Molecular dissection of the interaction between p27 and Kip1 ubiquitylation-promoting complex, the ubiquitin ligase that regulates proteolysis of p27 in G1 phase

Shuhei Kotoshibai, Takumi Kamura, Taichi Hara, Noriko Ishida, Keiichi I. Nakayama

Research output: Contribution to journalArticle

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Abstract

The cyclin-dependent kinase (CDK) inhibitor p27 is degraded at the G 0-G1 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 ubiquitin-dependent degradation of p27 at G1 phase. We have now investigated the structural requirements for the interactions of KPC1 with KPC2 and p27. The NH2-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 E-CDK2 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 E-CDK2 competes with KPC1 for access to the CDK inhibitory domain of p27. These results suggest that free p27 is recognized by the NH2-terminal region of KPC1, which also associates with KPC2, and that p27 is then polyubiquitylated by the COOH-terminal RING-finger domain of KPC1.

Original languageEnglish
Pages (from-to)17694-17700
Number of pages7
JournalJournal of Biological Chemistry
Volume280
Issue number18
DOIs
Publication statusPublished - 2005 May 6
Externally publishedYes

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Proteolysis
Dissection
Cyclin E
Ubiquitination
G1 Phase
Ligases
Ubiquitin
Cyclin-Dependent Kinases
Cyclin-Dependent Kinase Inhibitor p27
Degradation
RING Finger Domains
Proteasome Endopeptidase Complex
Phosphotransferases
Cells
Cell Cycle

ASJC Scopus subject areas

  • Biochemistry

Cite this

Molecular dissection of the interaction between p27 and Kip1 ubiquitylation-promoting complex, the ubiquitin ligase that regulates proteolysis of p27 in G1 phase. / Kotoshibai, Shuhei; Kamura, Takumi; Hara, Taichi; Ishida, Noriko; Nakayama, Keiichi I.

In: Journal of Biological Chemistry, Vol. 280, No. 18, 06.05.2005, p. 17694-17700.

Research output: Contribution to journalArticle

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