Raf exists in a native heterocomplex with hsp90 and p50 that can be reconstituted in a cell-free system

L. F. Stancato, Y. H. Chow, K. A. Hutchison, G. H. Perdew, R. Jove, W. B. Pratt

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Abstract

Recently, we have demonstrated that the tyrosine kinase pp60(v-src) can undergo cell-free assembly into a heterocomplex with rabbit hsp90 and p50 when the immunoadsorbed protein is incubated with rabbit reticulocyte lysate (Hutchison, K. A., Brott, B. K., De Leon, J. H., Perdew, G. H., Jove, R., and Pratt, W. B. (1992) J. Biol. Chem. 267, 2902-2908). Using a baculovirus system to express a high level of human c-Raf serine/threonine kinase in Sf9 insect cells, we show here that immunoadsorbed c-Raf undergoes similar lysate-mediated assembly into a heterocomplex with hsp90 and p50. As with pp60(v-src) and steroid receptors, binding of c-Raf to hsp90 occurs in an ATP-dependent and K+-dependent manner and the resulting heterocomplex is stabilized by molybdate. With a very rapid and gentle procedure of Sf9 cell cytosol preparation and c-Raf immunoadsorption, we show coimmunoadsorption of the insect homologue of hsp90. The same procedures permit detection of a native complex of v-Raf with rat hsp90 and p50 in stably transfected rat 3Y1 fibroblasts, and v-Raf is also assembled into a heterocomplex with rabbit hsp90 and p50 by reticulocyte lysate. Using the 22W mutant of c-Raf in which the NH2-terminal half has been deleted, we show that the catalytic domain of the kinase is sufficient for both formation of the native heterocomplex in mouse NIH 3T3 cells and cell-free reconstitution of the heterocomplex by rabbit reticulocyte lysate. Although the native Raf-heat shock protein heterocomplex is less stable than native pp60(v-src) and glucocorticoid receptor heterocomplexes, by analogy with these proteins its detection may have important implications regarding the mechanism of Raf trafficking through the cytoplasm.

Original languageEnglish (US)
Pages (from-to)21711-21716
Number of pages6
JournalJournal of Biological Chemistry
Volume268
Issue number29
StatePublished - 1993

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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