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 microM), whereas ILK was insensitive to AV25 (at concentrations as high as 100 microM). 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.
Wilson
David P
DP
Smooth Muscle Research Group and Department of Biochemistry and Molecular Biology, University of Calgary Faculty of Medicine, 3330 Hospital Drive N.W., Calgary, Alberta, Canada T2N 4N1.
Sutherland
Cindy
C
Borman
Meredith A
MA
Deng
Jing Ti
JT
Macdonald
Justin A
JA
Walsh
Michael P
MP
D000818:Animals; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D020930:MAP Kinase Kinase Kinases; D052998:Microcystins; D009119:Muscle Contraction / Q000187:drug effects / Q000502:physiology; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism / Q000502:physiology*; D009218:Myosins / Q000378:metabolism*; D010456:Peptides, Cyclic / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D051381:Rats; D000099200:Scaffold Protein ILK
D002118:Calcium; D020930:MAP Kinase Kinase Kinases; D052998:Microcystins; D009218:Myosins; D010456:Peptides, Cyclic; D017346:Protein Serine-Threonine Kinases; D000099200:Scaffold Protein ILK; C078588:microcystin; C090757:mitogen-activated protein kinase kinase kinase 12