, p27(kip1) (p27), cyclin E-cyclin dependent kinase 2, and the retinoblastoma protein, is closed through a newly identified, conserved E2F site in the Skp2 promoter. Interference with the loop, by knockin of a Skp2-resistant p27 mutant (p27(T187A)), delays passage through the restriction point but does not interfere with S phase entry under continuous serum stimulation. Skp2 knock down inhibits S phase entry in nontransformed mouse embryonic fibroblasts but not in human papilloma virus-E7 expressing fibroblasts. We propose that the essential role for Skp2-dependent degradation of p27 is in the formation of an autoinduction loop that selectively controls the transition to mitogen-independence, and that Skp2-dependent proteolysis may be dispensable when pocket proteins are constitutively inactivated.     

p27(Kip1) ubiquitination and degradation is regulated by the SCF(Skp2) complex through phosphorylated Thr187 in p27.

Many tumorigenic processes affect cell-cycle progression by their effects on the levels of the cyclin-dependent kinase inhibitor p27(Kip1) [1,2]. The phosphorylation- and ubiquitination-dependent proteolysis of p27 is implicated in control of the G1-S transition in the cell cycle [3-6]. To determine the factors that control p27 stability, we established a cell-free extract assay that recapitulates the degradation of p27. Phosphorylation of p27 at Thr187 was essential for its degradation. Degradation was also dependent on SCF(Skp2), a protein complex implicated in targeting phosphorylated proteins for ubiquitination [7-10]. Immunodepletion of components of the complex - Cul-1, Skp1, or Skp2 - from the extract abolished p27 degradation, while addition of purified SCF(Skp2) to Skp2- depleted extract restored the capacity to degrade p27. A specific association was observed between Skp2 and a p27 carboxy-terminal peptide containing phosphorylated Thr187, but not between Skp2 and the non-phosphorylated peptide. Skp2-dependent associations between Skp1 or Cul-1 and the p27 phosphopeptide were also detected. Isolated SCF(Skp2) contained an E3 ubiquitin ligase activity towards p27. Our data thus suggest that SCF(Skp2) specifically targets p27 for degradation during cell-cycle progression.     

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.     

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).     

Cyclin-dependent kinases phosphorylate human Cdt1 and induce its degradation

Eukaryotic cells tightly control DNA replication so that replication origins fire only once during S phase within the same cell cycle. Cell cycle-regulated degradation of the replication licensing factor Cdt1 plays important roles in preventing more than one round of DNA replication per cell cycle. We have previously shown that the SCF(Skp2)-mediated ubiquitination pathway plays an important role in Cdt1 degradation. In this study, we demonstrate that human Cdt1 is a substrate of Cdk2 and Cdk4 both in vivo and in vitro. Overexpression of cyclin-dependent kinase inhibitors such as p21 and p27 dramatically suppresses the phosphorylation of Cdt1, disrupts the interaction of Cdt1 with the F-box protein Skp2, and blocks the degradation of Cdt1. Further analysis reveals that Cdt1 interacts with cyclin/cyclin-dependent kinase (Cdk) complexes through a cyclin/Cdk binding consensus site, located at the N terminus of Cdt1. A Cdt1 mutant carrying four amino acid substitutions at the Cdk binding site dramatically reduces associations with cyclin/Cdk complexes. This mutant is not phosphorylated, fails to bind Skp2 and is more stable than wild-type Cdt1. These data suggest that cyclin/Cdk-mediated Cdt1 phosphorylation is required for the association of Cdt1 with the SCF(Skp2) ubiquitin ligase and thus is important for the cell cycle dependent degradation of Cdt1 in mammalian cells.     

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.     

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.     

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).     

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.     

The cell-cycle regulatory protein Cks1 is required for SCF(Skp2)-mediated ubiquitinylation of p27

The cyclin-dependent kinase (CDK) inhibitor p27 is degraded in late G1 phase by the ubiquitin pathway, allowing CDK activity to drive cells into S phase. Ubiquitinylation of p27 requires its phosphorylation at Thr 187 (refs 3, 4) and subsequent recognition by S-phase kinase associated protein 2 (Skp2; refs 5-8), a member of the F-box family of proteins that associates with Skp1, Cul-1 and ROC1/Rbx1 to form an SCF ubiquitin ligase complex. However, in vitro ligation of p27 to ubiquitin could not be reconstituted by known purified components of the SCFSkp2 complex. Here we show that the missing factor is CDK subunit 1 (Cks1), which belongs to the highly conserved Suc1/Cks family of proteins that bind to some CDKs and phosphorylated proteins and are essential for cell-cycle progression. Human Cks1, but not other members of the family, reconstitutes ubiquitin ligation of p27 in a completely purified system, binds to Skp2 and greatly increases binding of T187-phosphorylated p27 to Skp2. Our results represent the first evidence that an SCF complex requires an accessory protein for activity as well as for binding to its phosphorylated substrate.     

APC/C(Cdc20) controls the ubiquitin-mediated degradation of p21 in prometaphase

During the G1/S transition, p21 proteolysis is mediated by Skp2; however, p21 reaccumulates in G2 and is degraded again in prometaphase. How p21 degradation is controlled in mitosis remains unexplored. We found that Cdc20 (an activator of the ubiquitin ligase APC/C) binds p21 in cultured cells and identified a D box motif in p21 necessary for APC/C(Cdc20)-mediated ubiquitylation of p21. Overexpression of Cdc20 or Skp2 destabilized wild-type p21; however, only Skp2, but not Cdc20, was able to destabilize a p21(D box) mutant. Silencing of Cdc20 induced an accumulation of p21, increased the fraction of p21 bound to Cdk1, and inhibited Cdk1 activity in p21(+/+) prometaphase cells, but not in p21(-/-) cells. Thus, in prometaphase Cdc20 positively regulates Cdk1 by mediating the degradation of p21. We propose that the APC/C(Cdc20)-mediated degradation of p21 contributes to the full activation of Cdk1 necessary for mitotic events and prevents mitotic slippage during spindle checkpoint activation.