Key result
In gastric smooth muscle cells, activation of PI3Kγ by muscarinic m2 receptors causes ILK-dependent phosphorylation of paxillin and activation of Cdc42, leading to actin polymerization.
In gastric smooth muscle cells, muscarinic m2 receptor activation induces actin polymerization via a PI3Kγ and ILK-dependent pathway involving paxillin, Cdc42, and N-WASp.
Identifies a PI3Kγ-ILK pathway in gastric smooth muscle; leaves open relevance to human motility disorders or therapy.
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/αPix [Cool2/PAK-interacting exchange factor alpha], Cool1/βPix [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 PI3Kγ 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 PI3Kγ 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.
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Mahavadi et al. (2018) studied this question. Muscarinic m2 receptor activation was evaluated on Actin polymerization and associated protein phosphorylation/activation. In gastric smooth muscle cells, activation of PI3Kγ by muscarinic m2 receptors causes ILK-dependent phosphorylation of paxillin and activation of Cdc42, leading to actin polymerization.
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