PTEN regulates the ubiquitin-dependent degradation of the CDK inhibitor p27(KIP1) through the ubiquitin E3 ligase SCF(SKP2)

The PTEN tumor suppressor acts as a phosphatase for phosphatidylinositol-3,4,5-trisphosphate (PIP3) [1, 2]. We have shown previously that PTEN negatively controls the G1/S cell cycle transition and regulates the levels of p27(KIP1), a CDK inhibitor [3, 4]. Recently, we and others have identified an ubiquitin E3 ligase, the SCF(SKP2) complex, that mediates p27 ubiquitin-dependent proteolysis [5-7]. Here we report that PTEN and the PI 3-kinase pathway regulate p27 protein stability. PTEN-deficiency in mouse embryonic stem (ES) cells causes a decrease of p27 levels with concomitant increase of SKP2, a key component of the SCF(SKP2) complex. Conversely, in human glioblastoma cells, ectopic PTEN expression leads to p27 accumulation, which is accompanied by a reduction of SKP2. We found that ectopic expression of SKP2 alone is sufficient to reverse PTEN-induced p27 accumulation, restore the kinase activity of cyclin E/CDK2, and partially overcome the PTEN-induced G1 cell cycle arrest. Consistently, recombinant SCF(SKP2) complex or SKP2 protein alone can rescue the defect in p27 ubiquitination in extracts prepared from cells treated with a PI 3-kinase inhibitor. Our findings suggest that SKP2 functions as a critical component in the PTEN/PI 3-kinase pathway for the regulation of p27(KIP1) and cell proliferation.     

Association of human CUL-1 and ubiquitin-conjugating enzyme CDC34 with the F-box protein p45(SKP2): evidence for evolutionary conservation in the subunit composition of the CDC34-SCF pathway.

In normal and transformed cells, the F-box protein p45(SKP2) is required for S phase and forms stable complexes with p19(SKP1) and cyclin A-cyclin-dependent kinase (CDK)2. Here we identify human CUL-1, a member of the cullin family, and the ubiquitin-conjugating enzyme CDC34 as additional partners of p45(SKP2) in vivo. CUL-1 also associates with cyclin A and p19(SKP1) in vivo and, with p45(SKP2), they assemble into a large multiprotein complex. In Saccharomyces cerevisiae, a complex of similar molecular composition (an F-box protein, a member of the cullin family and a homolog of p19(SKP1)) forms a functional E3 ubiquitin protein ligase complex, designated SCFCDC4, that facilitates ubiquitination of a CDK inhibitor by CDC34. The data presented here imply that the p45(SKP2)-CUL-1-p19(SKP1) complex may be a human representative of an SCF-type E3 ubiquitin protein ligase. We propose that all eukaryotic cells may use a common ubiquitin conjugation apparatus to promote S phase. Finally, we show that multiprotein complex formation involving p45(SKP2)-CUL-1 and p19(SKP1) is governed, in part, by periodic, S phase-specific accumulation of the p45(SKP2) subunit and by the p45(SKP2)-bound cyclin A-CDK2. The dependency of p45(SKP2)-p19(SKP1) complex formation on cyclin A-CDK2 may ensure tight coordination of the activities of the cell cycle clock with those of a potential ubiquitin conjugation pathway.     

Degradation of cyclin A does not require its phosphorylation by CDC2 and cyclin-dependent kinase 2

Many cyclins are degraded by the ubiquitination/proteasome pathways involving the anaphase-promoting complex and SCF complexes. These degradations are frequently dependent on phosphorylation by cyclin-dependent kinases (CDKs), providing a self-limiting mechanism for CDK activity. Here we present evidence from in vitro and in vivo assay systems that the degradation of human cyclin A can be inhibited by kinase-inactive mutants of CDK2 and CDC2. One obvious interpretation of these results is that like other cyclins, CDK-dependent phosphorylation of the cyclin A may be involved in cyclin A degradation. Our data indicated that CDK2 can phosphorylate cyclin A on Ser-154. Site-directed mutagenesis of Ser-154 abolished the phosphorylation by recombinant CDK2 in vitro and the majority of cyclin A phosphorylation in the cell. Activation of CDK2 and binding to SKP2 or p27(KIP1) were not affected by the phosphorylation of Ser-154. Surprising, in marked contrast to cyclin E, where phosphorylation of Thr-380 by CDK2 is required for proteolysis, degradation of cyclin A was not affected by Ser-154 phosphorylation. It is likely that the stabilization of cyclin A by the kinase-inactive CDKs was mainly due to a cell cycle effect. These data suggest an important difference between the regulation of cyclin A and cyclin E.     

