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

被引:602
作者
Vlach, J [1 ]
Hennecke, S [1 ]
Amati, B [1 ]
机构
[1] SWISS INST EXPT CANC RES,CH-1066 EPALINGES,SWITZERLAND
关键词
CDK; CKI; cyclin; p27; proteasome;
D O I
10.1093/emboj/16.17.5334
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The p27(Kip1) protein associates with G(1)-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 demonstrate that each contact is critical for kinase inhibition and induction of G(1) 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.
引用
收藏
页码:5334 / 5344
页数:11
相关论文
共 71 条
  • [1] Adams PD, 1996, MOL CELL BIOL, V16, P6623
  • [2] Agrawal D, 1996, MOL CELL BIOL, V16, P4327
  • [3] CLOSING THE CELL-CYCLE CIRCLE IN YEAST - G2 CYCLIN PROTEOLYSIS INITIATED AT MITOSIS PERSISTS UNTIL THE ACTIVATION OF G1 CYCLINS IN THE NEXT CYCLE
    AMON, A
    IRNIGER, S
    NASMYTH, K
    [J]. CELL, 1994, 77 (07) : 1037 - 1050
  • [4] SKP1 connects cell cycle regulators to the ubiquitin proteolysis machinery through a novel motif, the F-box
    Bai, C
    Sen, P
    Hofmann, K
    Ma, L
    Goebl, M
    Harper, JW
    Elledge, SJ
    [J]. CELL, 1996, 86 (02) : 263 - 274
  • [5] G2 cyclins are required for the degradation of G1 cyclins in yeast
    Blondel, M
    Mann, C
    [J]. NATURE, 1996, 384 (6606) : 279 - 282
  • [6] The proteolysis of mitotic cyclins in mammalian cells persists from the end of mitosis until the onset of S phase
    Brandeis, M
    Hunt, T
    [J]. EMBO JOURNAL, 1996, 15 (19) : 5280 - 5289
  • [7] Decreased levels of the cell-cycle inhibitor p27(Kip1) protein: Prognostic implications in primary breast cancer
    Catzavelos, C
    Bhatacharya, N
    Ung, YC
    Wilson, JA
    Roncari, L
    Sandhu, C
    Shaw, P
    Yeger, H
    MoravaProtzner, I
    Kapusta, L
    Franssen, E
    Pritchard, KI
    Slingerland, JM
    [J]. NATURE MEDICINE, 1997, 3 (02) : 227 - 230
  • [8] THE UBIQUITIN-PROTEASOME PROTEOLYTIC PATHWAY
    CIECHANOVER, A
    [J]. CELL, 1994, 79 (01) : 13 - 21
  • [9] Turnover of cyclin E by the ubiquitin-proteasome pathway is regulated by cdk2 binding and cyclin phosphorylation
    Clurman, BE
    Sheaff, RJ
    Thress, K
    Groudine, M
    Roberts, JM
    [J]. GENES & DEVELOPMENT, 1996, 10 (16) : 1979 - 1990
  • [10] Requirement of p27(Kip1) for restriction point control of the fibroblast cell cycle
    Coats, S
    Flanagan, WM
    Nourse, J
    Roberts, JM
    [J]. SCIENCE, 1996, 272 (5263) : 877 - 880