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Papers In Press, published online ahead of print April 30, 2007
Biochemistry, Virginia Commonwealth University, Richmond, VA 23298-0614
Corresponding Author: cechalfant{at}vcu.edu
Previously, ceramide-1-phosphate (C1P) was demonstrated to be a potent and specific activator of cPLA2alpha via interaction with the C2 domain. In this study, we hypothesized that the specific interaction site for C1P was localized to the cationic beta-groove (R57, K58, R59) of the C2 domain of cPLA2alpha. In this regard, mutants of this region of cPLA2alpha were generated ((R57A/K58A/R59A), (R57A/R59A), (K58A/R59A), (R57A/K58A), (R57A), (K58A), and (R59A)) and examined for C1P affinity by surface plasmon resonance (SPR). The triple (R57A/ K58A/ R59A), the double mutants (R57A/R59A), (K58A/R59A), (R57A/K58A), and the single mutant (R59A) demonstrated significantly reduced affinity for C1P containing vesicles compared to wild-type cPLA2alpha. Examining these mutants for enzymatic activity demonstrated that these five mutants of cPLA2alpha also showed a significant reduction in the ability of C1P to: 1) increase the Vmax of the reaction; and 2) significantly decrease the dissociation constant (KsA) of the reaction as compared to the wild-type enzyme. The mutational effect was specific for C1P as all of the cationic mutants of cPLA2alphademonstrated normal basal activity as well as normal affinities for PC and PtdIns(4,5)P2 compared to wild-type cPLA2alpha. This study, for the first time, demonstrates a novel C1P interaction site mapped to the cationic beta-groove of the C2 domain of cPLA2alpha.
J. Biol. Chem, 10.1074/jbc.M701396200
Submitted on February 16, 2007
Accepted on April 30, 2007
Ceramide-1-phosphate binds group IVA cytosolic phospholipase A2 via a novel site in the C2 domain
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