Raf exists in a native heterocomplex with hsp90 and p50 that can be reconstituted in a cell-free system.
Recently, we have demonstrated that the tyrosine kinase pp60v-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 pp60v-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 pp60v-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.
Stancato
L F
LF
Department of Pharmacology, University of Michigan Medical School, Ann Arbor 48109.
Chow
Y H
YH
Hutchison
K A
KA
Perdew
G H
GH
Jove
R
R
Pratt
W B
WB
D016475:3T3 Cells; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D016466:CHO Cells; D002384:Catalysis; D002474:Cell-Free System; D002478:Cells, Cultured; D006224:Cricetinae; D006360:Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D007313:Insecta; D051379:Mice; D027384:Oncogene Proteins v-raf; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf; D011817:Rabbits; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000378:metabolism; D015682:Retroviridae Proteins, Oncogenic / Q000235:genetics / Q000378:metabolism*
D006360:Heat-Shock Proteins; D011506:Proteins; D011518:Proto-Oncogene Proteins; D011965:Receptors, Glucocorticoid; D015682:Retroviridae Proteins, Oncogenic; D011505:Protein-Tyrosine Kinases; D027384:Oncogene Proteins v-raf; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf