Noncatalytic Requirement for Cyclin A-cdk2 in p27 Turnover

Abstract

Ubiquitin-dependent proteolysis makes a major contribution to decreasing the levels of p27. Ubiquitindependent proteolysis of p27 kip1 is growth and cell cycle regulated in two ways: first, skp2, a component of the E3-ubiquitin ligase, is growth regulated, and second, a kinase must phosphorylate the threonine-187 position on p27 so that it can be recognized by skp2. In vitro, p27 is phosphorylated by cyclin E-and cyclin A-associated cdk2 as well as by cyclin B1-cdk1. Having analyzed the effect of different cyclin-cyclin-dependent kinase complexes on ubiquitination of p27 in a reconstitution assay system, we now report a noncatalytic requirement for cyclin A-cdk2. Multiparameter flow cytometric analysis also indicates that p27 turnover correlates best with the onset of S phase, once the levels of cyclin A become nearly maximal. Finally, increasing the amount of both cyclin E-cdk2 and skp2 was less efficient at promoting p27 ubiquitination than was increasing the amount of cyclin A-cdk2 alone in extracts prepared from cultures of >93%-purified G 1 cells. Together these lines of evidence suggest that cyclin A-cdk2 plays an ancillary noncatalytic role in the ubiquitination of p27 by the SCF skp2 complex.

A pathway in quiescent cells that controls p27Kip1 stability, subcellular localization, and tumor suppression

“…This is not surprising given the very high level of redundancy among different cyclins and CDKs (Pagano and Jackson 2004;Sherr and Roberts 2004), and may thus suggest that any cyclin-CDK complex that can bind p27 can mediate this function. Our data also indicate that these cyclin-CDK complexes do not need to be enzymatically active to promote p27 degradation in G0, possibly providing a structural scaffold for p27 recognition by ubiquitin ligases as reported by others (Zhu et al 2004). Interestingly, cyclin-CDK binding was dispensable for p27 turnover only during the G1 phase, which may reflect p27 degradation by the cytoplasmic KPC pathway, recently found to mediate the degradation of free p27 (Kamura et al 2004;Kotoshiba et al 2005).…”

“…In contrast, p27 CK − protein levels were strikingly different from wild-type p27. As expected, the relative abundance of the p27 CK − protein was increased in S-phase cells, consistent with the requirement for phosphorylation of p27 by cyclin E-CDK2 and binding to cyclin-CDK for ubiquitination by the SCF-Skp2 E3 ligase (Sheaff et al 1997;Vlach et al 1997;Montagnoli et al 1999;Zhu et al 2004). Surprisingly, the p27 CK − protein was also elevated in quiescence, suggesting that cyclin-CDK-p27 heterotrimer assembly also sets the level of p27 in nondividing cells.…”

“…Thus, a p27 CK − mutant that cannot bind to cyclins and CDKs was still a substrate for phosphorylation on Thr187, but was not efficiently degraded (Vlach et al 1997;Montagnoli et al 1999). Supporting this idea, others have found a noncatalytic requirement for cyclin A-CDK2 complexes for p27 turnover (Zhu et al 2004).…”

Mutant B-RAF signaling and cyclin D1 regulate Cks1/S-phase kinase-associated protein 2-mediated degradation of p27Kip1 in human melanoma cells

“…Third, both Skp2 and Cks1 levels are decreased by knockdown of either B-RAF or cyclin D1. The mechanism of B-RAF-cyclin D1 effects on p27 Kip1 appears distinct from the Rb-mediated competition for Skp2 binding that also enhances p27 Kip1 levels (Ji et al, 2004) and a non-catalytic role for Cdk2 in the regulation of Skp2-p27 Kip1 complexes ( Zhu et al, 2004). As expression of Skp2 is regulated in a cell cycle-dependent manner (Bashir et al, 2004;Wei et al, 2004), knockdown of B-RAF or cyclin D1 likely prevents the expression of Skp2 in late G1/S.…”

p27kip1 Protein Levels Reflect a Nexus of Oncogenic Signaling during Cell Transformation

“…Phosphorylation-200 ng of His-tagged human p27 was phosphorylated by incubation with 200 ng of purified cyclin B1-CDK1 complex as described (11) and [␥-…”