GELDANAMYCIN-INDUCED DESTABILIZATION OF RAF-1 INVOLVES THE PROTEASOME

The Raf-1-MEK-MAPK pathway plays an important role in transducing extr acellular growth factor signaling into altered nuclear transcription f actor function, The benzoquinone ansamycin Geldanamycin (GA) specifica lly binds to the heat shock protein HSP90 and alters its complex with Raf-1. This leads to a decrease in Raf-1 levels and to disruption of t he Raf-1-MEK-MAPK signaling pathway, The enhanced degradation of Raf-1 protein was prevented by inhibitors of the proteasome, while inhibiti on of lysosomal or other proteases was ineffective, Raf-1 that was pro tected from GA-induced degradation was of higher molecular weight and showed a laddering pattern consistent with its polyubiquitination. Unl ike Raf-1 in untreated cells, the protein was insoluble in Triton X100 - or NP40-based buffers, Signaling through this pathway was inhibited by GA, concomitant with loss of Raf-1 protein, but was restored if Raf -1 was protected from GA-induced degradation by proteasome inhibitors. (C) 1997 Academic Press.