The F-box protein SKP2 binds to the phosphorylated threonine 380 in cyclin E and regulates ubiquitin-dependent degradation of cyclin E

Cyclin E is required for S phase entry. The subsequent ubiquitin-dependent degradation of cyclin E contributes to an orderly progression of the S phase. It has been shown that phosphorylation of threonine 380 (Thr380) in cyclin E provides a signal for its ubiquitin-dependent proteolysis. We report that SKP2, an F-box protein and a substrate-targeting component of the SCF(SKP2) ubiquitin E3 ligase complex, mediates cyclin E degradation. In vitro, SKP2 specifically interacted with the cyclin E peptide containing the phosphorylated-Thr380 but not with a cognate nonphosphorylated peptide. In vivo, expression of SKP2 induced cyclin E polyubiquitination and degradation. Conversion of Thr380 into nonphosphorylatable amino acids caused significant resistance of cyclin E to SKP2. The presence of the CDK inhibitor p27(Kip1) also prevented the SKP2-dependent degradation of cyclin E. Our findings suggest that SKP2 regulates cyclin E stability, thus contributing to the control of S phase progression.     

The CDK Subunit CKS2 Counteracts CKS1 to Control Cyclin A/CDK2 Activity in Maintaining Replicative Fidelity and Neurodevelopment

CKS proteins are evolutionarily conserved cyclin-dependent kinase (CDK) subunits whose functions are incompletely understood. Mammals have two CKS proteins. CKS1 acts as a cofactor to the ubiquitin ligase complex SCF(SKP2) to promote degradation of CDK inhibitors, such as p27. Little is known about the role of the closely related CKS2. Using a Cks2(-/-) knockout mouse model, we show that CKS2 counteracts CKS1 and stabilizes p27. Unopposed CKS1 activity in Cks2(-/-) cells leads to loss of p27. The resulting unrestricted cyclin A/CDK2 activity is accompanied by shortening of the cell cycle, increased replication fork velocity, and DNA damage. In?vivo, Cks2(-/-) cortical progenitor cells are limited in their capacity to differentiate into mature neurons,?a phenotype akin to animals lacking p27. We propose that?the balance between CKS2 and CKS1 modulates p27 degradation, and with it cyclin A/CDK2 activity, to safeguard replicative fidelity and control neuronal differentiation.     

Triggering ubiquitination of a CDK inhibitor at origins of DNA replication

To ensure proper timing of the G1-S transition in the cell cycle, the cyclin E-Cdk2 complex, which is responsible for the initiation of DNA replication, is restrained by the p21(Cip1)/p27(Kip1)/p57(Kip2) family of CDK (cyclin-dependent kinase) inhibitors in humans and by the related p27(Xic1) protein in Xenopus. Activation of cyclin E-Cdk2 is linked to the ubiquitination of human p27(Kip1) or Xenopus p27(Xic1) by SCF (for Skp1-Cullin-F-box protein) ubiquitin ligases. For human p27(Kip1), ubiquitination requires direct phosphorylation by cyclin E-Cdk2. We show here that Xic1 ubiquitination does not require phosphorylation by cyclin E-Cdk2, but it does require nuclear accumulation of the Xic1-cyclin E-Cdk2 complex and recruitment of this complex to chromatin by the origin-recognition complex together with Cdc6 replication preinitiation factors; it also requires an activation step necessitating cyclin E-Cdk2-kinase and SCF ubiquitin-ligase activity, and additional factors associated with mini-chromosome maintenance proteins, including the inactivation of geminin. Components of the SCF ubiquitin-ligase complex, including Skp1 and Cul1, are also recruited to chromatin through cyclin E-Cdk2 and the preinitiation complex. Thus, activation of the cyclin E-Cdk2 kinase and ubiquitin-dependent destruction of its inhibitor are spatially constrained to the site of a properly assembled preinitiation complex.     

Association of the cell cycle regulatory proteins p45(SKP2) and CksHs1. Functional effect on CDK2 complex formation and kinase activity

In mammalian cells, CDK2 is part of a multiprotein complex that includes Cyclin A or E and cell cycle regulatory proteins such as p21(Cip1), PCNA, p27(Kip1), p45(SKP2), p19(SKP1), and CksHs1/CksHs2. While the role of some of these proteins has been well studied, the function of other proteins in the complex remains unclear. In this study, we showed that the carboxyl-terminal region of p45(SKP2) associates directly with CksHs1 and that CksHs1 negatively regulated the interaction between p45(SKP2) and CDK2. Moreover, we showed that overexpression of CksHs1 inhibits CDK2 kinase activity and that additional expression of p45(SKP2) overcame this inhibition and restored CDK2 kinase activity. We proposed that the association of CksHs1 and p45(SKP2) prevented CksHs1 from binding CDK2 and negatively regulating the CDK2 kinase activity.     

Cyclin T: three forms for different roles in physiological and pathological functions

Cyclins are members of family of proteins involved in the cell cycle regulation. They are regulatory subunits of complexes with proteins called cyclin-dependent kinases (CDKs). There are three forms of cyclin T: cyclin T1, cyclin T2a, and T2b. All cyclin T contain an N-terminal "cyclin homology box," the most conserved region among different members of the cyclin family that serves to bind CDK9. In addition to the N-terminal cyclin domain, cyclin T contains a putative coiled-coil motif, a His-rich motif, and a C-terminal PEST sequence. The CDK9/cyclin T complex is able to activate gene expression in a catalytic-dependent manner, phosphorylating the carboxy-terminal domain (CTD) of RNA polymerase II. In addition, only cyclin T1 supports interactions between Tat and TAR. The interaction of Tat with cyclin T1 alters the conformation of Tat to enhance the affinity and specificity of the Tat:TAR interaction. On the other hand, CDK9/cyclin T2 complexes are involved in the regulation of terminal differentiation in muscle cells.     

