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J. Biol. Chem., Vol. 282, Issue 36, 26503-26516, September 7, 2007

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B- and C-RAF Display Essential Differences in Their Binding to Ras

THE ISOTYPE-SPECIFIC N TERMINUS OF B-RAF FACILITATES RAS BINDING^*

Andreas Fischer¹, Mirko Hekman¹, Jürgen Kuhlmann, Ignacio Rubio^¶, Stefan Wiese^||2, and Ulf R. Rapp³

From the Institut für Medizinische Strahlenkunde und Zellforschung, University of Wuerzburg, 97078 Wuerzburg, Germany, Max Planck Institute for Molecular Physiology, 44227 Dortmund, Germany, ^¶Institute of Molecular Cell Biology, Center for Molecular Biomedicine, Friedrich-Schiller-University Jena, 07747 Jena, Germany, and ^||Institute for Clinical Neurobiology, University of Wuerzburg, 97080 Wuerzburg, Germany

Recruitment of RAF kinases to the plasma membrane was initially proposed to be mediated by Ras proteins via interaction with the RAF Ras binding domain (RBD). Data reporting that RAF kinases possess high affinities for particular membrane lipids support a new model in which Ras-RAF interactions may be spatially restricted to the plane of the membrane. Although the coupling features of Ras binding to the isolated RAF RBD were investigated in great detail, little is known about the interactions of the processed Ras with the functional and full-length RAF kinases. Here we present a quantitative analysis of the binding properties of farnesylated and nonfarnesylated H-Ras to both full-length B- and C-RAF in the presence and absence of lipid environment. Although isolated RBD fragments associate with high affinity to both farnesylated and nonfarnesylated H-Ras, the full-length RAF kinases revealed fundamental differences with respect to Ras binding. In contrast to C-RAF that requires farnesylated H-Ras, cytosolic B-RAF associates effectively and with significantly higher affinity with both farnesylated and nonfarnesylated H-Ras. To investigate the potential farnesyl binding site(s) we prepared several N-terminal fragments of C-RAF and found that in the presence of cysteine-rich domain only the farnesylated form of H-Ras binds with high association rates. The extreme N terminus of B-RAF turned out to be responsible for the facilitation of lipid independent Ras binding to B-RAF, since truncation of this region resulted in a protein that changed its kinase properties and resembles C-RAF. In vivo studies using PC12 and COS7 cells support in vitro results. Co-localization measurements using labeled Ras and RAF documented essential differences between B- and C-RAF with respect to association with Ras. Taken together, these data suggest that the activation of B-RAF, in contrast to C-RAF, may take place both at the plasma membrane and in the cytosolic environment.

Received for publication, August 4, 2006 , and in revised form, July 5, 2007.

^* This work was supported by Deutsche Forschungsgemeinschaft Grants SFB 487 and SFB 581. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S3.

¹ These two authors contributed equally to this work.

² Present address: Group for Molecular Cell Biology, Ruhr-University, 44801 Bochum, Germany.

³ To whom correspondence should be addressed. Tel.: 49-931-201-45141; Fax: 49-931-201-45835; E-mail: rappur{at}mail.uni-wuerzburg.de.

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