Physical Association with Ras Enhances Activation of Membrane-bound Raf (RafCAAX)*
- From the Department of Cell Biology and Neuroscience, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9039
Abstract
The transforming activity of artificially membrane-targeted Raf1 suggests that Ras-mediated recruitment of Raf1 to the plasma membrane is an important step in Raf1 activation. Cellular Ras is concentrated in the caveolae, a microdomain of the plasma membrane that is highly enriched in caveolin, glycosylphosphatidylinositol-anchored proteins, and signal transduction molecules. Growth factor stimulation recruits Raf1 to this membrane domain. Whether Ras simply promotes Raf1 association with caveolae membranes or also modulates subsequent activation events is presently unclear. We have identified a ras variant, ras12V,37G, that does not interact with Raf1 but does interact with a mutant raf1, raf1(257L). To examine the role of Ras in the activation of membrane-bound Raf1, raf1CAAX, and raf1(257L)CAAX, membrane-targeted variants of Raf1 and raf1(257L), respectively, were expressed in fibroblasts with or without coexpression of ras12V,37G. Cell fractionation localized both raf1CAAX and raf1(257L)CAAX to caveolae membranes independent of ras12V,37G expression; however, coexpression of ras12V,37G enhanced the activation of raf(257L)CAAX, but not raf1CAAX, as monitored by induction of cellular transformation, increased Raf kinase activity, and induction of activated MAP kinase. These results suggest that the Ras/Raf1 interaction plays a role in Raf1 activation that is distinct from membrane recruitment.
Footnotes
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↵* This work was supported by National Institutes of Health Grants HL20948 and GM43169 (to R. G. A.) and CA71443 (to M. A. W.) and by the Perot Family Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ To whom correspondence should be addressed. Tel.: 214-648-2861; Fax: 214-648-8694; E-mail: white08@utsw.swmed.ed.
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↵1 The abbreviations used are: MAP, mitogen-activated protein; MEK, mitogen-activated or extracellular signal-regulated kinase kinase; Tricine,N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; PAGE, polyacrylamide gel electrophoresis.
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- Received January 27, 1997.
