Muscarinic m2 receptor-mediated actin polymerization via PI3 kinase γ and integrin-linked kinase in gastric smooth muscle

Background Actin polymerization plays an important role in smooth muscle contraction. Integrin-linked kinase (ILK) was shown to mediate actin polymerization in airway smooth muscle. The role of ILK in actin polymerization in response to m2 receptor activation was not in gastric smooth muscle. Methods Phosphorylation of paxillin, neuronal Wiskott-Aldrich syndrome protein (N-WASp), and association of paxillin with GEF proteins (Cool2/alpha Pix [Cool2/PAK-interacting exchange factor alpha], Cool1/beta Pix [Cool1/PAK-interacting exchange factor beta], and DOCK 180 [Dedicator of cytokinesis]) and N-WASp with Arp2/3 complex were measured by western blot. Activation of Cdc42 was determined using an antibody for activated Cdc42. Actin polymerization was measured as an increase in F-actin/G-actin ratio. Results Phosphorylation of paxillin, an association of paxillin with GEF proteins, Cdc42 activity, and actin polymerization were increased in response to m2 receptor activation in gastric smooth muscle cells. The increases in paxillin phosphorylation, Cdc42 activity, and actin polymerization were inhibited by a PI3K gamma inhibitor (AS-605240), ILK siRNA, and ILK dominant negative mutant (ILK [R211]). Increase in actin polymerization was also inhibited by Cdc42 dominant negative mutant (Cdc42 [T17N]). Increases in the association of paxillin with GEF proteins, phosphorylation of N-WASp and its association with Arp2/3 complex were inhibited by ILK (R211). Conclusion In gastric smooth muscle cells, activation of PI3K gamma by muscarinic m2 receptors causes ILK-dependent phosphorylation of paxillin, an association of paxillin with Cdc42 GEF proteins and activation of Cdc42, which, in turn, causes phosphorylation of N-WASp and its association with Arp2/3 complex leading to actin polymerization.