Structural and biochemical analyses of native and phosphatase-treated protein kinase C indicate that protein kinase C is processed by three phosphorylations. Firstly, trans-phosphorylation on the activation loop (T500) renders it catalytically competent to autophosphorylate. Secondly, a subsequent autophosphorylation on the carboxyl terminus (T641) maintains catalytic competence. Thirdly, a second autophosphorylation on the carboxyl terminus (S660) regulates the enzyme's subcellular localization. The conservation of each of these residues (or an acidic residue) in conventional, novel and atypical protein kinase Cs underscores the essential role for each in regulating the protein kinase C family.

The contributions of phospholipid headgroup structure, diacylglycerol, and Ca2+ in regulating the interaction of protein kinase C beta II with membranes or detergent/lipid mixed micelles were examined. Binding measurements revealed that, in the absence of diacylglycerol, protein kinase C displays no significant selectivity for headgroup structure other than change: the enzyme binds with equal affinity to phosphatidyl-L-serine, phosphatidyl-D-serine, and other monoanionic lipids such as phosphatidylglycerol. In contrast, selectivity for headgroup occurs in the presence of diacylglycerol. This second messenger increases the affinity of protein kinase C for phosphatidyl-L-serine-containing membranes or micelles by 2 orders of magnitude, but has only moderate effects on the affinity of protein kinase C for surfaces containing other anionic lipids. Ca2+ does not affect the diacylglycerol-mediated increase in protein kinase C's affinity for phosphatidylserine, but does increase the enzyme's affinity for acidic phospholipids. Lastly, ionic strength studies reveal that electrostatic interactions are the primary driving force in the interaction of protein kinase C with membranes. In the absence of either diacylglycerol or phosphatidylserine, these interactions are sufficiently weak that little binding occurs at physiological ionic strength; thus, protein kinase C is unlikely to translocate to the plasma membranes in the absence of diacylglycerol, even if intracellular Ca2+ levels are high. Our data reveal that, although there is no specificity for binding acidic lipids in the absence of diacylglycerol, specific structural elements of the L-serine headgroup are required for the high-affinity binding of protein kinase C to diacylglycerol-containing membranes.

The regulation of conventional protein kinase Cs by Ca 2؉ 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 Ca 2؉ 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 ␤II, where the concentration of Ca 2؉ promoting half-maximal membrane binding is approximately 40-fold higher than the apparent K m for Ca 2؉ for activation. In addition, the Ca 2؉ requirement for activation of protein kinase C ␤II is an order of magnitude greater than that for the alternatively spliced protein kinase C ␤I; these isozymes differ only in 50 amino acids at the carboxyl terminus, revealing that residues in the carboxyl terminus influence the enzyme's Ca 2؉ regulation. In the second part, we use proteases as conformational probes to show that Ca 2؉ -dependent membrane binding and Ca 2؉-dependent activation involve two distinct sets of structural changes in protein kinase C ␤II. 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 Ca 2؉ 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 Ca 2؉ -dependent affinity for acidic membranes and show that, surprisingly, some of these determinants are in the carboxyl terminus of the enzyme, distal from the Ca 2؉

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