Abstract
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.
Highlights
Introduction of Plasmids Encoding RecombinantProteins or ODNs into Tracheal Smooth Muscle Tissues—Plasmids encoding wild type CrkII or SH3N CrkII mutant have been previously described [35]. cDNA constructs for wild type Cdc42 and the Asn-17 Cdc42 dominant negative mutants have been previously described (36 –38)
Contractile stimulation initiates the interaction of neuronal WiskottAldrich syndrome protein (N-Wiskott-Aldrich syndrome protein (WASp)) with the Arp2/3 complex in this tissue, and this requires activation of the small GTPase Cdc42 [25]
Activation of the Arp2/3 complex by NWASp is required for actin polymerization and tension development in response to contractile stimulation with acetylcholine in tracheal smooth muscle tissues [15]
Summary
Introduction of Plasmids Encoding RecombinantProteins or ODNs into Tracheal Smooth Muscle Tissues—Plasmids encoding wild type CrkII or SH3N CrkII mutant (cysteine is substituted for tryptophan 109) have been previously described [35]. cDNA constructs for wild type Cdc and the Asn-17 Cdc dominant negative mutants have been previously described (36 –38). Proteins or ODNs into Tracheal Smooth Muscle Tissues—Plasmids encoding wild type CrkII or SH3N CrkII mutant (cysteine is substituted for tryptophan 109) have been previously described [35]. CDNA constructs for wild type Cdc and the Asn-17 Cdc dominant negative mutants have been previously described (36 –38). Escherichia coli (Bluescript) transformed with these plasmids were grown in LB medium, and plasmids were purified by alkaline lysis with SDS (maxipreparation) or by a kit from Invitrogen Antisense ODNs with the following sequence were designed to suppress endogenous CrkII expression in canine tracheal muscle tissues: 5Ј-TCACTCCACTACCCTGCC-3Ј. The following sequence of sense oligonucleotides was used as a control: 5Ј-GGCAGGGTAGTGGAGTGA-3Ј. The ODNs were fully phosphorothiolated to enhance their intracellular stability in smooth muscle cells and were synthesized and purified by GenoMechanix, L.L.C., Gainsville, FL
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