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

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.