Substrate-dependent activation requirements and kinetic properties of protein kinase C

Protein kinase C (PKC) requires basic amino acids around the phosphorylated Ser or Thr, Previous studies of the effector requirements of PKCs alpha, beta and gamma with two commonly used substrates, MBP3-14 (AQKRP (S) under bar QRSKYL) and peptide epsilon (ERMRPRKRQG (S) under bar VRRRV), revealed that MBP3-14 phosphorylation required Ca2+, phosphatidylserine and diacylglycerol, while peptide epsilon supported high levels of phosphatidylserine-dependent activity in the absence of Ca2+ or diacylglycerol. Since the Arg versus Lys content is much larger in peptide epsilon than in MBP3-14, we examined the role of these amino acids in conferring substrate-dependent effector requirements for PKC activation. We substituted Lys for Arg in peptide epsilon (peptide epsilon[R --> K]) and Arg for Lys in MBP3-14 (MBP3-14[K --> R]) and analyzed the effector requirements and kinetic properties of PKCs alpha, beta and gamma with the parent and modified peptides, In general, significant Ca2+ and diacylglycerol dependence was observed with peptide epsilon[R --> K] as compared to peptide epsilon. On the other hand, the effector requirements with MBP3-14 [K --> R] were the same as with MBP3-14, presumably due to a subthreshold Arg content. Both K-m and V-max determined in the presence of Ca2+, phosphatidylserine and diacylglycerol were increased by the peptide epsilon modification for all three isoenzymes, while the only effect of MBP3-14 modification was a decrease in K(m )for PKC beta, K-m and V-max values for peptide epsilon and peptide epsilon[R --> K] phosphorylation by PKC alpha were also determined in the absence of Ca2+ or diacylglycerol, While diacylglycerol had no effect, Ca2+ decreased the K-m for both substrates to a similar extent, Overall, the degree of effector dependence did not correlate with absolute K-m values. The mechanism of PKC activation by Arg-rich substrates, therefore, does not involve their ability to bind to the active site. (C) 1998 Federation of European Biochemical Societies.