Destabilization of Raf-1 by Geldanamycin Leads to Disruption of the Raf-1–MEK–Mitogen-Activated Protein Kinase Signalling Pathway

Abstract

The serine/threonine kinase Raf-1 functions downstream of Ras in a signal transduction cascade which transmits mitogenic stimuli from the plasma membrane to the nucleus. Raf-1 integrates signals coming from extracellular factors and, in turn, activates its substrate, MEK kinase. MEK activates mitogen-activated protein kinase (MAPK), which phosphorylates other kinases as well as transcription factors. Raf-1 exists in a complex with HSP90 and other proteins. The benzoquinone ansamycin geldanamycin (GA) binds to HSP90 and disrupts the Raf-1-HSP90 multimolecular complex, leading to destabilization of Raf-1. In this study, we examined whether Raf-1 destabilization is sufficient to block the Raf-1-MEK-MAPK signalling pathway and whether GA specifically inactivates the Raf-1 component of this pathway. Using the model system of NIH 3T3 cells stimulated with phorbol 12-myristate 13-acetate (PMA), we show that GA does not affect the ability of protein kinase C␣ to be activated by phorbol esters, but it does block activation of MEK and MAPK. Further, GA does not decrease the activity of constitutively active MEK in transiently transfected cells. Finally, disruption of the Raf-1-MEK-MAPK signalling pathway by GA prevents both the PMA-induced proliferative response and PMA-induced activation of a MAPK-sensitive nuclear transcription factor. Thus, we demonstrate that interaction between HSP90 and Raf-1 is a sine qua non for Raf stability and function as a signal transducer and that the effects observed cannot be attributed to a general impairment of protein kinase function.

Targeting of the protein chaperone, HSP90, by the transformation suppressing agent, radicicol

“…Previous studies have shown that HSP90 was required for the stabilization and appropriate intracellular localization of Raf (Schulte et al, 1995(Schulte et al, , 1996. The observations of (Soga et al, 1998) suggested that radicicol treatment of K-Ras transformed cells resulted in the destabilization of Raf-1.…”

“…Molecular chaperones such as HSP90 play a major role in cellular homeostasis by aiding in the proper folding and localization of a number of key proteins (reviewed in Johnson and Craig, 1997). The association of Raf-1 with the molecular chaperones HSP90 and cdc37/p50 appears to play a critical role in maintaining the stability and membrane localization of Raf (Schulte et al, 1995(Schulte et al, , 1996. In particular, HSP90 binding agents such as the benzoquinone ansamycin, geldanamycin, promotes ± 4), pre-incubated ®rst with geldanamycin followed by KT8529 (lanes 6 ± 9) or pre-incubated ®rst with UCS1006 followed by KT8529 (lanes 11 ± 14) as indicated at the bottom of the autoradiograms and as described in the Materials and methods section.…”

“…The ability of radicicol to bind to HSP90 and cause the destabilization of Raf (Soga et al, 1998) is reminiscent of geldanamycin Schulte et al, 1995Schulte et al, , 1996Stancato et al, 1997). To compare the HSP90 binding ability of radicicol and geldanamycin, the ability of geldanamycin to block the speci®c binding of radicicol to HSP90 was examined ( Figure 5).…”

“…These ®ndings suggest the possibility that radicicol, by binding to HSP90, may destabilize the Raf-1-HSP90 complex in a manner reminiscent of the benzoquinone ansamycin, geldanamycin (Schulte et al, 1996). In this regard, radicicol is the ®rst non-ansamycin antibiotic to associate speci®cally with HSP90.…”

“…Besides Ras, other proteins that interact with Raf, and perhaps regulate it, include Mitogen-or extracellular-regulated kinase (MEK1/2); proteins of the 14-3-3 family; molecular chaperones such as HSP90 and Cdc37/p50; and the protein Kinase Suppressor of Ras (KSR) (reviewed in Morrison and Cutler, 1997). Interaction of HSP90 and Cdc37/p50 appears to be important for the proper intracellular localization and stability of Raf (Schulte et al, 1995(Schulte et al, , 1996Stancato et al, 1997;reviewed in Hunter and Poon, 1997).…”