Ca2+ Differentially Regulates Conventional Protein Kinase Cs' Membrane Interaction and Activation

Abstract

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؉

Prolonged glucose infusion into conscious rats inhibits early steps in insulin signalling and induces translocation of GLUT4 and protein kinase C in skeletal muscle

“…However, the effect on PKCbI failed to reach statistical significance in the latter study. PKC isoform b was reported to be activated under hyperglycaemic conditions in breast cancer cells [61], in C 2 C 12 myotubes and rat skeletal muscle [17,18], in rat heart muscle [30] and in human skeletal muscle cells [62]. Overexpression of PKC-bI and PKC-bII but not PKC-d, -e, -q or -z inhibited the tyrosine kinase activity of the insulin-stimulated insulin receptor [57].…”

Role of the Ca2+/Phosphatidylserine Binding Region of the C2 Domain in the Translocation of Protein Kinase Cα to the Plasma Membrane

“…Thus, the C1 and the C2 domains are involved in membrane translocation and subsequent enzyme activation (4). Many studies performed to date suggest a sequential mechanism in the activation of PKC␣ by Ca 2ϩ in which membrane association is followed by increased catalytic activity (5)(6)(7)(8)(9). However, it is still not clear whether these domains function in a concerted way or as independent modules.…”

Irreversible Inactivation of Protein Kinase C by Glutathione

“…The susceptibility of PKC isozymes to limited trypsinolysis serves as an indicator of conformational changes that involve alterations in the exposure of the hinge region (8,41,42). Fig.…”