The adapter protein CrkII regulates neuronal Wiskott-Aldrich syndrome protein, actin polymerization, and tension development during contractile stimulation of smooth muscle

Actin polymerization has been shown to occur in tracheal smooth muscle tissues and cells in response to contractile stimulation, and there is evidence that the polymerization of actin is required for contraction. In tracheal smooth muscle, agonist-induced actin polymerization is mediated by activation of neuronal Wiskott-Aldrich syndrome protein (N-WASp) and the Arp (actin-related protein) 2/3 complex, and activation of the small GTPase Cdc42 regulates the activation of N-WASp. In the present study, the role of the adapter protein CrkII in the regulation of N-WASp and Cdc42 activation, actin polymerization, and tension development in smooth muscle tissues was evaluated. Stimulation of tracheal smooth muscle tissues with acetylcholine increased the association of CrkII with N-WASp. Plasmids encoding wild type CrkII or a CrkII mutant lacking the SH3 effector-binding ability, CrkII SH3N, were introduced into tracheal smooth muscle tissues, and the tissues were incubated for 2 days to allow for protein expression. Expression of the CrkII SH3N mutant in smooth muscle tissues inhibited the association of CrkII with N-WASp and the activation of Cdc42. The CrkII SH3N mutant also inhibited the increase in the association of N-WASp with Arp2, a major component of the Arp2/3 complex, in response to contractile stimulation, indicating inhibition of N-WASp activation. Expression of the CrkII SH3N mutant also inhibited tension generation and actin polymerization in response to contractile stimulation; however, it did not inhibit myosin light chain phosphorylation. These results suggest that CrkII plays a critical role in the regulation of N-WASp activation, perhaps by regulating the activation of Cdc42, and that it thereby regulates actin polymerization and active tension generation in tracheal smooth muscle. These studies suggest a novel signaling pathway for the regulation of N-WASp activation and active contraction in smooth muscle tissues.