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 language | English |
|---|---|
| Pages (from-to) | 5334-5344 |
| Number of pages | 11 |
| Journal | EMBO Journal |
| Volume | 16 |
| Issue number | 17 |
| DOIs | |
| Publication status | Published - Sep 1 1997 |
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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 journal › Article
}
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 -