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https://hdl.handle.net/2440/34775
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Type: | Journal article |
Title: | Integrin-linked kinase is responsible for Ca2+-independent myosin diphosphorylation and contraction of vascular smooth muscle |
Author: | Wilson, D. Sutherland, C. Borman, M. Deng, J. MacDonald, J. Walsh, M. |
Citation: | Biochemical Journal, 2005; 392(3):641-648 |
Publisher: | Portland Press |
Issue Date: | 2005 |
ISSN: | 0264-6021 1470-8728 |
Statement of Responsibility: | David P. Wilson, Cindy Sutherland, Meredith A. Borman, Jing Ti Deng, Justin A. MacDonald and Michael P. Walsh |
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 mM), whereas ILK was insensitive to AV25 (at concentrations as high as 100 mM). 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. |
Keywords: | Integrin-linked kinase microcystin myosin light-chain phosphatase myosin phosphorylation vascular smooth muscle zipper-interacting protein kinase |
DOI: | 10.1042/BJ20051173 |
Published version: | http://dx.doi.org/10.1042/bj20051173 |
Appears in Collections: | Aurora harvest 6 Molecular and Biomedical Science publications |
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