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(Received for publication, June 28, 1996, and in revised form, October 3, 1996)
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From the We have expressed the mitogenic
signaling proteins Src, Ras, Raf-1, Mek (MAP kinase kinase), and Erk
(MAP kinase) in baculovirus-infected Sf9 insect cells in order to study
a potential role for the chaperone hsp90 in formation of multiprotein
complexes. One such complex obtained by immunoadsorption with anti-Ras
antibody of cytosol prepared from cells simultaneously expressing Ras,
Raf, Mek, and Erk contained Ras, Raf, and Erk. To detect directly the
protein-protein interactions involved in forming multiprotein
complexes, we combined cytosols from single infections in
vitro in all possible combinations of protein pairs. We detected
complexes between Ras·Raf, Ras·Src, Raf·Mek, and Raf·Src, but
no complex containing Erk was obtained by mixing cytosols. Thus,
cellular factors appear to be required for assembly of the
Erk-containing multiprotein complex. One cellular factor thought to be
involved in signaling protein complex formation is the chaperone hsp90,
and we show that Src, Raf, and Mek are each complexed with insect
hsp90. Treatment 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 Ras, although it did
decrease the level of cytosolic Raf. To study geldanamycin action, we
treated rat 3Y1 fibroblasts 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 3Y1 cells. To determine if
geldanamycin affects mitogenic response, we treated HeLa cells with
epidermal growth factor (EGF) and showed that geldanamycin treatment
decreased EGF signaling and decreased the level of Raf protein without
affecting the EGF-mediated increase in Raf kinase activity. We conclude
that hsp90 is not required for forming complexes between the mitogenic
signaling proteins or for Raf kinase activity and that EGF signaling is decreased indirectly by geldanamycin because the antibiotic increases degradation of Raf and perhaps other components of the signaling pathway.
Department of Pharmacology, The University
of Michigan Medical School, Ann Arbor, Michigan 48109 and the
§ Molecular Oncology Program, Moffitt Cancer Center,
Tampa, Florida 33612
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