ASSOCIATION OF HSP90 WITH CELLULAR SRC-FAMILY KINASES IN A CELL-FREE SYSTEM CORRELATES WITH ALTERED KINASE STRUCTURE AND FUNCTION

Following synthesis in the cytoplasm, the transforming proteins encode d by the retroviral oncogenes src, yes,fps,fes, and fgr form complexes with hsp90 and the hsp90 cohort p50. These cytoplasmic complexes are intermediates in the production of the mature membrane-associated kina se. However, soluble complexes between the nascent cellular homologs o f these proteins and hsp90-p50 have not been readily detected [Brugge, J.S. (1986) Curr. Top. Microbiol. Immunol. 123, 1-22 and references t herein]. In this paper, we have utilized protein synthesis in reticulo cyte lysate to determine whether three cellular members of the src fam ily of tyrosine kinases, myeloid-specific p59(fgr) B cell-specific p59 (fgr), and p56(lck), form complexes with hsp90. Following their synthe sis, fast- and slow-sedimenting forms of these proteins can be separat ed on glycerol gradients. Anti-hsp90 monoclonal antibodies co-immunoad sorb the fast-sedimenting, but not the slow-sedimenting, forms of thes e kinases from gradient fractions. These hsp90 complexes can be detect ed in the complete absence of detergent. Conversely, an unrelated prot ein, firefly luciferase, does not form stable complexes with hsp90 fol lowing synthesis in reticulocyte lysate. Anti-p56(lck) antibodies spec ifically co-immunoadsorb hsp90 from protein synthesis reactions progra mmed with lck RNA. The fast-sedimenting, complex-bound form of p56(lck ) is deficient in autophosphorylation activity and phosphorylates an e xogenous substrate, acid-treated enolase, less efficiently than does t he monomeric form. Fast-sedimenting p56(lck) is hypersentitive to limi ted proteolysis by chymotrypsin. These results demonstrate that cellul ar members of the src family of tyrosine kinases, like the oncogenic v iral members, form specific and stable complexes with hsp90 and provid e an initial characterization of the structural and functional differe nces between hsp90-associated versus monomeric kinases.