The present study characterized the signalling pathways initiated by the bioactive lipid, LPA (lysophosphatidic acid) in smooth muscle. Expression of LPA3 receptors, but not LPA1 and LPA2, receptors was demonstrated by Western blot analysis. LPA stimulated phosphoinositide hydrolysis, PKC (protein kinase C) and Rho kinase (Rho-associated kinase) activities: stimulation of all three enzymes was inhibited by expression of the Gαq, but not the Gαi, minigene. Initial contraction and MLC20 (20 kDa regulatory light chain of myosin II) phosphorylation induced by LPA were abolished by inhibitors of PLC (phospholipase C)-β (U73122) or MLCK (myosin light-chain kinase; ML-9), but were not affected by inhibitors of PKC (bisindolylmaleimide) or Rho kinase (Y27632). In contrast, sustained contraction, and phosphorylation of MLC20 and CPI-17 (PKC-potentiated inhibitor 17 kDa protein) induced by LPA were abolished selectively by bisindolylmaleimide. LPA-induced activation of IKK2 {IκB [inhibitor of NF-κB (nuclear factor κB)] kinase 2} and PKA (protein kinase A; cAMP-dependent protein kinase), and degradation of IκBα were blocked by the RhoA inhibitor (C3 exoenzyme) and in cells expressing dominant-negative mutants of IKK2(K44A) or RhoA(N19RhoA). Phosphorylation by Rho kinase of MYPT1 (myosin phosphatase targeting subunit 1) at Thr696 was masked by phosphorylation of MYPT1 at Ser695 by PKA derived from IκB degradation via RhoA, but unmasked in the presence of PKI (PKA inhibitor) or C3 exoenzyme and in cells expressing IKK2(K44A). We conclude that LPA induces initial contraction which involves activation of PLC-β and MLCK and phosphorylation of MLC20, and sustained contraction which involves activation of PKC and phosphorylation of CPI-17 and MLC20. Although Rho kinase was activated, phosphorylation of MYPT1 at Thr696 by Rho kinase was masked by phosphorylation of MYPT1 at Ser695 via cAMP-independent PKA derived from the NF-κB pathway.
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Research Article|
April 14 2008
Gq-dependent signalling by the lysophosphatidic acid receptor LPA3 in gastric smooth muscle: reciprocal regulation of MYPT1 phosphorylation by Rho kinase and cAMP-independent PKA
Wimolpak Sriwai;
Wimolpak Sriwai
1Departments of Physiology and Medicine, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, VA 23298, U.S.A.
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Huiping Zhou;
Huiping Zhou
1Departments of Physiology and Medicine, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, VA 23298, U.S.A.
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Karnam S. Murthy
Karnam S. Murthy
1
1Departments of Physiology and Medicine, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, VA 23298, U.S.A.
1To whom correspondence should be addressed (email skarnam@hsc.vcu.edu).
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Publisher: Portland Press Ltd
Received:
September 20 2007
Revision Received:
January 29 2008
Accepted:
February 01 2008
Accepted Manuscript online:
February 01 2008
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2008 Biochemical Society
2008
Biochem J (2008) 411 (3): 543–551.
Article history
Received:
September 20 2007
Revision Received:
January 29 2008
Accepted:
February 01 2008
Accepted Manuscript online:
February 01 2008
Citation
Wimolpak Sriwai, Huiping Zhou, Karnam S. Murthy; Gq-dependent signalling by the lysophosphatidic acid receptor LPA3 in gastric smooth muscle: reciprocal regulation of MYPT1 phosphorylation by Rho kinase and cAMP-independent PKA. Biochem J 1 May 2008; 411 (3): 543–551. doi: https://doi.org/10.1042/BJ20071299
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