Molecular Dissection of the Interaction between p27 and Kip1 Ubiquitylation-promoting Complex, the Ubiquitin Ligase That Regulates Proteolysis of p27 in G1 Phase

Abstract

The cyclin-dependent kinase (CDK) inhibitor p27 is degraded at the G 0 -G 1 transition of the cell cycle by the ubiquitin-proteasome pathway in a Skp2-independent manner. We recently identified a novel ubiquitin ligase, KPC (Kip1 ubiquitylation-promoting complex), consisting of KPC1 and KPC2, which regulates the ubiquitindependent degradation of p27 at G 1 phase. We have now investigated the structural requirements for the interactions of KPC1 with KPC2 and p27. The NH 2 -terminal region of KPC1 was found to be responsible for binding to KPC2 and to p27. KPC1 mutants that lack this region failed to mediate polyubiquitylation of p27 in vitro and expression of one such mutant delayed p27 degradation in vivo. We also generated a series of deletion mutants of p27 and found that KPC failed to polyubiquitylate a p27 mutant that lacks the CDK inhibitory domain. Interestingly, the cyclin E⅐CDK2 complex prevented both the interaction of KPC with p27 as well as KPC-mediated polyubiquitylation of p27. A complex of cyclin E with a kinase-negative mutant of CDK2 also exhibited these inhibitory effects, suggesting that cyclin E⅐CDK2 competes with KPC1 for access to the CDK inhibitory domain of p27. These results suggest that free p27 is recognized by the NH 2 -terminal region of KPC1, which also associates with KPC2, and that p27 is then polyubiquitylated by the COOH-terminal RING-finger domain of KPC1.

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

“…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).…”

Transforming Growth Factor-β, Estrogen, and Progesterone Converge on the Regulation of p27Kip1 in the Normal and Malignant Endometrium

“…A plethora of studies show that the fate of p27 and resulting functional consequences are tightly regulated by cell cycledependent phosphorylation of the molecule by different kinases at specific sites that dictate both its degradation and nuclear retention (15,16,19,20,(43)(44)(45). The most prominent means for posttranslational regulation of nuclear levels of p27, particularly in cancer, is through ubiquitin-mediated degradation of p27 by the 26S proteasome, which is signaled by phosphorylation of p27 on Thr 187 by cyclin E/cdk2 and other kinases during S-G 2 phase of the cell cycle (16,19,46).…”

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

“…A Thr187 phosphorylation-independent ubiquitination pathway, involving Kip1 ubiquitination-promoting complex (KPC), has been implicated in p27 degradation at the G 0 /G 1 transition (Kamura et al , 2004 ). In contrast to SCF Skp2 -mediated ubiquitination, which targets p27 within Cdk/cyclin complexes, KPC only targets free cytoplasmic p27 (Kotoshiba et al , 2005 ). In addition, the Cul4A-DDB1 E3 ligase, when complexed with DDB1 and Artemis, mediates ubiquitination and proteasomal degradation of nuclear p27 in cultured cells at the G 1 to S transition, independent of phosphorylation of Thr157 or Thr187 (Yan et al , 2011 ).…”