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J. Biol. Chem., Vol. 282, Issue 36, 26575-26590, September 7, 2007

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Unique N-region Determines Low Basal Activity and Limited Inducibility of A-RAF Kinase

THE ROLE OF N-REGION IN THE EVOLUTIONARY DIVERGENCE OF RAF KINASE FUNCTION IN VERTEBRATES^*

Angela Baljuls, Thomas Mueller, Hannes C. A. Drexler, Mirko Hekman, and Ulf R. Rapp¹

From the Institute for Medical Radiation and Cell Research and Institute of Physiological Chemistry, University of Wuerzburg, 97078 Wuerzburg, Germany

In mammals the RAF family of serine/threonine kinases consists of three members, A-, B-, and C-RAF. A prominent feature of RAF isoforms regards differences in basal and inducible kinase activities. To elucidate the nature of these differences, we studied the role of the nonconserved residues within the N-region (Negative-charge regulatory region). The nonconserved amino acids in positions -3 and +1 relative to the highly conserved serine 299 in A-RAF and serine 338 in C-RAF have so far not been considered as regulatory residues. Here we demonstrate the essential role of these residues in the RAF activation process. Substitution of tyrosine 296 in A-RAF to arginine led to a constitutively active kinase. In contrast, substitution of glycine 300 by serine (mimicking B- and C-RAF) acts in an inhibitory manner. Consistent with these data, the introduction of glycine in the analogous position of C-RAF (S339G mutant) led to a constitutively active C-RAF kinase. Based on the three-dimensional structure of the catalytic domain of B-RAF and using the sequences of the N-regions of A- and C-RAF, we searched by molecular modeling for the putative contact points between these two moieties. A tight interaction between the N-region residue serine 339 of C-RAF and arginine 398 of the catalytic domain was identified and proposed to inhibit the kinase activity of RAF proteins, because abrogation of this interaction contributes to RAF activation. Furthermore, tyrosine 296 in A-RAF favors a spatial orientation of the N-region segment, which enables a tighter contact to the catalytic domain, whereas a glutamine residue at this position in C-RAF abrogates this interaction. Considering this observation, we suggest that tyrosine 296, which is unique for A-RAF, is a major determinant of the low activating potency of this RAF isoform.

Received for publication, March 21, 2007 , and in revised form, July 2, 2007.

^* This work was supported by Deutsche Forschungsgemeinschaft Grant GK 639, project A5, and 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 Figs. S1 and S2.

¹ To whom correspondence should be addressed: University of Wuerzburg, Institute for Medical Radiation and Cell Research, Versbacher Strasse 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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