Regulation of protein kinase C by the cytoskeletal protein calponin

Previous studies from this laboratory have shown that, upon agonist activat ion, calponin co-immunoprecipitates and co-localizes with protein kinase CE (PKC epsilon) in vascular smooth muscle cells. In the present study we dem onstrate 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 Ca region of both PKC epsilon and PKC alpha with possible involvement of C1B. The C2 region of PKC epsilo n binds to the calponin repeats with a requirement for the region between a mino acids 160 and 182. We have also found that calponin can directly activ ate PKC autophosphorylation. By using anti-phospho-antibodies to residue Se r-660 of PKC beta II, we found that calponin, in a lipid-independent manner , increased auto-phosphorylation of PKCa, -epsilon, and -beta II severalfol d compared with control conditions. Similarly, calponin was found to increa se the amount of P-32-labeled phosphate incorporated into PKC from [gamma-P -32]ATP. We also observed that calponin addition strongly increased the inc orporation of radiolabeled phosphate into an exogenous PKC peptide substrat e, suggesting an activation of enzyme activity. Thus, these results raise t he possibility that calponin may function in smooth muscle to regulate PKC activity by facilitating the phosphorylation of PRC.