Activity of Plasma Membrane-recruited Raf-1 Is Regulated by Ras via the Raf Zinc Finger*
- From the Queensland Cancer Fund Laboratory of Experimental Oncology, Department of Pathology, University of Queensland Medical School, Brisbane 4006, Australia
Abstract
Ras recruits Raf to the plasma membrane for activation by a combination of tyrosine phosphorylation and other as yet undefined mechanism(s). We show here that the Raf zinc finger is not required for plasma membrane recruitment of Raf by Ras but is essential for full activation of Raf at the plasma membrane. Membrane targeting cannot compensate for the absence of the zinc finger. One facet of the zinc finger activation defect is revealed using a constitutively activated Raf mutant. Targeting Raf Y340D,Y341D to the plasma membrane increments activity, but full activation requires coexpression with activated Ras. This sensitivity to regulation by Ras at the plasma membrane is abrogated by mutations in the Raf zinc finger but is unaffected by mutation of the minimal Ras binding domain. These data show for the first time that Ras has two separate roles in Raf activation: recruitment of Raf to the plasma membrane through an interaction with the minimal Ras binding domain and activation of membrane-localized Raf via a mechanism that requires the Raf zinc finger.
Footnotes
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↵* This work was supported the National Health and Medical Research Council of Australia.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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↵‡ Also supported by the Royal Children’s Hospital Foundation. To whom correspondence should be addressed: Dept. of Pathology, University of Queensland Medical School, Herston Rd., Brisbane 4006, Australia. Tel.: 61 7 3365 5340; Fax: 61 7 3365 5511; E-mail:j.hancock{at}mailbox.uq.edu.au.
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↵1 The abbreviations used are: MEK, mitogen-activated protein kinase kinase; RBD, Ras binding domain; CRD, cysteine-rich domain; PCR, polymerase chain reaction; PAGE; polyacrylamide gel electrophoresis; ERK, extracellular-regulated protein kinase; MBP, myelin basic protein; RafCAAX, Raf with the C-terminal membrane targeting sequences of K-ras(B); DD, Y340D,Y341D double mutant; FF, Y340F,Y341F double mutant; CCSS, C165S,C168S double mutant.
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↵2 S. Roy and J. F. Hancock, unpublished data.
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- Received March 28, 1997.
- Revision received May 12, 1997.
