Phosphorylation-dependent degradation of the cyclin-dependent kinase inhibitor p27(Kip1)

Jaromir Vlach, Silke Hennecke, Bruno Amati

Research output: Contribution to journalArticle

Abstract

The p27(Kip1) protein associates with G1-specific cyclin-CDK complexes and inhibits their catalytic activity. p27(Kip1) is regulated at various levels, including translation, degradation by the ubiquitin/proteasome pathway and non-covalent sequestration. Here, we describe point mutants of p27 deficient in their interaction with either cyclins (p27(c-)), CDKs (p27(k-)) or both (p27(ck-)), and demostrate that each contact is critical for kinase inhibition and induction of G1 arrest. Through its intact cyclin contact, p27(k-) associated with active cyclin E-CDK2 and, unlike wild type p27, p27(c-) or p27(ck-), was efficiently phosphorylated by CDK2 on a conserved C-terminal CDK target site (TPKK). Retrovirally expressed p27(k-) was rapidly degraded through the proteasome in Rat1 cells, but was stabilized by secondary mutation of the TPKK site to VPKK. In this experimental setting, exogenous wild-type p27 formed inactive ternary complexes with cellular cyclin E-CDK2, was not degraded through the proteasome, and was not further stabilized by the VPKK mutation, p27(ck-), which was not recruited to cyclin E-CDK2, also remained stable in vivo. Thus, selective degradation of p27(k-) depended upon association with active cyclin E-CDK2 and subsequent phosphorylation. Altogether, these data show that p27 must be phosphorylated by CDK2 on the TPKK site in order to be degraded by the proteasome. We propose that cellular p27 must also exist transiently in a cyclin-bound non-inhibitory conformation in vivo.

Original languageEnglish
Pages (from-to)5334-5344
Number of pages11
JournalEMBO Journal
Volume16
Issue number17
DOIs
Publication statusPublished - Sep 1 1997

Fingerprint

Cyclin-Dependent Kinase Inhibitor p27
Cyclin E
Phosphorylation
Cyclins
Proteasome Endopeptidase Complex
Degradation
Cyclin G1
Mutation
Ubiquitin
Conformations
Catalyst activity
Phosphotransferases
Association reactions

Keywords

  • CDK
  • CKI
  • Cyclin
  • p27
  • Proteasome

ASJC Scopus subject areas

  • Cell Biology
  • Genetics

Cite this

Phosphorylation-dependent degradation of the cyclin-dependent kinase inhibitor p27(Kip1). / Vlach, Jaromir; Hennecke, Silke; Amati, Bruno.

In: EMBO Journal, Vol. 16, No. 17, 01.09.1997, p. 5334-5344.

Research output: Contribution to journalArticle

Vlach, Jaromir ; Hennecke, Silke ; Amati, Bruno. / Phosphorylation-dependent degradation of the cyclin-dependent kinase inhibitor p27(Kip1). In: EMBO Journal. 1997 ; Vol. 16, No. 17. pp. 5334-5344.
@article{7498f952951642c6a011b110431b66aa,
title = "Phosphorylation-dependent degradation of the cyclin-dependent kinase inhibitor p27(Kip1)",
abstract = "The p27(Kip1) protein associates with G1-specific cyclin-CDK complexes and inhibits their catalytic activity. p27(Kip1) is regulated at various levels, including translation, degradation by the ubiquitin/proteasome pathway and non-covalent sequestration. Here, we describe point mutants of p27 deficient in their interaction with either cyclins (p27(c-)), CDKs (p27(k-)) or both (p27(ck-)), and demostrate that each contact is critical for kinase inhibition and induction of G1 arrest. Through its intact cyclin contact, p27(k-) associated with active cyclin E-CDK2 and, unlike wild type p27, p27(c-) or p27(ck-), was efficiently phosphorylated by CDK2 on a conserved C-terminal CDK target site (TPKK). Retrovirally expressed p27(k-) was rapidly degraded through the proteasome in Rat1 cells, but was stabilized by secondary mutation of the TPKK site to VPKK. In this experimental setting, exogenous wild-type p27 formed inactive ternary complexes with cellular cyclin E-CDK2, was not degraded through the proteasome, and was not further stabilized by the VPKK mutation, p27(ck-), which was not recruited to cyclin E-CDK2, also remained stable in vivo. Thus, selective degradation of p27(k-) depended upon association with active cyclin E-CDK2 and subsequent phosphorylation. Altogether, these data show that p27 must be phosphorylated by CDK2 on the TPKK site in order to be degraded by the proteasome. We propose that cellular p27 must also exist transiently in a cyclin-bound non-inhibitory conformation in vivo.",
keywords = "CDK, CKI, Cyclin, p27, Proteasome",
author = "Jaromir Vlach and Silke Hennecke and Bruno Amati",
year = "1997",
month = "9",
day = "1",
doi = "10.1093/emboj/16.17.5334",
language = "English",
volume = "16",
pages = "5334--5344",
journal = "EMBO Journal",
issn = "0261-4189",
publisher = "Nature Publishing Group",
number = "17",

