Definition of protein kinase sequence motifs that trigger high affinity binding of Hsp90 and Cdc37.
Hsp90 cooperates with its co-chaperone Cdc37 to provide obligatory support to numerous protein kinases involved in the regulation of cellular signal transduction pathways. In this report, the crystal structure of the Src family tyrosine kinase Lck was used to guide the creation of kinase constructs to determine features recognized by Hsp90 and its "kinase-specific" co-chaperone Cdc37. Two parameters were assayed: the ability and extent to which the constructs bound to Hsp90 and Cdc37, and the ability of the constructs to trigger salt-resistant high affinity complexes with Hsp90 and Cdc37 independent of the presence of molybdate. Although Hsp90 interacted with both the N-terminal and C-terminal lobes (NL and CL, respectively) of the catalytic domains of the kinases, the lobes themselves were not sufficient to trigger the high affinity binding of Hsp90. Only constructs containing a complete N- or C-terminal lobe and part of the adjacent lobe bound to Hsp90 and Cdc37 in salt-stable complexes independent of molybdate. The two minimum constructs that bound Hsp90 and Cdc37 contained the alpha-C-helix and the beta4- and beta5-strands of the NL through to end of the CL and the NL through to the alpha-E-helix and the amino acids that cap the helix. Cdc37 interacted with only the NL and minimally required the alpha-C-helix and beta4- and beta5-strands of this lobe of Lck. The results indicate that the high affinity binding activity of Hsp90 is triggered through its interaction with adjacent subdomain structures of kinase catalytic domains. Furthermore, the alpha-C-helix and part of its adjoining loop connection to the beta4-strand appear to be the primary determinants recognized by Cdc37.
Prince
Thomas
T
Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, Oklahoma 74078-3035, USA.
Matts
Robert L
RL
D002384:Catalysis; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003461:Crystallography; D029721:Drosophila Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018841:HSP90 Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018832:Molecular Chaperones / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008982:Molybdenum; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
D018797:Cell Cycle Proteins; D029721:Drosophila Proteins; D018841:HSP90 Heat-Shock Proteins; D018832:Molecular Chaperones; C044659:molybdate; D008982:Molybdenum; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck)