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 encoded 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 kinase. However, soluble complexes between the nascent cellular homologs of these proteins and hsp90-p50 have not been readily detected [Brugge, J.S. (1986) Curr. Top. Microbiol. Immunol. 123, 1-22 and references therein]. In this paper, we have utilized protein synthesis in reticulocyte lysate to determine whether three cellular members of the src family of tyrosine kinases, myeloid-specific p59fgr, B cell-specific p59fgr, and p56lck, form complexes with hsp90. Following their synthesis, fast- and slow-sedimenting forms of these proteins can be separated on glycerol gradients. Anti-hsp90 monoclonal antibodies co-immunoadsorb the fast-sedimenting, but not the slow-sedimenting, forms of these kinases from gradient fractions. These hsp90 complexes can be detected in the complete absence of detergent. Conversely, an unrelated protein, firefly luciferase, does not form stable complexes with hsp90 following synthesis in reticulocyte lysate. Anti-p56lck antibodies specifically co-immunoadsorb hsp90 from protein synthesis reactions programmed with lckRNA. The fast-sedimenting, complex-bound form of p56lck is deficient in autophosphorylation activity and phosphorylates an exogenous substrate, acid-treated enolase, less efficiently than does the monomeric form. Fast-sedimenting p56lck is hypersentitive to limited proteolysis by chymotrypsin.(ABSTRACT TRUNCATED AT 250 WORDS)
Hartson
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SD
Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater 74078-0454.
Matts
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RL
D001665:Binding Sites; D002474:Cell-Free System; D006360:Heat-Shock Proteins / Q000378:metabolism*; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D015688:Oncogene Protein pp60(v-src) / Q000378:metabolism*; D011487:Protein Conformation; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D013329:Structure-Activity Relationship; D019061:src-Family Kinases
D006360:Heat-Shock Proteins; D011518:Proto-Oncogene Proteins; D011505:Protein-Tyrosine Kinases; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D015688:Oncogene Protein pp60(v-src); C053102:proto-oncogene proteins c-fgr; D019061:src-Family Kinases