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      p21‐Activated kinase (Pak) regulates airway smooth muscle contraction by regulating paxillin complexes that mediate actin polymerization

      1 , 1 , , 1

      The Journal of Physiology

      John Wiley and Sons Inc.

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          Abstract

          Key points

          • In airway smooth muscle, tension development caused by a contractile stimulus requires phosphorylation of the 20 kDa myosin light chain (MLC), which activates crossbridge cycling and the polymerization of a pool of submembraneous actin.

          • The p21‐activated kinases (Paks) can regulate the contractility of smooth muscle and non‐muscle cells, and there is evidence that this occurs through the regulation of MLC phosphorylation.

          • We show that Pak has no effect on MLC phosphorylation during the contraction of airway smooth muscle, and that it regulates contraction by mediating actin polymerization.

          • We find that Pak phosphorylates the adhesion junction protein, paxillin, on Ser273, which promotes the formation of a signalling complex that activates the small GTPase, cdc42, and the actin polymerization catalyst, neuronal Wiskott–Aldrich syndrome protein (N‐WASP).

          • These studies demonstrate a novel role for Pak in regulating the contractility of smooth muscle by regulating actin polymerization.

          Abstract

          The p21‐activated kinases (Pak) can regulate contractility in smooth muscle and other cell and tissue types, but the mechanisms by which Paks regulate cell contractility are unclear. In airway smooth muscle, stimulus‐induced contraction requires phosphorylation of the 20 kDa light chain of myosin, which activates crossbridge cycling, as well as the polymerization of a small pool of actin. The role of Pak in airway smooth muscle contraction was evaluated by inhibiting acetylcholine (ACh)‐induced Pak activation through the expression of a kinase inactive mutant, Pak1 K299R, or by treating tissues with the Pak inhibitor, IPA3. Pak inhibition suppressed actin polymerization and contraction in response to ACh, but it did not affect myosin light chain phosphorylation. Pak activation induced paxillin phosphorylation on Ser273; the paxillin mutant, paxillin S273A, inhibited paxillin Ser273 phosphorylation and inhibited actin polymerization and contraction. Immunoprecipitation analysis of tissue extracts and proximity ligation assays in dissociated cells showed that Pak activation and paxillin Ser273 phosphorylation triggered the formation of an adhesion junction signalling complex with paxillin that included G‐protein‐coupled receptor kinase‐interacting protein (GIT1) and the cdc42 guanine exchange factor, βPIX (Pak interactive exchange factor). Assembly of the Pak–GIT1–βPIX–paxillin complex was necessary for cdc42 and neuronal Wiskott–Aldrich syndrome protein (N‐WASP) activation, actin polymerization and contraction in response to ACh. RhoA activation was also required for the recruitment of Pak to adhesion junctions, Pak activation, paxillin Ser273 phosphorylation and paxillin complex assembly. These studies demonstrate a novel role for Pak in the regulation of N‐WASP activation, actin dynamics and cell contractility.

          Key points

          • In airway smooth muscle, tension development caused by a contractile stimulus requires phosphorylation of the 20 kDa myosin light chain (MLC), which activates crossbridge cycling and the polymerization of a pool of submembraneous actin.

          • The p21‐activated kinases (Paks) can regulate the contractility of smooth muscle and non‐muscle cells, and there is evidence that this occurs through the regulation of MLC phosphorylation.

          • We show that Pak has no effect on MLC phosphorylation during the contraction of airway smooth muscle, and that it regulates contraction by mediating actin polymerization.

          • We find that Pak phosphorylates the adhesion junction protein, paxillin, on Ser273, which promotes the formation of a signalling complex that activates the small GTPase, cdc42, and the actin polymerization catalyst, neuronal Wiskott–Aldrich syndrome protein (N‐WASP).

          • These studies demonstrate a novel role for Pak in regulating the contractility of smooth muscle by regulating actin polymerization.

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          Author and article information

          Journal
          J Physiol
          J. Physiol. (Lond.)
          10.1111/(ISSN)1469-7793
          TJP
          jphysiol
          The Journal of Physiology
          John Wiley and Sons Inc. (Hoboken )
          0022-3751
          1469-7793
          29 May 2016
          01 September 2016
          : 594
          : 17 ( doiID: 10.1113/tjp.2016.594.issue-17 )
          : 4879-4900
          Affiliations
          [ 1 ] Department of Cellular and Integrative Physiology Indiana University School Medicine Indianapolis IN 46202‐5120 USA
          Author notes
          [* ] Corresponding author S. J. Gunst: Department of Cellular and Integrative Physiology, Indiana University School of Medicine, 635 Barnhill Dr., Indianapolis, IN 46202‐5120, USA. Email: sgunst@ 123456iupui.edu
          Article
          PMC5009781 PMC5009781 5009781 TJP7236
          10.1113/JP272132
          5009781
          27038336
          © 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society
          Page count
          Pages: 22
          Categories
          Cellular and Molecular Physiology
          Regulatory Pathways
          Smooth Muscle
          Research Paper
          Molecular and cellular
          Editor's Choice
          Custom metadata
          2.0
          tjp7236
          1 September 2016
          Converter:WILEY_ML3GV2_TO_NLMPMC version:4.9.4 mode:remove_FC converted:01.09.2016

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