Ca2+-independent Smooth Muscle Contraction: A NOVEL FUNCTION FOR INTEGRIN-LINKED KINASE

Jing Ti Deng,Jacquelyn E Van Lierop,Cindy Sutherland,Michael P Walsh

doi:10.1074/jbc.m011634200

Abstract

Smooth muscle contraction follows an increase in cytosolic Ca(2+) concentration, activation of myosin light chain kinase, and phosphorylation of the 20-kDa light chain of myosin at Ser(19). Several agonists acting via G protein-coupled receptors elicit a contraction without a change in [Ca(2+)](i) via inhibition of myosin light chain phosphatase and increased myosin phosphorylation. We showed that microcystin (phosphatase inhibitor)-induced contraction of skinned smooth muscle occurred in the absence of Ca(2+) and correlated with phosphorylation of myosin light chain at Ser(19) and Thr(18) by a kinase distinct from myosin light chain kinase. In this study, we identify this kinase as integrin-linked kinase. Chicken gizzard integrin-linked kinase cDNA was cloned, sequenced, expressed in E. coli, and shown to phosphorylate myosin light chain in the absence of Ca(2+) at Ser(19) and Thr(18). Subcellular fractionation revealed two distinct populations of integrin-linked kinase, including a Triton X-100-insoluble component that phosphorylates myosin in a Ca(2+)-independent manner. These results suggest a novel function for integrin-linked kinase in the regulation of smooth muscle contraction via Ca(2+)-independent phosphorylation of myosin, raise the possibility that integrin-linked kinase may also play a role in regulation of nonmuscle motility, and confirm that integrin-linked kinase is indeed a functional protein-serine/threonine kinase.

Highlights

integrin-linked kinase (ILK) is widespread in terms of its tissue distribution, being expressed in most mammalian cells, with highest expression levels in cardiac and skeletal muscle, and its sequence is highly conserved across species
We showed that microcystin-induced contraction of skinned smooth muscle occurred in the absence of Ca2؉ and correlated with phosphorylation of myosin light chain at Ser19 and Thr18 by a kinase distinct from myosin light chain kinase
Subcellular fractionation revealed two distinct populations of integrin-linked kinase, including a Triton X-100-insoluble component that phosphorylates myosin in a Ca2؉-independent manner. These results suggest a novel function for integrinlinked kinase in the regulation of smooth muscle contraction via Ca2؉-independent phosphorylation of myosin, raise the possibility that integrin-linked kinase may play a role in regulation of nonmuscle motility, and confirm that integrin-linked kinase is a functional protein-serine/threonine kinase

Summary

Introduction

ILK is widespread in terms of its tissue distribution, being expressed in most mammalian cells, with highest expression levels in cardiac and skeletal muscle, and its sequence is highly conserved across species. Regulation of nonmuscle motility involves myosin phosphorylation [9], similar to the regulation of smooth muscle contraction. The possibility arises, that ILK may play a role in regulation of nonmuscle motile processes such as cell migration, chemotaxis, cytokinesis, and fibroblast contraction mediating orientation of collagen fibers in connective tissue. Kolodney et al [46] recently demonstrated that contraction of chick embryo fibroblasts in response to fetal bovine serum stimulation involves the Ca2ϩindependent phosphorylation of myosin II. We observed no effect of phosphatidylinositol 3,4,5-trisphosphate on the phosphorylation of myosin by partially purified smooth muscle ILK (results not shown)

Objectives

Results

Conclusion