Affiliation:
1. Smooth Muscle Research Group and Department of Biochemistry and Molecular Biology, University of Calgary Faculty of Medicine, 3330 Hospital Drive N.W., Calgary, Alberta, Canada T2N 4N1
Abstract
Smooth muscle contraction is activated by phosphorylation at Ser-19 of LC20 (the 20 kDa light chains of myosin II) by Ca2+/calmodulin-dependent MLCK (myosin light-chain kinase). Diphosphorylation of LC20 at Ser-19 and Thr-18 is observed in smooth muscle tissues and cultured cells in response to various contractile stimuli, and in pathological circumstances associated with hypercontractility. MLCP (myosin light-chain phosphatase) inhibition can lead to LC20 diphosphorylation and Ca2+-independent contraction, which is not attributable to MLCK. Two kinases have emerged as candidates for Ca2+-independent LC20 diphosphorylation: ILK (integrin-linked kinase) and ZIPK (zipper-interacting protein kinase). Triton X-100-skinned rat caudal arterial smooth muscle was used to investigate the relative importance of ILK and ZIPK in Ca2+-independent, microcystin (phosphatase inhibitor)-induced LC20 diphosphorylation and contraction. Western blotting and in-gel kinase assays revealed that both kinases were retained in this preparation. Ca2+-independent contraction of calmodulin-depleted tissue in response to microcystin was resistant to MLCK inhibitors [AV25 (a 25-amino-acid peptide derived from the autoinhibitory domain of MLCK), ML-7, ML-9 and wortmannin], protein kinase C inhibitor (GF109203X) and Rho-associated kinase inhibitors (Y-27632 and H-1152), but blocked by the non-selective kinase inhibitor staurosporine. ZIPK was inhibited by AV25 (IC50 0.63±0.05 μM), whereas ILK was insensitive to AV25 (at concentrations as high as 100 μM). AV25 had no effect on Ca2+-independent, microcystin-induced LC20 mono- or di-phosphorylation, with a modest effect on force. We conclude that direct inhibition of MLCP in the absence of Ca2+ unmasks ILK activity, which phosphorylates LC20 at Ser-19 and Thr-18 to induce contraction. ILK is probably the kinase responsible for myosin diphosphorylation in vascular smooth muscle cells and tissues.
Subject
Cell Biology,Molecular Biology,Biochemistry
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