Abstract
The activation of the small GTPase RhoA is necessary for ACh-induced actin polymerization and airway smooth muscle (ASM) contraction, but the mechanism by which it regulates these events is unknown. Actin polymerization in ASM is catalyzed by the actin filament nucleation activator, N-WASp and the polymerization catalyst, Arp2/3 complex. Activation of the small GTPase cdc42, a specific N-WASp activator, is also required for actin polymerization and tension generation. We assessed the mechanism by which RhoA regulates actin dynamics and smooth muscle contraction by expressing the dominant negative mutants RhoA T19N and cdc42 T17N, and non-phosphorylatable paxillin Y118/31F and paxillin ΔLD4 deletion mutants in SM tissues. Their effects were evaluated in muscle tissue extracts and freshly dissociated SM cells. Protein interactions and cellular localization were analyzed using proximity ligation assays (PLA), immunofluorescence, and GTPase and kinase assays. RhoA inhibition prevented ACh-induced cdc42 activation, N-WASp activation and the interaction of N-WASp with the Arp2/3 complex at the cell membrane. ACh induced paxillin phosphorylation and its association with the cdc42 GEFS, DOCK180 and α/βPIX. Paxillin tyrosine phosphorylation and its association with βPIX were RhoA-dependent, and were required for cdc42 activation. The ACh-induced recruitment of paxillin and FAK to the cell membrane was dependent on RhoA. We conclude that RhoA regulates the contraction of ASM by catalyzing the assembly and activation of cytoskeletal signaling modules at membrane adhesomes that initiate signaling cascades that regulate actin polymerization and tension development in response to contractile agonist stimulation. Our results suggest that the RhoA-mediated assembly of adhesome complexes is a fundamental step in the signal transduction process in response to agonist -induced smooth muscle contraction.
Highlights
RhoA GTPase regulates airway smooth muscle (SM) contraction and actin polymerization by an unknown mechanism
RhoA Inactivation Inhibits ACh-induced N-WASp Activation in Smooth Muscle Tissues—We expressed RhoA T19N protein in tracheal muscle tissues to determine whether RhoA activation is necessary for the ACh-induced activation of N-WASp, 33998 JOURNAL OF BIOLOGICAL CHEMISTRY
Paxillin Tyrosine Phosphorylation Is Required for cdc42 and N-WASP Activation—We evaluated the role of paxillin phosphorylation in regulating the ACh-induced activation of cdc42 and N-WASp by expressing the non-phosphorylatable paxillin mutant, paxillin Y118/31F, in tracheal muscle tissues [31]
Summary
RhoA GTPase regulates airway smooth muscle (SM) contraction and actin polymerization by an unknown mechanism. Results: Agonist stimulation induces the RhoA-dependent recruitment of paxillin, vinculin, FAK, and cdc GEFs to adhesomes, which catalyze cdc and N-WASP activation. The activation of the small GTPase RhoA is necessary for ACh-induced actin polymerization and airway smooth muscle (ASM) contraction, but the mechanism by which it regulates these events is unknown. We assessed the mechanism by which RhoA regulates actin dynamics and smooth muscle contraction by expressing the dominant negative mutants RhoA T19N and cdc T17N, and non-phosphorylatable paxillin Y118/31F and paxillin ⌬LD4 deletion mutants in SM tissues. We conclude that RhoA regulates the contraction of ASM by catalyzing the assembly and activation of cytoskeletal signaling modules at membrane adhesomes that initiate signaling cascades that regulate actin polymerization and tension development in response to contractile agonist stimulation. Our results suggest that the RhoA-mediated assembly of adhesome complexes is a fundamental step in the signal transduction process in response to agonist -induced smooth muscle contraction
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