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J. Biol. Chem., Vol. 283, Issue 40, 27239-27254, October 3, 2008

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Positive Regulation of A-RAF by Phosphorylation of Isoform-specific Hinge Segment and Identification of Novel Phosphorylation Sites^*

Angela Baljuls, Werner Schmitz, Thomas Mueller^¶, René P. Zahedi^||, Albert Sickmann^||, Mirko Hekman, and Ulf R. Rapp¹

From the Institute for Medical Radiation and Cell Research, Institute of Physiological Chemistry, ^¶Julius-von-Sachs Institute for Biosciences, and ^||Rudolf Virchow Center/Deutsche Forschungsgemeinschaft Research Center for Experimental Biomedicine, University of Wuerzburg, 97078 Wuerzburg, Germany

In mammals the RAF family of serine/threonine kinases consists of three members, A-, B-, and C-RAF. Activation of RAF kinases involves a complex series of phosphorylations. Although the most prominent phosphorylation sites of B- and C-RAF are well characterized, little is known about regulatory phosphorylation of A-RAF. Using mass spectrometry, we identified here a number of novel in vivo phosphorylation sites in A-RAF. In particular, we found that Ser-432 participates in MEK binding and is indispensable for A-RAF signaling. On the other hand, phosphorylation within the activation segment does not contribute to epidermal growth factor-mediated activation. Furthermore, we show that the potential 14-3-3 binding domains in A-RAF are phosphorylated independently of its activation status. Of importance, we identified a novel regulatory domain in A-RAF (referred to as IH-segment) positioned between amino acids 248 and 267 that contains seven putative phosphorylation sites. Three of these sites, serines 257, 262, and 264, regulate A-RAF activation in a stimulatory manner. The spatial model of the A-RAF fragment, including residues between Ser-246 and Glu-277, revealed a switch of charge at the molecular surface of the IH-region upon phosphorylation, suggesting a mechanism in which the high accumulation of negative charges may lead to an electrostatic destabilization of protein-membrane interaction resulting in depletion of A-RAF from the plasma membrane. Together, we provide here for the first time a detailed analysis of in vivo A-RAF phosphorylation status and demonstrate that regulation of A-RAF by phosphorylation exhibits unique features compared with B- and C-RAF.

Received for publication, March 5, 2008 , and in revised form, July 23, 2008.

^* This work was supported by Deutsche Forschungsgemeinschaft Grant SFB 487, projects C3 and B2. 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 Table S1 and Figs. S1 and S2.

¹ To whom correspondence should be addressed: University of Wuerzburg, Institute for Medical Radiation and Cell Research, Versbacher Str. 5, 97078 Wuerzburg, Germany. Tel.: 49-931-201-45141; Fax: 49-931-201-45835; E-mail: rappur{at}mail.uni-wuerzburg.de.

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