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Substrate‐dependent activation requirements and kinetic properties of protein kinase C
Author(s) -
Andrea Jacquelyn E,
Sutherland Cindy,
Winter Christopher K,
Walsh Michael P
Publication year - 1998
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(98)00567-5
Subject(s) - diacylglycerol kinase , peptide , protein kinase c , phosphatidylserine , effector , biochemistry , phosphorylation , chemistry , phospholipid , membrane
Protein kinase C (PKC) requires basic amino acids around the phosphorylated Ser or Thr. Previous studies of the effector requirements of PKCs α, β and γ with two commonly used substrates, MBP 3‐14 (AQKRP QRSKYL) and peptide ϵ (ERMRPRKRQG VRRRV), revealed that MBP 3‐14 phosphorylation required Ca 2+ , phosphatidylserine and diacylglycerol, while peptide ϵ supported high levels of phosphatidylserine‐dependent activity in the absence of Ca 2+ or diacylglycerol. Since the Arg versus Lys content is much larger in peptide ϵ than in MBP 3‐14 , we examined the role of these amino acids in conferring substrate‐dependent effector requirements for PKC activation. We substituted Lys for Arg in peptide ϵ (peptide ϵ[R→K]) and Arg for Lys in MBP 3‐14 (MBP 3‐14 [K→R]) and analyzed the effector requirements and kinetic properties of PKCs α, β and γ with the parent and modified peptides. In general, significant Ca 2+ and diacylglycerol dependence was observed with peptide ϵ[R→K] as compared to peptide ϵ. On the other hand, the effector requirements with MBP 3‐14 [K→R] were the same as with MBP 3‐14 , presumably due to a subthreshold Arg content. Both K m and V max determined in the presence of Ca 2+ , phosphatidylserine and diacylglycerol were increased by the peptide ϵ modification for all three isoenzymes, while the only effect of MBP 3‐14 modification was a decrease in K m for PKCβ. K m and V max values for peptide ϵ and peptide ϵ[R→K] phosphorylation by PKCα were also determined in the absence of Ca 2+ or diacylglycerol. While diacylglycerol had no effect, Ca 2+ 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.