Rho kinase collaborates with p21-activated kinase to regulate actin polymerization and contraction in airway smooth muscle

Abstract

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.

Keywords: Rho-associated protein kinase; actin polymerization; cytoskeleton; smooth muscle contraction.