Browsing by Subject "Actin polymerization"

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    A novel role for RhoA GTPase in the regulation of airway smooth muscle contraction
    (Canadian Science Publishing, 2015-02) Zhang, Wenwu; Huang, Youliang; Wu, Yidi; Gunst, Susan J.; Cellular and Integrative Physiology, School of Medicine
    Recent studies have demonstrated a novel molecular mechanism for the regulation of airway smooth muscle (ASM) contraction by RhoA GTPase. In ASM tissues, both myosin light chain (MLC) phosphorylation and actin polymerization are required for active tension generation. RhoA inactivation dramatically suppresses agonist-induced tension development and completely inhibits agonist-induced actin polymerization, but only slightly reduces MLC phosphorylation. The inhibition of MLC phosphatase does not reverse the effects of RhoA inactivation on contraction or actin polymerization. Thus, RhoA regulates ASM contraction through its effects on actin polymerization rather than MLC phosphorylation. Contractile stimulation of ASM induces the recruitment and assembly of paxillin, vinculin, and focal adhesion kinase (FAK) into membrane adhesion complexes (adhesomes) that regulate actin polymerization by catalyzing the activation of cdc42 GTPase by the G-protein-coupled receptor kinase-interacting target (GIT) - p21-activated kinase (PAK) - PAK-interacting exchange factor (PIX) complex. Cdc42 is a necessary and specific activator of the actin filament nucleation activator, N-WASp. The recruitment and activation of paxillin, vinculin, and FAK is prevented by RhoA inactivation, thus preventing cdc42 and N-WASp activation. We conclude that RhoA regulates ASM contraction by catalyzing the assembly and activation of membrane adhesome signaling modules that regulate actin polymerization, and that the RhoA-mediated assembly of adhesome complexes is a fundamental step in the signal transduction process in response to a contractile agonist.
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    Rho kinase collaborates with p21-activated kinase to regulate actin polymerization and contraction in airway smooth muscle
    (Wiley, 2018-08) Zhang, Wenwu; Bhetwal, Bhupal P.; Gunst, Susan J.; Cellular and Integrative Physiology, School of Medicine
    KEY POINTS: The mechanisms by which Rho kinase (ROCK) regulates airway smooth muscle contraction were determined in tracheal smooth muscle tissues. ROCK may mediate smooth muscle contraction by inhibiting myosin regulatory light chain (RLC) phosphatase. ROCK can also regulate F-actin dynamics during cell migration, and actin polymerization is critical for airway smooth muscle contraction. Our results show that ROCK does not regulate airway smooth muscle contraction by inhibiting myosin RLC phosphatase or by stimulating myosin RLC phosphorylation. We find that ROCK regulates airway smooth muscle contraction by activating the serine-threonine kinase Pak, which mediates the activation of Cdc42 and neuronal Wiskott-Aldrich syndrome protein (N-WASp). N-WASP transmits signals from Cdc42 to the Arp2/3 complex for the nucleation of actin filaments. These results demonstrate a novel molecular function for ROCK in the regulation of Pak and Cdc42 activation that is critical for the processes of actin polymerization and contractility in airway smooth muscle. ABSTRACT: Rho kinase (ROCK), a RhoA GTPase effector, can regulate the contraction of airway and other smooth muscle tissues. In some tissues, ROCK can inhibit myosin regulatory light chain (RLC) phosphatase, which increases the phosphorylation of myosin RLC and promotes smooth muscle contraction. ROCK can also regulate cell motility and migration by affecting F-actin dynamics. Actin polymerization is stimulated by contractile agonists in airway smooth muscle tissues and is required for contractile tension development in addition to myosin RLC phosphorylation. We investigated the mechanisms by which ROCK regulates the contractility of tracheal smooth muscle tissues by expressing a kinase-inactive mutant of ROCK, ROCK-K121G, in the tissues or by treating them with the ROCK inhibitor H-1152P. Our results show no role for ROCK in the regulation of non-muscle or smooth muscle myosin RLC phosphorylation during contractile stimulation in this tissue. We found that ROCK regulates airway smooth muscle contraction by mediating activation of p21-activated kinase (Pak), a serine-threonine kinase, to promote actin polymerization. Pak catalyses paxillin phosphorylation on Ser273 and coupling of the GIT1-βPIX-Pak signalling module to paxillin, which activates the guanine nucleotide exchange factor (GEF) activity of βPIX towards Cdc42. Cdc42 is required for the activation of neuronal Wiskott-Aldrich syndrome protein (N-WASp), which transmits signals from Cdc42 to the Arp2/3 complex for the nucleation of actin filaments. Our results demonstrate a novel molecular function for ROCK in the regulation of Pak and Cdc42 activation that is critical for the processes of actin polymerization and contractility in airway smooth muscle.