p50(cdc37) acting in concert with Hsp90 is required for Raf-1 function

Genetic screens in Drosophila have identified p50(cdc37) to be an essential component of the sevenless receptor/ mitogen-activated kinase protein (MAP K) signaling pathway, but neither the function nor the target of p50(cdc37) in this pathway has been defined. In this study, we examined the role of p 50(cdc37) and its Hsp90 chaperone partner in Raf/MeK/MAPK signaling biochem ically. We found that coexpression of wild-type p50(cdc37) with Raf-1 resul ted in robust and dose-dependent activation of Raf-1 in Sf9 cells. In addit ion, p50(cdc37) greatly potentiated v-Src-mediated Raf-1 activation. Moreov er, we found that p50cdc37 is the primary determinant of Hsp90 recruitment to Raf-1. Overexpression of a p50(cdc37) mutant which is unable to recruit Hsp90 into the Raf-1 complex inhibited Raf-1 and MAPK activation by growth factors. Similarly, pretreatment,vith geldanamycin (GA), an Hsp90-specific inhibitor, prevented both the association of Raf-1 with the p50(cdc37)-Hsp9 0 heterodimer and Raf-1 kinase activation by serum. Activation of Raf-1 via baculovirus coexpression with oncogenic Src or Ras in Sf9 cells was also s trongly inhibited by dominant negative p50(cdc37) or by GA. Thus, formation of a ternary Raf-1-p50(cdc37)-Hsp90 complex is crucial for Raf-1 activity and MAPK pathway signaling. These results provide the first biochemical evi dence for the requirement of the p50(cdc37)-Hsp90 complex in protein kinase regulation and for Raf-1 function in particular.