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

Wenwu Zhang, Youliang Huang, Susan Gunst

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

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. 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.

Original languageEnglish (US)
Pages (from-to)4879-4900
Number of pages22
JournalJournal of Physiology
Volume594
Issue number17
DOIs
StatePublished - Sep 1 2016

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p21-Activated Kinases
Paxillin
Muscle Contraction
Polymerization
Smooth Muscle
Actins
Phosphorylation
Myosin Light Chains
Acetylcholine
Proteins
G-Protein-Coupled Receptor Kinases
Receptor-Interacting Protein Serine-Threonine Kinases
Muscle Tonus
Tissue Extracts
Monomeric GTP-Binding Proteins
Muscle Development

ASJC Scopus subject areas

  • Physiology

Cite this

p21-Activated kinase (Pak) regulates airway smooth muscle contraction by regulating paxillin complexes that mediate actin polymerization. / Zhang, Wenwu; Huang, Youliang; Gunst, Susan.

In: Journal of Physiology, Vol. 594, No. 17, 01.09.2016, p. 4879-4900.

Research output: Contribution to journalArticle

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