Abstract
The inhibition of myosin phosphatase evokes smooth muscle contraction in the absence of Ca(2+), yet the underlying mechanisms are not understood. To this end, we have cloned smooth muscle zipper-interacting protein (ZIP) kinase cDNA. ZIP kinase is present in various smooth muscle tissues including arteries. Triton X-100 skinning did not diminish ZIP kinase content, suggesting that ZIP kinase associates with the filamentous component in smooth muscle. Smooth muscle ZIP kinase phosphorylated smooth muscle myosin as well as the isolated 20-kDa myosin light chain in a Ca(2+)/calmodulin-independent manner. ZIP kinase phosphorylated myosin light chain at both Ser(19) and Thr(18) residues with the same rate constant. The actin-activated ATPase activity of myosin increased significantly following ZIP kinase-induced phosphorylation. Introduction of ZIP kinase into Triton X-100-permeabilized rabbit mesenteric artery provoked a Ca(2+)-free contraction. A protein phosphatase inhibitor, microcystin LR, also induced contraction in the absence of Ca(2+), which was accompanied by an increase in both mono- and diphosphorylation of myosin light chain. The observed sensitivity of the microcystin-induced contraction to various protein kinase inhibitors was identical to the sensitivity of isolated ZIP kinase to these inhibitors. These results suggest that ZIP kinase is responsible for Ca(2+) independent myosin phosphorylation and contraction in smooth muscle.
Highlights
Three subunits, a myosin-binding large subunit (MBS), a 20kDa small subunit, and a catalytic subunit of the type 1 protein serine/threonine phosphatase family [5]
We found that zipper-interacting protein (ZIP) kinase is expressed in smooth muscle tissues and that it can phosphorylate myosin in a Ca2ϩ-independent manner and thereby induce Ca2ϩ-free contraction of permeabilized smooth muscle
3) ZIP kinase is expressed in smooth muscle and is present in Triton X-100-permeabilized strips
Summary
Proteins—Smooth muscle myosin [25] and myosin light chain kinase [26] were prepared from turkey gizzards as described previously. Xenopus oocyte calmodulin [28] and smooth muscle MLC20 were expressed in Escherichia coli and purified as described [29, 30]. Biochemical Procedure—Phosphorylation of smooth muscle myosin or isolated MLC20 was carried out using 1 g/ml GST-ZIPK in the presence of 30 mM Tris-HCl, pH 7.5, 1 mM MgCl2, 100 mM NaCl, 1 mM EGTA, 1 mM DTT, 1 M microcystin LR, 0.1 mM [␥-32P]ATP (3000 Ci/mmol; PerkinElmer Life Sciences) for the indicated period, and the reaction was terminated by 10% trichloroacetic acid. Proteins were extracted in buffer containing 20 mM Tris, 23 mM glycine, 10 mM DTT, 9 M urea, and 0.002% bromphenol blue and subjected to urea/glycerol PAGE, followed by immunoblot using 20-kDa myosin light chain antibody or a phosphorylation site-specific antibody for phosphorylated 20-kDa myosin light chain at Ser19 [38]
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