The unimportance of being (protein kinase C) epsilon¹

^a Department of Molecular Physiology and Biological Physics, University of Virginia, Health Sciences Center, Charlottesville, Virginia 22906-0011, USA
^b Department of Medicine, University of Virginia, Health Sciences Center, Charlottesville, Virginia 22906-0011, USA
^c Department of Pathology, University of Virginia, Health Sciences Center, Charlottesville, Virginia 22906-0011, USA
^d Department of Physiology, Catholic University of Louvain, Brussels, Belgium
^e School of Nursing, Aarhus, Denmark

The purpose of our study was to determine the mechanism through which phorbol esters and smooth muscle myosin phosphatase inhibitors can induce contraction of smooth muscle in the absence of Ca²⁺. Protein kinase C- (PKC-) was previously implicated in this process based largely on its supposed absence in the ferret portal vein, and a correlation was drawn between the presence of this isoform and the ability of smooth muscle to contract independently of Ca²⁺ and phosphorylation of the 20 kDa regulatory light chains of myosin (MLC₂₀). We demonstrate here, with two antibodies, one to the NH₂ terminus and the other to the COOH terminus of PKC-, that is present in both ferret portal vein and rabbit portal vein smooth muscle, neither of which exhibits phorbol ester-induced contraction in the absence of Ca²⁺. However, in the presence of clamped submaximal Ca²⁺, phorbol ester increased 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-, 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 microcystin. Contractions induced by microcystin in Ca²⁺-free solution were associated with increased phosphorylation of myosin light chain kinase (MLCK). Activation of MLCK by autophosphorylation in the absence of Ca²⁺ occurs in vitro (1). We conclude that PKC- is neither necessary nor sufficient for Ca²⁺-independent regulation of myosin II in smooth muscle, but contractions induced by agents that inhibit smooth muscle myosin phosphatase in the absence of Ca²⁺ may be mediated by MLCK autophosphorylated or activated by another Ca²⁺-independent kinase.—Walker, L. A., Gailly, P., Jensen, P. E., Somlyo, A. V., Somlyo, A. P. The unimportance of being (protein kinase C) epsilon. FASEB J. 12, 813–821 (1998)

Key Words: calcium sensitization • PKC • myosin light chain kinase • phosphorylation

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