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 reticuloc yte 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 ser ine/threonine kinase in Sf9 insect cells, we show here that immunoadso rbed c-Raf undergoes similar lysate-mediated assembly into a heterocom plex with hsp90 and p50. As with pp60v-src and steroid receptors, bind ing of c-Raf to hsp90 occurs in an ATP-dependent and K+-dependent mann er 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 homolog ue of hsp90. The same procedures permit detection of a native complex of v-Raf with rat hsp90 and p50 in stably transfected rat 3Y1 fibrobla sts, and v-Raf is also assembled into a heterocomplex with rabbit hsp9 0 and p50 by reticulocyte lysate. Using the 22W mutant of c-Raf in whi ch the NH2-terminal half has been deleted, we show that the catalytic domain of the kinase is sufficient for both formation of the native he terocomplex 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 pro teins its detection may have important implications regarding the mech anism of Raf trafficking through the cytoplasm.