DESTABILIZATION OF RAF-1 BY GELDANAMYCIN LEADS TO DISRUPTION OF THE RAF-1-MEK-MITOGEN-ACTIVATED PROTEIN-KINASE SIGNALING PATHWAY

The serine/threonine kinase Raf-1 functions downstream of Ras in a sig nal transduction cascade which transmits mitogenic stimuli from the pl asma membrane to the nucleus. Raf-1 integrates signals coming from ext racellular factors and, in turn, activates its substrate, MEK kinase. MEK activates mitogen-activated protein kinase (MAPK), which phosphory lates other kinases as well as transcription factors. Raf-1 exists in a complex with HSP90 and other proteins. The benzoquinone ansamycin ge ldanamycin (GA) binds to HSP90 and disrupts the Raf-1-HSP90 multimolec ular complex, leading to destabilization of Raf-1. In this study, we e xamined whether Raf-1 destabilization is sufficient to block the Raf-1 -MEK-MAPK signalling pathway and whether GA specifically inactivates t he Raf-1 component of this pathway. Using the model system of NIH 3T3 cells stimulated with phorbol 12-myristate 13-acetate (PMA), we show t hat GA does not affect the ability of protein kinase Co to be activate d by phorbol esters, but it does block activation of MEK and MAPK. Fur ther, GA does not decrease the activity of constitutively active MEK i n transiently transfected cells. Finally, disruption of the Raf-1-MEK- MAPK signalling pathway by GA prevents both the PMA-induced proliferat ive response and PMA-induced activation of a MAPK-sensitive nuclear tr anscription factor. Thus, we demonstrate that interaction between HSP9 0 and Raf-1 is a sine qua non for Raf stability and function as a sign al transducer and that the effects observed cannot be attributed to a general impairment of protein kinase function.