PROTEIN-KINASE C-CATALYZED CALPONIN PHOSPHORYLATION IN SWINE CAROTID ARTERIAL HOMOGENATE

Calponin, a thin filament-associated protein, inhibits actin-activated myosin ATPase activity, and this inhibition is reversed by phosphoryl ation. Calponin phosphorylation by protein kinase C and Ca2+/calmoduli n-dependent protein kinase II has been shown in purified protein syste ms but has been difficult to demonstrate in more physiological prepara tions. We have previously shown that calponin is phosphorylated in a c ell-free homogenate of swine carotid artery. The goal of this study wa s to determine whether protein kinase C and/or Ca2+/calmodulin-depende nt protein kinase Ii catalyzes calponin phosphorylation. Ca2+-dependen t calponin phosphorylation was not inhibited by calmodulin antagonists . In contrast, both Ca2+- and phorbol dibutyrate/1-oleoyl-2-acetyl-sn- glycerol-dependent calponin phosphorylation were inhibited by the pseu dosubstrate inhibitor of protein kinase C and staurosporine. Our resul ts also demonstrate that stimulation with either Ca2+, phorbol dibutyr ate, or 1-oleoyl-2-acelyl-sn-glycerol activates endogenous protein kin ase C. We interpret our results as clearly demonstrating that the phys iological kinase for calponin phosphorylation is protein kinase C and not Ca2+/calmodulin-dependent protein kinase II. We also present data showing that the direct measurement of P-32 incorporation into calponi n and the indirect measurement of calponin phosphorylation using noneq uilibrium ph gradient gel electrophoresis provide similar quantitative values of calponin phosphorylation. J. Cell. Physiol. 176:545-552, 19 98. (C) 1998 Wiley-Liss, Inc.