Phosphorylation of the myosin phosphatase target subunit by integrin-linked kinase

Andrea Murányi, Justin A. MacDonald, Jing Ti Deng, David P. Wilson, Timothy A J Haystead, Michael P. Walsh, F. Erdődi, Enikö Kiss, Yue Wu, David J. Hartshorne

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Abstract

A mechanism proposed for regulation of myosin phosphatase (MP) activity is phosphorylation of the myosin phosphatase target subunit (MYPT1). Integrin-linked kinase (ILK) is associated with the contractile machinery and can phosphorylate myosin at the myosin light-chain kinase sites. The possibility that ILK may also phosphorylate and regulate MP was investigated. ILK was associated with the MP holoenzyme, shown by Western blots and in-gel kinase assays. MYPT1 was phosphorylated by ILK and phosphorylation sites in the N- and C-terminal fragments of MYPT1 were detected. From sequence analyses, three sites were identified: a primary site at Thr709, and two other sites at Thr695 and Thr495. One of the sites for cAMP-dependent protein kinase (PKA) was Ser694. Assays with the catalytic subunit of type 1 phosphatase indicated that only the C-terminal fragment of MYPT1 phosphorylated by zipper-interacting protein kinase, and ILK inhibited activity. The phosphorylated N-terminal fragment activated phosphatase activity and phosphorylation by PKA was without effect. Using full-length MYPT1 constructs phosphorylated by various kinases it was shown that Rho kinase gave marked inhibition; ILK produced an intermediate level of inhibition, which was considerably reduced for the Thr695 → Ala mutant; and PKA had no effect. In summary, phosphorylation of the various sites indicated that Thr695 was the major inhibitory site, Thr709 had only a slight inhibitory effect and Ser694 had no effect. The findings that ILK phosphorylated both MYPT1 and myosin and the association of ILK with MP suggest that ILK may influence cytoskeletal structure or function.

Original languageEnglish
Pages (from-to)211-216
Number of pages6
JournalBiochemical Journal
Volume366
Issue number1
DOIs
Publication statusPublished - Aug 15 2002

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Myosin-Light-Chain Phosphatase
Phosphorylation
Protein Kinases
Myosins
Phosphoric Monoester Hydrolases
Assays
Phosphotransferases
Myosin-Light-Chain Kinase
integrin-linked kinase
rho-Associated Kinases
Holoenzymes
Fasteners
Cyclic AMP-Dependent Protein Kinases
Machinery
Sequence Analysis
Catalytic Domain
Western Blotting
Gels
Association reactions

Keywords

  • Myosin dephosphorylation
  • Regulation of myosin phosphatase
  • Smooth-muscle contraction

ASJC Scopus subject areas

  • Biochemistry

Cite this

Murányi, A., MacDonald, J. A., Deng, J. T., Wilson, D. P., Haystead, T. A. J., Walsh, M. P., ... Hartshorne, D. J. (2002). Phosphorylation of the myosin phosphatase target subunit by integrin-linked kinase. Biochemical Journal, 366(1), 211-216. https://doi.org/10.1042/BJ20020401

Phosphorylation of the myosin phosphatase target subunit by integrin-linked kinase. / Murányi, Andrea; MacDonald, Justin A.; Deng, Jing Ti; Wilson, David P.; Haystead, Timothy A J; Walsh, Michael P.; Erdődi, F.; Kiss, Enikö; Wu, Yue; Hartshorne, David J.

In: Biochemical Journal, Vol. 366, No. 1, 15.08.2002, p. 211-216.

Research output: Contribution to journalArticle

Murányi, A, MacDonald, JA, Deng, JT, Wilson, DP, Haystead, TAJ, Walsh, MP, Erdődi, F, Kiss, E, Wu, Y & Hartshorne, DJ 2002, 'Phosphorylation of the myosin phosphatase target subunit by integrin-linked kinase', Biochemical Journal, vol. 366, no. 1, pp. 211-216. https://doi.org/10.1042/BJ20020401
Murányi A, MacDonald JA, Deng JT, Wilson DP, Haystead TAJ, Walsh MP et al. Phosphorylation of the myosin phosphatase target subunit by integrin-linked kinase. Biochemical Journal. 2002 Aug 15;366(1):211-216. https://doi.org/10.1042/BJ20020401
Murányi, Andrea ; MacDonald, Justin A. ; Deng, Jing Ti ; Wilson, David P. ; Haystead, Timothy A J ; Walsh, Michael P. ; Erdődi, F. ; Kiss, Enikö ; Wu, Yue ; Hartshorne, David J. / Phosphorylation of the myosin phosphatase target subunit by integrin-linked kinase. In: Biochemical Journal. 2002 ; Vol. 366, No. 1. pp. 211-216.
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AU - Haystead, Timothy A J

AU - Walsh, Michael P.

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AB - A mechanism proposed for regulation of myosin phosphatase (MP) activity is phosphorylation of the myosin phosphatase target subunit (MYPT1). Integrin-linked kinase (ILK) is associated with the contractile machinery and can phosphorylate myosin at the myosin light-chain kinase sites. The possibility that ILK may also phosphorylate and regulate MP was investigated. ILK was associated with the MP holoenzyme, shown by Western blots and in-gel kinase assays. MYPT1 was phosphorylated by ILK and phosphorylation sites in the N- and C-terminal fragments of MYPT1 were detected. From sequence analyses, three sites were identified: a primary site at Thr709, and two other sites at Thr695 and Thr495. One of the sites for cAMP-dependent protein kinase (PKA) was Ser694. Assays with the catalytic subunit of type 1 phosphatase indicated that only the C-terminal fragment of MYPT1 phosphorylated by zipper-interacting protein kinase, and ILK inhibited activity. The phosphorylated N-terminal fragment activated phosphatase activity and phosphorylation by PKA was without effect. Using full-length MYPT1 constructs phosphorylated by various kinases it was shown that Rho kinase gave marked inhibition; ILK produced an intermediate level of inhibition, which was considerably reduced for the Thr695 → Ala mutant; and PKA had no effect. In summary, phosphorylation of the various sites indicated that Thr695 was the major inhibitory site, Thr709 had only a slight inhibitory effect and Ser694 had no effect. The findings that ILK phosphorylated both MYPT1 and myosin and the association of ILK with MP suggest that ILK may influence cytoskeletal structure or function.

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