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J. Biol. Chem., Vol. 282, Issue 28, 20467-20474, July 13, 2007

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Ceramide-1-phosphate Binds Group IVA Cytosolic Phospholipase a₂ via a Novel Site in the C2 Domain^*

Robert V. Stahelin¹, Preeti Subramanian^¶1, Mohsin Vora^¶, Wonhwa Cho^||, and Charles E. Chalfant^¶**2

From the ^¶Department of Biochemistry, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, Virginia 23298-0614, the ^||Department of Chemistry, University of Illinois at Chicago, Chicago, Illinois 60607-7061, the Department of Biochemistry and Molecular Biology, Indiana University School of Medicine-South Bend and Department of Chemistry and Biochemistry and The Walther Center for Cancer Research, University of Notre Dame, South Bend, Indiana 46617, and ^**Research and Development, Hunter Holmes McGuire Veterans Affairs Medical Center, Richmond, Virginia 23249

Previously, ceramide-1-phosphate (C1P) was demonstrated to be a potent and specific activator of group IV cytosolic phospholipase A₂ (cPLA₂) via interaction with the C2 domain. In this study, we hypothesized that the specific interaction site for C1P was localized to the cationic -groove (Arg⁵⁷, Lys⁵⁸, Arg⁵⁹) of the C2 domain of cPLA₂. In this regard, mutants of this region of cPLA₂ were generated (R57A/K58A/R59A, R57A/R59A, K58A/R59A, R57A/K58A, R57A, K58A, and R59A) and examined for C1P affinity by surface plasmon resonance. The triple mutants (R57A/K58A/R59A), the double mutants (R57A/R59A, K58A/R59A, and R57A/K58A), and the single mutant (R59A) demonstrated significantly reduced affinity for C1P-containing vesicles as compared with wild-type cPLA₂. Examining these mutants for enzymatic activity demonstrated that these five mutants of cPLA₂ also showed a significant reduction in the ability of C1P to: 1) increase the V_max of the reaction; and 2) significantly decrease the dissociation constant (K ^A_s) of the reaction as compared with the wild-type enzyme. The mutational effect was specific for C1P as all of the cationic mutants of cPLA₂ demonstrated normal basal activity as well as normal affinities for phosphatidylcholine and phosphatidylinositol-4,5-bisphosphate as compared with wild-type cPLA₂. This study, for the first time, demonstrates a novel C1P interaction site mapped to the cationic -groove of the C2 domain of cPLA₂.

Received for publication, February 16, 2007 , and in revised form, March 22, 2007.

^* This work was supported by grants from the Veterans Affairs (Veterans Affairs Merit Review I) (to C. E. C.), from National Institutes of Health Grants HL072925 (to C. E. C.), CA117950 (to C. E. C.), GM52598 (to W. C.), GM53987 (to W. C.), and GM68849 (to W. C.) and American Heart Association AHA 5-30693 predoctoral fellowship (to P. S.). This work was also supported by an Indiana University Biomedical Research Grant (to R. V. S.). 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 two supplemental figures and a supplemental table.

¹ Both authors contributed equally to the manuscript.

² To whom correspondence should be addressed: Dept. of Biochemistry, Rm. 2-016, Sanger Hall, Virginia Commonwealth University, 1101 East Marshall St., P. O. Box 980614, Richmond, VA 23298-0614. Tel.: 804-828-9526; Fax: 804-828-1473; E-mail: cechalfant{at}vcu.edu.

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