THE UNIMPORTANCE OF BEING (PROTEIN-KINASE-C) EPSILON

The purpose of our study was to determine the mechanism through which phorbol esters and smooth muscle myosin phosphatase inhibitors can ind uce contraction of smooth muscle in the absence of Ca2+. Protein kinas e C-epsilon (PKC-epsilon) was previously implicated in this process ba sed largely on its supposed absence in the ferret portal vein, and a c orrelation was drawn between the presence of this isoform and the abil ity of smooth muscle to contract independently of Ca2+ and phosphoryla tion of the 20 kDa regulatory light chains of myosin (MLC20). We demon strate here, with two antibodies, one to the NH2 terminus and the othe r to the COOH terminus of PKC-epsilon, that Eis present in both ferret portal vein and rabbit portal vein smooth muscle, neither of which ex hibits phorbol ester-induced contraction in the absence of Ca2+, Howev er, in the presence of clamped submaximal Ca2+, phorbol ester increase d MLC20, phosphorylation from 17.7 +/- 1.7% to 46.4 +/- 3.6% in ferret portal vein smooth muscle and evoked an increase in force, Prolonged (48 h) incubation of ferret portal vein with phorbol esters completely down-regulated PKC-epsilon, as shown by Western blots, and abolished the phorbol ester-evoked contraction at submaximal Ca2+, but not Ca2+- independent, contractions induced by the phosphatase inhibitor microcy stin, Contractions induced by microcystin in Ca2+-free solution were a ssociated with increased phosphorylation of myosin light chain kinase (MLCK), Activation of MLCK by autophosphorylation in the absence of Ca 2+ occurs in vitro (1). We conclude that PKC-epsilon is neither necess ary nor sufficient for Ca2+-independent regulation of myosin II in smo oth muscle, but contractions induced by agents: that inhibit smooth mu scle myosin phosphatase in the absence of Ca2+ may be mediated by MLCK autophosphorylated or activated by another Ca2+-independent kinase.-W alker, L. A., Gailly, P., Jensen, P. E., Somlyo, A. V., Somlyo, A. P. The unimportance of being (protein kinase C) epsilon.