Regulation of PKC Autophosphorylation by Calponin in Contractile Vascular Smooth Muscle Tissue

Abstract

Protein kinase C (PKC) is a key enzyme involved in agonist-induced smooth muscle contraction. In some cases, regulatory phosphorylation of PKC is required for full activation of the enzyme. However, this issue has largely been ignored with respect to PKC-dependent regulation of contractile vascular smooth muscle (VSM) contractility. The first event in PKC regulation is a transphosphorylation by PDK at a conserved threonine in the activation loop of PKC, followed by the subsequent autophosphorylation at the turn motif and hydrophobic motif sites. In the present study, we determined whether phosphorylation of PKC is a regulated process in VSM and also investigated a potential role of calponin in the regulation of PKC. We found that calponin increases the level of in vitro PKCα phosphorylation at the PDK and hydrophobic sites, but not the turn motif site. In vascular tissues, phosphorylation of the PKC hydrophobic site, but not turn motif site, as well as phosphorylation of PDK at S241 increased in response to phenylephrine. Calponin knockdown inhibits autophosphorylation of cellular PKC in response to phenylephrine, confirming results with recombinant PKC. Thus these results show that autophosphorylation of PKC is regulated in dVSM and calponin is necessary for autophosphorylation of PKC in VSM.