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

Hak Rim Kim,Cynthia Gallant,Kathleen G Morgan

doi:10.1155/2013/358643

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.

Highlights

In recent years, it has become clear that multiple, redundant signaling pathways are responsible for the fine-tuning of smooth muscle contractility [1]
In the last decade, accumulating evidence from in vitro protein chemistry and cellular studies of nonmuscle cell types has indicated that regulatory phosphorylation of protein kinase C (PKC) itself is required for full activation of PKC [4], but, this issue has largely been ignored with respect to PKC regulation in contractile dVSM
The main findings of the present study are that autophosphorylation of PKCα and PKCε is regulated by an α-agonist in dVSM and that the smooth muscle differentiation marker, CaP, is necessary for this autophosphorylation

Summary

Introduction

It has become clear that multiple, redundant signaling pathways are responsible for the fine-tuning of smooth muscle contractility [1]. This complexity allows close control of important physiologic processes such as blood pressure and blood flow; it raises the question as to how the cell regulates the spatially precise activity of overlapping pathways [2, 3]. The conventional PKCα and the novel PKCε are the best studied isoforms in the contractile, fully differentiated vascular smooth muscle cell (dVSMC) [7,8,9,10]. In the last decade, accumulating evidence from in vitro protein chemistry and cellular studies of nonmuscle cell types has indicated that regulatory phosphorylation of PKC itself is required for full activation of PKC [4], but, this issue has largely been ignored with respect to PKC regulation in contractile dVSM

Methods

Results

Conclusion