Integrin-linked kinase (ILK) has been implicated in Ca2+- independent contraction of smooth muscle via its ability to phosphorylate myosin. We investigated the possibility that this kinase might also phosphorylate and regulate the myosin light-chain phosphatase inhibitor proteins CPI-17 [protein kinase C (PKC)-dependent phosphatase inhibitor of 17kDa] and PHI-1 (phosphatase holoenzyme inhibitor-1), known substrates of PKC. Both phosphatase inhibitors were phosphorylated by ILK in an in-gel kinase assay and in solution. A Thr→Ala mutation at Thr38 of CPI-17 and Thr57 of PHI-1 eliminated phosphorylation by ILK. Phosphopeptide mapping, phospho amino acid analysis and immunoblotting using phospho-specific antibodies indicated that ILK predominantly phosphorylated the site critical for potent inhibition, i.e. Thr38 of CPI-17 or Thr57 of PHI-1. CPI-17 and PHI-1 thiophosphorylated by ILK at Thr38 or Thr57 respectively inhibited myosin light-chain phosphatase (MLCP) activity bound to myosin, whereas the site-specific mutants CPI-17-Thr38Ala and PHI-1-Thr57Ala, treated with ILK under identical conditions, like the untreated wild-type proteins had no effect on the phosphatase. Consistent with these effects, both thiophospho-CPI-17 and -PHI-1 induced Ca2+ sensitization of contraction of Triton X-100-demembranated rat-tail arterial smooth muscle, whereas CPI-17-Thr38Ala and PHI-1-Thr57Ala treated with ILK in the presence of adenosine 5′-[γ-thio]triphosphate failed to evoke a contractile response. We conclude that ILK may activate smooth-muscle contraction both directly, via phosphorylation of myosin, and indirectly, via phosphorylation and activation of CPI-17 and PHI-1, leading to inhibition of MLCP.
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October 2002
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Research Article|
October 15 2002
Phosphorylation of the myosin phosphatase inhibitors, CPI-17 and PHI-1, by integrin-linked kinase
Jing Ti DENG;
Jing Ti DENG
∗Smooth Muscle Research Group and Canadian Institutes of Health Research Group in Regulation of Vascular Contractility, University of Calgary Faculty of Medicine, 3330 Hospital Drive N.W., Calgary, Alberta T2N 4N1, Canada,
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Cindy SUTHERLAND;
Cindy SUTHERLAND
∗Smooth Muscle Research Group and Canadian Institutes of Health Research Group in Regulation of Vascular Contractility, University of Calgary Faculty of Medicine, 3330 Hospital Drive N.W., Calgary, Alberta T2N 4N1, Canada,
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David L. BRAUTIGAN;
David L. BRAUTIGAN
†Center for Cell Signaling, University of Virginia School of Medicine, Box 800577-MSB 7225, Charlottesville, VA 22908, U.S.A.
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Masumi ETO;
Masumi ETO
†Center for Cell Signaling, University of Virginia School of Medicine, Box 800577-MSB 7225, Charlottesville, VA 22908, U.S.A.
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Michael P. WALSH
Michael P. WALSH
1
∗Smooth Muscle Research Group and Canadian Institutes of Health Research Group in Regulation of Vascular Contractility, University of Calgary Faculty of Medicine, 3330 Hospital Drive N.W., Calgary, Alberta T2N 4N1, Canada,
1To whom correspondence should be addressed (e-mail walsh@ucalgary.ca).
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Publisher: Portland Press Ltd
Received:
April 03 2002
Revision Received:
June 24 2002
Accepted:
July 29 2002
Accepted Manuscript online:
July 29 2002
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2002
2002
Biochem J (2002) 367 (2): 517–524.
Article history
Received:
April 03 2002
Revision Received:
June 24 2002
Accepted:
July 29 2002
Accepted Manuscript online:
July 29 2002
Citation
Jing Ti DENG, Cindy SUTHERLAND, David L. BRAUTIGAN, Masumi ETO, Michael P. WALSH; Phosphorylation of the myosin phosphatase inhibitors, CPI-17 and PHI-1, by integrin-linked kinase. Biochem J 15 October 2002; 367 (2): 517–524. doi: https://doi.org/10.1042/bj20020522
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