P50(CDC37) 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

Several protein kinases (e.g. pp60(src), v-Raf) exist in heterocomplex es with hsp90 and a 50-kDa protein that is the mammalian homolog of th e yeast cell cycle control protein Cdc37. In contrast, unliganded ster oid receptors exist in heterocomplexes with hsp90 and a tetratri-copep tide repeat (TPR) domain protein, such as an immunophilin. Although p5 0(cdc37) and TPR domain proteins bind directly to hsp90, p50(cdc37) is , not present in native steroid receptor.hsp90 heterocomplexes. To obt ain some insight as to how v-Raf selects predominantly hsp90.p50(cdc37 ) heterocomplexes, rather than hsp90 TPR protein heterocomplexes, we h ave examined the binding of p50(cdc37) to hsp90 and to Raf. We show th at p50(cdc37) exists in separate hsp90 heterocomplexes from the TPR do main proteins and that intact TPR proteins compete for p50(cdc37) bind ing 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 sh ow 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 vari ety of protein kinase heterocomplexes immunoadsorbed from cytosols.