Ubiquitination of p21Cip1/WAF1 by SCFSkp2: substrate requirement and ubiquitination site selection

Multiple proteolytic pathways are involved in the degradation of the cyclin-dependent kinase inhibitor p21(Cip1/WAF1). Timed destruction of p21(Cip1/WAF1) plays a critical role in cell-cycle progression and cellular response to DNA damage. The SCF(Skp2) complex (consisting of Rbx1, Cul1, Skp1, and Skp2) is one of the E3 ubiquitin ligases involved in ubiquitination of p21(Cip1/WAF1). Little is known about how SCF(Skp2) recruits its substrates and selects particular acceptor lysine residues for ubiquitination. In this study, we investigated the requirements for SCF(Skp2) recognition of p21(Cip1/WAF1) and lysine residues that are ubiquitinated in vitro and inside cells. We demonstrate that ubiquitination of p21(Cip1/WAF1) requires a functional interaction between p21(Cip1/WAF1) and the cyclin E-Cdk2 complex. Mutation of both the cyclin E recruitment motif (RXL) and the Cdk2-binding motif (FNF) at the N terminus of p21(Cip1/WAF1) abolishes its ubiquitination by SCF(Skp2), while mutation of either motif alone has minimal effects, suggesting either contact is sufficient for substrate recruitment. Thus, SCF(Skp2) appears to recognize a trimeric complex consisting of cyclin E-Cdk2-p21(Cip1/WAF1). Furthermore, we show that p21(Cip1/WAF1) can be ubiquitinated at four distinct lysine residues located in the carboxyl-terminal region but not two other lysine residues in the N-terminal region. Any one of these four lysine residues can be targeted for ubiquitination in the absence of the others in vitro, and three of these four lysine residues are also ubiquitinated in vivo, suggesting that there is limited specificity in the selection of ubiquitination sites. Interestingly, mutation of the carboxyl-terminal proline to lysine enables ubiquitin conjugation at the carboxyl terminus of the substrate both in vitro and in vivo. Thus, our results highlight a unique property of the ubiquitination enzymatic reaction in that substrate ubiquitination site selection can be remarkably diverse and occur in distinct spatial areas.     

Phosphorylation of cyclin Y by CDK14 induces its ubiquitination and degradation

Cyclin Y, a membrane associated cyclin, is capable of binding and activating CDK14. Here we report that human cyclin Y (CCNY) is a phosphoprotein in vivo and that phosphorylation of CCNY by CDK14 triggers its ubiquitination and degradation. Inactivation of either CDK14 or Cul1 results in accumulation of CCNY. An in vivo and in vitro mapping of CCNY phosphorylation sites by mass spectrometry revealed that the flanking regions of the conserved cyclin box are heavily phosphorylated. Phosphorylation of CCNY at Serines 71 and 73 creates a putative phospho-degron that controls its association with an SCF complex. Mutation of serine to alanine at these two sites stabilized CCNY and enhanced the activity of CCNY/CDK14 on phosphorylation of LRP6. Our results provide insight into autoregulation of the cyclin Y/CDK14 pair in CDK14 activation and cyclin Y turnover which is a process that is involved in membrane proximal signaling.     

Cyclin T1 expression is regulated by multiple signaling pathways and mechanisms during activation of human peripheral blood lymphocytes

Stimulation of primary human T lymphocytes results in up-regulation of cyclin T1 expression, which correlates with phosphorylation of the C-terminal domain of RNA polymerase II (RNAP II). Up-regulation of cyclin T1 and concomitant stabilization of cyclin-dependent kinase 9 (CDK9) may facilitate productive replication of HIV in activated T cells. We report that treatment of PBLs with two mitogens, PHA and PMA, results in accumulation of cyclin T1 via distinct mechanisms. PHA induces accumulation of cyclin T1 mRNA and protein, which results from cyclin T1 mRNA stabilization, without significant change in cyclin T1 promoter activity. Cyclin T1 mRNA stabilization requires the activation of both calcineurin and JNK because inhibition of either precludes cyclin T1 accumulation. In contrast, PMA induces cyclin T1 protein up-regulation by stabilizing cyclin T1 protein, apparently independently of the proteasome and without accumulation of cyclin T1 mRNA. This process is dependent on Ca2+-independent protein kinase C activity but does not require ERK1/2 activation. We also found that PHA and anti-CD3 Abs induce the expression of both the cyclin/CDK complexes involved in RNAP II C-terminal domain phosphorylation and the G1-S cyclins controlling cell cycle progression. In contrast, PMA alone is a poor inducer of the expression of G1-S cyclins but often as potent as PHA in inducing RNAP II cyclin/CDK complexes. These findings suggest coordination in the expression and activation of RNAP II kinases by pathways that independently stimulate gene expression but are insufficient to induce S phase entry in primary T cells.     

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.