CA2-KINASE CS MEMBRANE INTERACTION AND ACTIVATION( DIFFERENTIALLY REGULATES CONVENTIONAL PROTEIN)

The regulation of conventional protein kinase Cs by Ca2+ was examined by determining how this cation affects the enzyme's 1) membrane bindin g and catalytic function and 2) conformation. In the first part, we sh ow that significantly lower concentrations of Ca2+ are required to eff ect half-maximal membrane binding than to half-maximally activate the enzyme, The disparity between binding and activation kinetics is most striking for protein kinase C beta II, where the concentration of Ca2 promoting half-maximal membrane binding is approximately 40-fold high er than the apparent K-m for Ca2+ for activation, In addition, the Ca2 + requirement for activation of protein kinase C beta II is an order o f magnitude greater than that for the alternatively spliced protein ki nase C beta I; these isozymes differ only in 50 amino acids at the car boxyl terminus, revealing that residues in the carboxyl terminus influ ence the enzyme's Ca2+ regulation, In the second part, we use protease s as conformational probes to show that Ca2+-dependent membrane bindin g and Ca2+-dependent activation involve two distinct sets of structura l changes in protein kinase C beta II. Three separate domains spanning the entire protein participate in these conformational changes, sugge sting significant interdomain interactions, A highly localized hinge m otion between the regulatory and catalytic halves of the protein accom panies membrane binding; release of the carboxyl terminus accompanies the low affinity membrane binding mediated by concentrations of Ca2+ t oo low to promote catalysis; and exposure of the amino-terminal pseudo substrate and mashing of the carboxyl terminus accompany catalysis. In summary, these data reveal that structural determinants unique to eac h isozyme of protein kinase C dictate the enzyme's Ca2+-dependent affi nity for acidic membranes and show that, surprisingly, some of these d eterminants are in the carboxyl terminus of the enzyme, distal from th e Ca2+-binding site in the amino-terminal regulatory domain.