Hsp90 Chaperone Machine Structure and Function

Icahn School Of Medicine At Mount Sinai, New York NY

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Abstract

DESCRIPTION (provided by applicant): Hsp90 is a molecular chaperone whose function is largely restricted to folding of signal transducing proteins, such as protein kinases and transcription factors. Hsp90 does not function by itself, but in association with many different co-chaperones or helpers. The goal of our studies is to understand the role of Hsp90 as a chaperone machine involved in protein kinase folding. Studies over the past decade have demonstrated general principles of Hsp90 function and organization, but little is known about their function in protein kinase folding, which also requires the chaperone called Cdc37. In addition, there are many novel co-chaperones that were not assigned roles in the original description of Hsp90 action. Our studies address these areas where knowledge is lacking in three specific aims. In aim 1 we will determine the chaperone requirements for protein kinase folding. We will first determine which yeast kinases interact with Hsp90 and Cdc37 using physical binding and functional assays. Binding will be determined by Western blot analysis of Hsp90 and Cdc37 that associate with individual kinases. Kinases that require Cdc37 for activity will be screened in a cdc37 mutant yeast strain. This analysis will reveal the extent to which chaperones interact with the yeast kinome. We will then determine whether co-chaperones that function with Hsp90 act in the same way for all kinases or whether they exhibit specificity for different kinases. The effect of stress on chaperone interactions will also be determined. In aim 2 we will determine the composition of the Hsp90 chaperone machine. Hsp90 exists in a series of sub-complexes containing different co-chaperones. In the first part of this aim we will characterize the composition of these sub-complexes and in the second part of the aim we will determine which Hsp90 co-chaperones exist in complexes with a protein kinase. We will then test our hypothesis that Stil and Cdc37 participate in assembly of different Hsp90:kinase complexes. In aim 3 we analyze the function of chaperones and co-chaperones in protein kinase folding and activation. In the first sub-aim we will assay protein kinase conformation and stability in different co-chaperone mutant strains by limited proteolysis and pulse chase analysis. In the second sub-aim we will assay for chaperone activity of novel co-chaperones that are important for protein kinase folding. Finally, we will distinguish between the role of Cdc37 in Cdk folding and cyclin binding.