Cdk2: A genuine protein kinase client of Hsp90 and Cdc37

被引:45
作者
Prince, T [1 ]
Sun, L [1 ]
Matts, RL [1 ]
机构
[1] Oklahoma State Univ, Dept Biochem & Mol Biol, Stillwater, OK 74078 USA
关键词
D O I
10.1021/bi051423m
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
Hsp90 and its cochaperone Cdc37 cooperate to provide requisite support to numerous protein kinases involved in cellular signal transduction. In this report, we studied the interactions of Hsp90 and Cdc37 with the cyclin-dependent kinase, Cdk2. Treatment of K562 cells with the Hsp90 inhibitor, geldanamycin, caused a 75% reduction in Cdk2 levels and reduced the levels of its activating kinase, Cdk7, by more than 60%, suggesting that both of these kinases may be Hsp90 clients. Using classical pull-down assays and the Hsp90 inhibitory agents geldanamycin and molybdate, Cdk2 is shown to be a genuine client of the Hsp90 chaperone complex. Subsequently, pull-down assays directed at helix alpha C of Cdk2 are shown to disrupt Hsp90 and Cdc37 binding and explain the initial difficulties in demonstrating these interactions. Mutant constructs containing deletions of secondary structural elements from the Nand C-termini of Cdk2 were prepared and assayed for their ability to coadsorb Hsp90 and Cdc37 in a salt-stable high-affinity manner with and without the addition of molybdate. Consistent with similar work done with the cyclin-dependent kinase relative Cdk4, the presence of the G-box motif of Cdk2 was shown to be critical for Cdc37 binding, whereas consistent with work done with the Src-family tyrosine kinase Lck, the presence of helix alpha C and the stabilization of helix alpha E were shown to be needed for Hsp90 binding.
引用
收藏
页码:15287 / 15295
页数:9
相关论文
共 55 条
[1]   A ROLE FOR HSP90 IN CELL-CYCLE CONTROL - WEE1 TYROSINE KINASE-ACTIVITY REQUIRES INTERACTION WITH HSP90 [J].
ALIGUE, R ;
AKHAVANNIAK, H ;
RUSSELL, P .
EMBO JOURNAL, 1994, 13 (24) :6099-6106
[2]   Hsp90 is essential for the synthesis and subsequent membrane association, but not the maintenance, of the Src-kinase p56lck [J].
Bijlmakers, MJJE ;
Marsh, M .
MOLECULAR BIOLOGY OF THE CELL, 2000, 11 (05) :1585-1595
[3]   Effects of phosphorylation of threonine 160 on cyclin-dependent kinase 2 structure and activity [J].
Brown, NR ;
Noble, MEM ;
Lawrie, AM ;
Morris, MC ;
Tunnah, P ;
Divita, G ;
Johnson, LN ;
Endicott, JA .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1999, 274 (13) :8746-8756
[4]   Physical interaction of mammalian CDC37 with CDK4 [J].
Dai, K ;
Kobayashi, R ;
Beach, D .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1996, 271 (36) :22030-22034
[5]   CRYSTAL-STRUCTURE OF CYCLIN-DEPENDENT KINASE-2 [J].
DEBONDT, HL ;
ROSENBLATT, J ;
JANCARIK, J ;
JONES, HD ;
MORGAN, DO ;
KIM, SH .
NATURE, 1993, 363 (6430) :595-602
[6]   Cdc37 promotes the stability of protein kinases Cdc28 and Cak1 [J].
Farrell, A ;
Morgan, DO .
MOLECULAR AND CELLULAR BIOLOGY, 2000, 20 (03) :749-754
[7]   A NOVEL CYCLIN ASSOCIATES WITH MO15/CDK7 TO FORM THE CDK-ACTIVATING KINASE [J].
FISHER, RP ;
MORGAN, DO .
CELL, 1994, 78 (04) :713-724
[8]   CDC37 IS REQUIRED FOR ASSOCIATION OF THE PROTEIN-KINASE CDC28 WITH G(1) AND MITOTIC CYCLINS [J].
GERBER, MR ;
FARRELL, A ;
DESHAIES, RJ ;
HERSKOWITZ, I ;
MORGAN, DO .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1995, 92 (10) :4651-4655
[9]   PROTEIN KINASES .6. THE EUKARYOTIC PROTEIN-KINASE SUPERFAMILY - KINASE (CATALYTIC) DOMAIN-STRUCTURE AND CLASSIFICATION [J].
HANKS, SK ;
HUNTER, T .
FASEB JOURNAL, 1995, 9 (08) :576-596
[10]   p50cdc37 is a nonexclusive hsp90 cohort which participates intimately in hsp90-mediated folding of immature kinase molecules [J].
Hartson, SD ;
Irwin, AD ;
Shao, JY ;
Scroggins, BT ;
Volk, L ;
Huang, WJ ;
Matts, RL .
BIOCHEMISTRY, 2000, 39 (25) :7631-7644