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Association of Hsp90 with cellular Src-family kinases in a cell-free system correlates with altered kinase structure
1Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater 74078-0454.
Abstract:
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)
Insights
Cellular src family tyrosine kinases, including p59fgr and p56lck, form complexes with heat shock protein 90 (hsp90) after synthesis. These hsp90 complexes are associated with reduced kinase activity.
Area of Science:
- Molecular Biology
- Cellular Signaling
- Protein Biochemistry
Background:
- Retroviral oncogenes like src form cytoplasmic complexes with hsp90 and p50.
- These complexes are intermediates for mature, membrane-associated kinases.
- Soluble complexes of nascent cellular homologs with hsp90-p50 were not readily detected previously.
Purpose of the Study:
- To investigate complex formation between cellular src family tyrosine kinases and hsp90.
- To determine if myeloid-specific p59fgr, B cell-specific p59fgr, and p56lck associate with hsp90 post-synthesis.
Main Methods:
- In vitro protein synthesis in reticulocyte lysate.
- Separation of synthesized proteins using glycerol gradients.
- Co-immunoadsorption using anti-hsp90 and anti-p56lck antibodies.
- Detection of protein complexes in the absence of detergent.
Main Results:
- Fast-sedimenting forms of p59fgr and p56lck were co-immunoadsorbed by anti-hsp90 antibodies.
- These hsp90 complexes were detected without detergent.
- Firefly luciferase did not form stable complexes with hsp90.
- The complex-bound form of p56lck showed deficient autophosphorylation and reduced substrate phosphorylation.
Conclusions:
- Cellular src family tyrosine kinases (p59fgr, p56lck) form soluble complexes with hsp90.
- These hsp90-bound kinases exhibit altered enzymatic activity.
- The findings suggest a role for hsp90 in regulating src family kinase function.