ABSTRACT
Genetic screens in Drosophila have identified p50(cdc37) to be an essential component of the sevenless receptor/mitogen-activated kinase protein (MAPK) 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 p50(cdc37) and its Hsp90 chaperone partner in Raf/Mek/MAPK signaling biochemically. We found that coexpression of wild-type p50(cdc37) with Raf-1 resulted in robust and dose-dependent activation of Raf-1 in Sf9 cells. In addition, p50(cdc37) greatly potentiated v-Src-mediated Raf-1 activation. Moreover, we found that p50(cdc37) 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 with geldanamycin (GA), an Hsp90-specific inhibitor, prevented both the association of Raf-1 with the p50(cdc37)-Hsp90 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 strongly 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 evidence for the requirement of the p50(cdc37)-Hsp90 complex in protein kinase regulation and for Raf-1 function in particular.
Subject(s)
Cell Cycle Proteins/metabolism , Drosophila Proteins , HSP90 Heat-Shock Proteins/metabolism , Molecular Chaperones , Proto-Oncogene Proteins c-raf/metabolism , Animals , Benzoquinones , COS Cells , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Cell Cycle Proteins/genetics , Cell Line , Chaperonins , Chickens , Dimerization , Enzyme Activation , Enzyme Inhibitors/pharmacology , Epidermal Growth Factor/metabolism , Epidermal Growth Factor/pharmacology , HSP90 Heat-Shock Proteins/genetics , Humans , Lactams, Macrocyclic , Proto-Oncogene Proteins c-raf/genetics , Quinones/pharmacology , Rabbits , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Signal Transduction , SpodopteraABSTRACT
Using a monoclonal antibody, IVd4, that recognizes a novel group of hyaluronan-binding proteins, we have immunoscreened a cDNA library constructed from embryonic chick heart muscle mRNA. One of the cDNAs isolated from the library encodes a 29.3-kDa protein homologous to Cdc37, an essential cell cycle regulatory factor previously characterized genetically in yeast and Drosophila; this is the first vertebrate CDC37 gene to be cloned to date. We also present evidence for the existence of a second chick isoform that is identical to the 29.3-kDa protein over the first 175 amino acids but is entirely different at the carboxyl terminus and lacks the IVd4 epitope. The avian Cdc37 binds hyaluronan, chondroitin sulfate and heparin in vitro, and both isoforms contain glycosaminoglycan-binding motifs previously described in several hyaluronan-binding proteins. These findings suggest a role for glycosaminoglycans in cell division control.
