THE HSP90-BINDING ANTIBIOTIC GELDANAMYCIN DECREASES RAF LEVELS AND EPIDERMAL GROWTH-FACTOR SIGNALING WITHOUT DISRUPTING FORMATION OF SIGNALING COMPLEXES OR REDUCING THE SPECIFIC ENZYMATIC-ACTIVITY OF RAF KINASE

We have expressed the mitogenic signaling proteins Src, Pus, Raf-l, Me k (MAP kinase kinase), and Erk (MAP kinase) in baculovirus-infected Sf 9 insect cells in order to study a potential role for the chaperone hs p90 in formation of multiprotein complexes. One such complex obtained by immunoadsorption with anti-Pas antibody of cytosol prepared fi om c ells simultaneously expressing Pas, Raf, Mek, and Erk contained Pas, R af, and Erk. To detect directly the protein-protein interactions invol ved in forming multiprotein complexes, we combined cytosols from singl e infections in vitro in all possible combinations of protein pairs, W e detected complexes between Ras Raf, Ras Src, Raf Mek, and Raf Src, b ut no complex containing Erk was obtained by mixing cytosols. Thus, ce llular factors appear to be required for assembly of the Erk-containin g multiprotein complex, One cellular factor thought to be involved in signaling protein complex formation is the chaperone hsp90, and we sho w that Src, Raf, and Mek are each complexed with insect hsp90. Treatme nt of Sf9 cells with geldanamycin, a benzoquinone ansamycin that binds to hsp90 and disrupts its function, did not decrease coadsorption of either Raf or Erk with Pas, although it did decrease the level of cyto solic Raf. To study geldanamycin action, we treated rat 3Y1 fibroblast s expressing v-Raf and showed that the antibiotic blocked assembly of Raf-hsp90 complexes at an intermediate stage of assembly where Raf is still bound to the p60 and hsp70 components of the assembly mechanism. As in Sf9 cells, Raf levels decline with geldanamycin treatment of 3Y 1 cells. To determine if geldanamycin affects mitogenic response, we t reated HeLa cells with epidermal growth factor (EGF) and showed that g eldanamycin treatment decreased EGF signaling and decreased the level of Raf protein without affecting the EGF-mediated increase in Raf kina se activity. We conclude that hsp90 is not required for forming comple xes between the mitogenic signaling proteins or for Raf kinase activit y and that EGF signaling is decreased indirectly by geldanamycin becau se the antibiotic increases degradation of Raf and perhaps other compo nents of the signaling pathway.