Cdk2-dependent Inhibition of p21 Stability via a C-terminal Cyclin-binding Motif

Abstract

p21 is a member of the Cip/Kip family of cyclin-dependent kinase (CDK) inhibitors that includes p21, p27, and p57. Recent studies have suggested that Cdk2 activity may promote p21 degradation through a pathway similar to that for p27, although the mechanism by which this occurs has not been clarified. In the current report, co-expression with cyclin E and Cdk2 stabilized p21 in a manner that required the CDK-binding site of p21 and a cyclin-binding site (cy1) located in the p21 N terminus. Strikingly, however, a kinase-dead Cdk2 mutant stabilized p21 to a greater extent than did wild-type Cdk2, consistent with the notion that Cdk2 activity can destabilize p21. The ability of wild-type Cdk2 to destabilize p21 required a potential Cdk2 phosphorylation site in p21 at serine 130 and an intact cyclin-binding motif (cy2) in the p21 C terminus. Finally, p21 was phosphorylated by Cdk2 at Ser-130 in vitro, and this ability of Cdk2 to phosphorylate p21 was dependent, in large part, on the presence of cy2. These results support a model in which active Cdk2 destabilizes p21 via the cy2 cyclinbinding motif and p21 phosphorylation.

Disorder-function relationships for the cell cycle regulatory proteins p21 and p27

“…Further, disorder within the CTD of p21 (Esteve et al , 2003 ;Yoon et al , 2009 ) may mediate signal transduction from step 1 to step 2 as does that of p27 ( Figure 3A). Interestingly, it has been reported that Ser130 phosphorylation by Cdk2/cyclin E is required for ubiquitination of p21 by SCF Skp2 and its subsequent proteasomal degradation (Bornstein et al , 2003 ;Zhu et al , 2005 ). This experimental evidence supports the hypothesis that the signaling conduit observed for p27 is conserved in p21 and that NRTKs control p21 activity and stability.…”

“…However, if Cdk/cyclin complexes are in excess of p21 molecules, they will additionally bind to the secondary C-terminal RxL motif. Zhu et al (2005) showed through cellular overexpression studies that this secondary interaction serves to recruit active Cdk2/cyclin E complexes for phosphorylation of p21 on Ser130. As discussed above, phosphorylation of Ser130 is a signal for p21 ubiquitination and 26S proteosomal degradation.…”

Oncogenic pathways impinging on the G2-restriction point

“…Under high-serum conditions, p21 CIP1 and p27 KIP1 are known to be unstable due to high-cyclindependent kinase activity (Morisaki et al, 1997;Zhu et al, 2005). Indeed, both p21 CIP1 and p27 KIP1 were subjected to protein degradation under high-serum conditions in the three cell types, as cycloheximide treatment resulted in reduced protein levels (Figure 4g, compare lanes 1 and 2, lanes 4 and 5 and lanes 7 and 8).…”

PCNA-dependent regulation of p21 ubiquitylation and degradation via the CRL4Cdt2 ubiquitin ligase complex

“…Therefore, the CRL4 Cdt2 ubiquitin ligase is redundant with SCF Skp2 in promoting the destruction of p21 in S-phase cells in a similar fashion to that observed for the replication factor Cdt1. Interestingly, whereas the CRL4 Cdt2 -dependent ubiquitylation of p21 depends on p21 interaction with PCNA and is stimulated by GSK3␤-dependent phosphorylation on Ser114, SCF Skp2 -dependent ubiquitylation of p21 is promoted by p21 binding to and phosphorylation by Cdk1/2 on Ser130 (Bornstein et al 2003;Wang et al 2005;Zhu et al 2005). Thus, it is possible that CRL4 Cdt2 preferentially frees PCNA from p21, while SCF Skp2 preferentially targets p21 in complex with Cdk1/2 (see model in Fig.…”