Ca2+ differentially regulates conventional protein kinase Cs' membrane interaction and activation.
The regulation of conventional protein kinase Cs by Ca2+ was examined by determining how this cation affects the enzyme's 1) membrane binding and catalytic function and 2) conformation. In the first part, we show that significantly lower concentrations of Ca2+ are required to effect 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 betaII, where the concentration of Ca2+ promoting half-maximal membrane binding is approximately 40-fold higher than the apparent Km for Ca2+ for activation. In addition, the Ca2+ requirement for activation of protein kinase C betaII is an order of magnitude greater than that for the alternatively spliced protein kinase C betaI; these isozymes differ only in 50 amino acids at the carboxyl terminus, revealing that residues in the carboxyl terminus influence the enzyme's Ca2+ regulation. In the second part, we use proteases as conformational probes to show that Ca2+dependent membrane binding and Ca2+-dependent activation involve two distinct sets of structural changes in protein kinase C betaII. Three separate domains spanning the entire protein participate in these conformational changes, suggesting significant interdomain interactions. A highly localized hinge motion between the regulatory and catalytic halves of the protein accompanies membrane binding; release of the carboxyl terminus accompanies the low affinity membrane binding mediated by concentrations of Ca2+ too low to promote catalysis; and exposure of the amino-terminal pseudosubstrate and masking of the carboxyl terminus accompany catalysis. In summary, these data reveal that structural determinants unique to each isozyme of protein kinase C dictate the enzyme's Ca2+-dependent affinity for acidic membranes and show that, surprisingly, some of these determinants are in the carboxyl terminus of the enzyme, distal from the Ca2+-binding site in the amino-terminal regulatory domain.
Keranen
L M
LM
Department of Pharmacology, University of California at San Diego, La Jolla, California 92093-0640, USA.
Newton
A C
AC
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000378:metabolism; D002118:Calcium / Q000502:physiology*; D002384:Catalysis; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism; D004789:Enzyme Activation; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011493:Protein Kinase C / Q000378:metabolism*
D000906:Antibodies; D007527:Isoenzymes; D011493:Protein Kinase C; D010450:Endopeptidases; D008274:Magnesium; D002118:Calcium