Regulation of protein kinase C betaII by its C2 domain.

Abstract

The C2 domain serves as a membrane-targeting module in a diverse group of proteins that includes the conventional protein kinase Cs. This work examines the mechanism by which the C2 domain targets protein kinase C to membranes. Molecular modeling identified two highly-charged surfaces on the C2 domain of protein kinase C betaIotaIota: the Ca2+ binding site which contains five aspartates and a basic face positioned behind the Ca2+ site that contains seven lysine residues. Both surfaces were mutated to assess their role in Ca2+-dependent membrane binding. Surprisingly, removal of four positive charges on the basic face had no effect on protein kinase C's lipid or Ca2+ sensitivity, revealing that the basic face does not provide determinants involved in lipid binding, nor is it positioned close enough to the membrane to enhance nonspecific recruitment by its electropositive face. In contrast, replacement of two negative charges with two positive charges in the Ca2+ binding site decreased protein kinase C's affinity both for Ca2+ and for anionic lipids by several orders of magnitude. The dramatic reduction in electronegative potential resulting from this mutation did not increase protein kinase C's affinity for acidic membranes in the absence of Ca2+, revealing that simple charge neutralization does not account for how Ca2+ increases protein kinase C's affinity for anionic membranes. Our data suggest that (1) the membrane interaction surface of the C2 domain is localized to the Ca2+-binding site, with the positive face positioned away from the membrane, and (2) the Ca2+ site does not serve as a simple electrostatic switch.