P50cdc37 Binds Directly to the Catalytic Domain of Raf as Well as to a Site on hsp90 That Is Topologically Adjacent to the Tetratricopeptide Repeat Binding Site

Adam M Silverstein,Nicholas Grammatikakis,Brent H Cochran,Michael Chinkers,William B Pratt

doi:10.1074/jbc.273.32.20090

Abstract

Several protein kinases (e.g. pp60(src), v-Raf) exist in heterocomplexes with hsp90 and a 50-kDa protein that is the mammalian homolog of the yeast cell cycle control protein Cdc37. In contrast, unliganded steroid receptors exist in heterocomplexes with hsp90 and a tetratricopeptide repeat (TPR) domain protein, such as an immunophilin. Although p50(cdc37) and TPR domain proteins bind directly to hsp90, p50(cdc37) is not present in native steroid receptor.hsp90 heterocomplexes. To obtain some insight as to how v-Raf selects predominantly hsp90.p50(cdc37) heterocomplexes, rather than hsp90.TPR protein heterocomplexes, we have examined the binding of p50(cdc37) to hsp90 and to Raf. We show that p50(cdc37) exists in separate hsp90 heterocomplexes from the TPR domain proteins and that intact TPR proteins compete for p50(cdc37) binding to hsp90 but a protein fragment containing a TPR domain does not. This suggests that the binding site for p50(cdc37) lies topologically adjacent to the TPR acceptor site on the surface of hsp90. Also, we show that p50(cdc37) binds directly to v-Raf, with the catalytic domain of Raf being sufficient. We propose that the combination of exclusive binding of p50(cdc37) versus a TPR domain protein to hsp90 plus direct binding of p50(cdc37) to Raf allows the protein kinase to select for the dominant hsp90.p50(cdc37) composition that is observed with a variety of protein kinase heterocomplexes immunoadsorbed from cytosols.

Highlights

Several protein kinases exist in heterocomplexes with hsp90 and a 50-kDa protein that is the mammalian homolog of the yeast cell cycle control protein Cdc37
We provide evidence that p50cdc37 cannot bind to hsp90 when the tetratricopeptide repeat (TPR) acceptor site on hsp90 is occupied by one of the TPR domain proteins, such as p60/Hop or phosphatase 5 (PP5)
P50cdc37 does bind to hsp90 when the small TPR domain fragment of PP5 occupies the TPR acceptor site and prevents binding of the TPR domain proteins

Summary

Introduction

Several protein kinases (e.g. pp60src, v-Raf) exist in heterocomplexes with hsp and a 50-kDa protein that is the mammalian homolog of the yeast cell cycle control protein Cdc. P50cdc and TPR domain proteins bind directly to hsp, p50cdc is not present in native steroid receptor1⁄7hsp heterocomplexes. It has been shown that CyP-40 and FKBP52 compete with each other for binding to hsp90 [21, 24], and that these immunophilins exist in independent receptor1⁄7hsp901⁄7FKBP52 and receptor1⁄7hsp901⁄7CyP-40 heterocomplexes [24, 25]. Another component of steroid receptor heterocomplexes is protein phosphatase 5 (PP5) [26], which contains four TPRs [27]. Because the binding of FKBP52 and CyP-40 to hsp is competed by fragments of PP5 [28] and CyP-40 [29] comprising the TPR domains, we have proposed that there is a common TPR acceptor site on hsp that binds a variety of TPR-containing proteins [29]

Methods

Results

Conclusion