US EPA

535467 

Journal Article 

Integrin-linked kinase is responsible for Ca2+-independent myosin diphosphorylation and contraction of vascular smooth muscle 

Wilson, DP; Sutherland, C; Borman, MA; Deng, JT; Macdonald, JA; Walsh, MP 

2005 

Yes 

Biochemical Journal
ISSN: 0264-6021
EISSN: 1470-8728 

392 

Pt 3 

641-648 

English 

16201970 

10.1042/bj20051173 

WOS:000234354700027 

Smooth muscle contraction is activated by phosphorylation at Set-19 of LC20 (the 20 kDa light chains of myosin 11) 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 (zipperinteracting 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 mu M), whereas ILK was insensitive to AV25 (at concentrations as high as 100 mu M). 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. 

Integrin-linked kinase; Microcystin; Myosin light-chain phosphatase; Myosin phosphorylation; Vascular smooth muscle; Zipper-interacting protein kinase; Calcium; Gels; Muscle; Pathology; Probability; Tissue; Integrin-linked kinase; Microcystin; Myosin light-chain phosphatase; Myosin phosphorylation; Vascular smooth muscle; Zipper-interacting protein kinase; Enzymes; 1 (5 chloro 1 naphthalenesulfonyl)hexahydro 1h 1,4 diazepine; 2 [1 (3 dimethylaminopropyl) 3 indolyl] 3 (3 indolyl)maleimide; 4 (1 aminoethyl) n (4 pyridyl)cyclohexanecarboxamide; calcium ion; calmodulin; cyanoginosin; integrin; integrin linked kinase; myosin; myosin light chain phosphatase; phosphatase; phosphotransferase; protein kinase; Rho kinase inhibitor; serine; staurosporine; threonine; triton x 100; wortmannin; article; enzyme assay; human; human cell; LC 50; muscle contraction; priority journal; protein phosphorylation; vascular smooth muscle; Western blotting; Animals; Calcium; Microcystins; Muscle Contraction; Muscle, Smooth, Vascular; Myosins; Peptides, Cyclic; Phosphorylation; Protein-Serine-Threonine Kinases; Rats