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

doi:10.1016/s0021-9258(20)80600-0

Abstract

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.

Highlights

From the Departments of $Pharmacology and $Microbiology and Immunology, The University ofMichigan Medical School, Ann Arbor, Michigan 48109 and the Wepartment of Foods and Nutrition, Purdue University, West Lafayette, Indiana47907
We have demonstrated that thetyrosine ki- heterocomplex with hsp90 and p50 until it localizes t o cell nase pp60v-a"cr an undergo cell-freeassembly into a het- membranes [2, 3], it is thought that hsp90 and p50 are someerocomplex with rabbit hsp90 and p50 when the how involved in the trafficking ofpp60""" through the cytoimmunoadsorbed protein is incubated with rabbit re- plasm tothe cell membrane.The steroid ticulocyte lysate
The same procedures permidt etection of a native the presence of pp60"~""or steroid receptors [7,8,9]. Both steroid complex of v-Raf with rat hsp9O and p50 in stably trans- receptor [10,11]and pp60""'" [12] heterocomplexes with hsp90 fected rat 3Y1 fibroblasts, and v-Raf is assembled have been reconstituted by an ATP-dependent, K+-dependinto a heterocomplex with rabbit hsp90 and p50 by re- ent [13, 14]2system in rabbitreticulocyte lysate

Summary

11 To whom correspondence should be addressed

Dept. of Pharmacology, The University of Michigan Medical School, 6322 Medical Science. Using the 22W munoadsorbed to protein A-Sepharose with 10% BuGR antibody as described by Hutchison et al [25].Prior to reconstitution withreticulocyte lysate, GR-associated proteins were stripped by incubating the immunopellets at 0 "C in 0.5 M NaCl as described by Hutchison et a f .(14).All other procedures were the sameas those for Raf. Gel Electrophoresisand WesternBlotting-The immunoadsorbed protein A-Sepharose pellets were heated in SDS sample buffer, and protant of c-Raf in which the NH2-terminal half hasbeen deleted, we show that the catalytdicomain of the kinaseis sufficient for both formation of the native heterocomplex and cell-free heterocomplex assembly. Gel Electrophoresisand WesternBlotting-The immunoadsorbed protein A-Sepharose pellets were heated in SDS sample buffer, and protant of c-Raf in which the NH2-terminal half hasbeen deleted, we show that the catalytdicomain of the kinaseis sufficient for both formation of the native heterocomplex and cell-free heterocomplex assembly These results are consistent with the notion that hsp anpd may be involved in Raf trafficking in the intactcell. The immunoblots were incubated a second time with the appropriate '251-labeled counterantibody, or in the case of p50, the protein

EXPERIMENTAL PROCEDURES

RESULTS

Methods