Cdk2: a genuine protein kinase client of Hsp90 and Cdc37

Biochemistry. 2005 Nov 22;44(46):15287-95. doi: 10.1021/bi051423m.

Abstract

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 alphaC 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 N- and 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 alphaC and the stabilization of helix alphaE were shown to be needed for Hsp90 binding.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Benzoquinones
  • Cell Cycle Proteins / metabolism*
  • Chaperonins
  • Cyclin A / metabolism
  • Cyclin-Dependent Kinase 2 / immunology
  • Cyclin-Dependent Kinase 2 / metabolism*
  • HSP90 Heat-Shock Proteins / antagonists & inhibitors
  • HSP90 Heat-Shock Proteins / metabolism*
  • Humans
  • K562 Cells
  • Lactams, Macrocyclic
  • Molecular Chaperones / metabolism*
  • Quinones / pharmacology
  • Rabbits
  • Reticulocytes / metabolism

Substances

  • Benzoquinones
  • CDC37 protein, human
  • Cell Cycle Proteins
  • Cyclin A
  • HSP90 Heat-Shock Proteins
  • Lactams, Macrocyclic
  • Molecular Chaperones
  • Quinones
  • Cyclin-Dependent Kinase 2
  • Chaperonins
  • geldanamycin