MUTAGENESIS OF THE C2 DOMAIN OF PROTEIN-KINASE-C-ALPHA - DIFFERENTIALROLES OF CA2-BINDING RESIDUES( LIGANDS AND MEMBRANE)

The C2 domains of conventional protein kinase C (PKC) have been implic ated in their Ca2+-dependent membrane binding. The C2 domain of PKC-al pha contains several Ca2+ ligands that bind multiple Ca2+ ions and oth er putative membrane binding residues. To understand the roles of indi vidual Ca2+ ligands and protein-bound Ca2+ ions in the membrane bindin g and activation of PKC-alpha, we mutated five putative Ca2+ Ligands ( D187N, D193N, D246N, D248N, and D254N) and measured the effects of mut ations on vesicle binding, enzyme activity and monolayer penetration o f PKC-alpha. Altered properties of these mutants indicate that individ ual Ca2+ ions and their ligands have different roles in the membrane b inding and activation of PKC-alpha. The binding of Ca2+ to Asp(187), A sp(193), and Asp(246) of PKC-alpha is important for the initial bindin g of protein to membrane surfaces, On the other hand, the binding of a nother Ca2+ to Asp(187), Asp(246), Asp(248) and Asp(254) induces the c onformational change of PKC-alpha, which in turn triggers its membrane penetration and activation. Among these Ca2+ ligands, Asp(246) was sh own to be most essential for both membrane binding and activation of P KC-alpha, presumably due to its coordination to multiple Ca2+ ions. Fu rthermore, to identify the residues in the C2 domain that are involved in membrane binding of PKC-alpha, we mutated four putative membrane b inding residues (Trp(245), Trp(247), Arg(249) and Arg(252)). Membrane binding and enzymatic properties of two double site mutants (W245A/W24 7A and R249A/R252A) indicate that Arg(249) and Arg(252) are involved i n electrostatic interactions of PRC-alpha with anionic membranes, wher eas Trp(245) and Trp247 participate in its penetration into membranes and resulting hydrophobic interactions. Taken together, these studies provide the first experimental evidence for the role of C2 domain of c onventional PKC as a membrane docking unit as web as a module that tri ggers conformational changes to activate the protein.