}

TY - JOUR

T1 - Phosphorylation-dependent degradation of the cyclin-dependent kinase inhibitor p27(Kip1)

AU - Vlach, Jaromir

AU - Hennecke, Silke

AU - Amati, Bruno

PY - 1997/9/1

Y1 - 1997/9/1

N2 - The p27(Kip1) protein associates with G1-specific cyclin-CDK complexes and inhibits their catalytic activity. p27(Kip1) is regulated at various levels, including translation, degradation by the ubiquitin/proteasome pathway and non-covalent sequestration. Here, we describe point mutants of p27 deficient in their interaction with either cyclins (p27(c-)), CDKs (p27(k-)) or both (p27(ck-)), and demostrate that each contact is critical for kinase inhibition and induction of G1 arrest. Through its intact cyclin contact, p27(k-) associated with active cyclin E-CDK2 and, unlike wild type p27, p27(c-) or p27(ck-), was efficiently phosphorylated by CDK2 on a conserved C-terminal CDK target site (TPKK). Retrovirally expressed p27(k-) was rapidly degraded through the proteasome in Rat1 cells, but was stabilized by secondary mutation of the TPKK site to VPKK. In this experimental setting, exogenous wild-type p27 formed inactive ternary complexes with cellular cyclin E-CDK2, was not degraded through the proteasome, and was not further stabilized by the VPKK mutation, p27(ck-), which was not recruited to cyclin E-CDK2, also remained stable in vivo. Thus, selective degradation of p27(k-) depended upon association with active cyclin E-CDK2 and subsequent phosphorylation. Altogether, these data show that p27 must be phosphorylated by CDK2 on the TPKK site in order to be degraded by the proteasome. We propose that cellular p27 must also exist transiently in a cyclin-bound non-inhibitory conformation in vivo.

AB - The p27(Kip1) protein associates with G1-specific cyclin-CDK complexes and inhibits their catalytic activity. p27(Kip1) is regulated at various levels, including translation, degradation by the ubiquitin/proteasome pathway and non-covalent sequestration. Here, we describe point mutants of p27 deficient in their interaction with either cyclins (p27(c-)), CDKs (p27(k-)) or both (p27(ck-)), and demostrate that each contact is critical for kinase inhibition and induction of G1 arrest. Through its intact cyclin contact, p27(k-) associated with active cyclin E-CDK2 and, unlike wild type p27, p27(c-) or p27(ck-), was efficiently phosphorylated by CDK2 on a conserved C-terminal CDK target site (TPKK). Retrovirally expressed p27(k-) was rapidly degraded through the proteasome in Rat1 cells, but was stabilized by secondary mutation of the TPKK site to VPKK. In this experimental setting, exogenous wild-type p27 formed inactive ternary complexes with cellular cyclin E-CDK2, was not degraded through the proteasome, and was not further stabilized by the VPKK mutation, p27(ck-), which was not recruited to cyclin E-CDK2, also remained stable in vivo. Thus, selective degradation of p27(k-) depended upon association with active cyclin E-CDK2 and subsequent phosphorylation. Altogether, these data show that p27 must be phosphorylated by CDK2 on the TPKK site in order to be degraded by the proteasome. We propose that cellular p27 must also exist transiently in a cyclin-bound non-inhibitory conformation in vivo.

KW - CDK

KW - CKI

KW - Cyclin

KW - p27

KW - Proteasome

UR - http://www.scopus.com/inward/record.url?scp=0030847760&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0030847760&partnerID=8YFLogxK

U2 - 10.1093/emboj/16.17.5334

DO - 10.1093/emboj/16.17.5334

M3 - Article

C2 - 9311993

AN - SCOPUS:0030847760

VL - 16

SP - 5334

EP - 5344

JO - EMBO Journal

JF - EMBO Journal

SN - 0261-4189

IS - 17

ER -