Regulation of protein kinase C by the cytoskeletal protein calponin

Abstract

Previous studies from this laboratory have shown that, upon agonist activation, calponin co-immunoprecipitates and co-localizes with protein kinase Cε (PKCε) in vascular smooth muscle cells. In the present study we demonstrate that calponin binds directly to the regulatory domain of PKC both in overlay assays and, under native conditions, by sedimentation with lipid vesicles. Calponin was found to bind to the C2 region of both PKCε and PKCα with possible involvement of C1B. The C2 region of PKCε binds to the calponin repeats with a requirement for the region between amino acids 160 and 182. We have also found that calponin can directly activate PKC autophosphorylation. By using anti-phospho-antibodies to residue Ser-660 of PKCβII, we found that calponin, in a lipid-independent manner, increased auto-phosphorylation of PKCα, -ε, and -βII severalfold compared with control conditions. Similarly, calponin was found to increase the amount of 32P-labeled phosphate incorporated into PKC from [γ-32P]ATP. We also observed that calponin addition strongly increased the incorporation of radiolabeled phosphate into an exogenous PKC peptide substrate, suggesting an activation of enzyme activity. Thus, these results raise the possibility that calponin may function in smooth muscle to regulate PKC activity by facilitating the phosphorylation of PKC.