We investigated whether Rho activation is required for Ca2+-insensitive paxillin phosphorylation, myosin light chain (MLC) phosphorylation, and contraction in tracheal muscle. Tyrosine-phosphorylated proteins have been implicated in the Ca2+-insensitive contractile activation of smooth muscle tissues. The contractile activation of tracheal smooth muscle increases tyrosine phosphorylation of the cytoskeletal proteins paxillin and focal adhesion kinase. Paxillin is implicated in integrin-mediated signal transduction pathways that regulate cytoskeletal organization and cell motility. In fibroblasts and other nonmuscle cells, paxillin tyrosine phosphorylation depends on the activation of Rho and is inhibited by cytochalasin, an inhibitor of actin polymerization. In permeabilized muscle strips, we found that ACh induced Ca2+-insensitive contraction, MLC phosphorylation, and paxillin tyrosine phosphorylation. Ca2+-insensitive contraction and MLC phosphorylation induced by ACh were inhibited by C3 transferase, an inhibitor of Rho activation; however, C3 transferase did not inhibit paxillin tyrosine phosphorylation. Ca2+-insensitive paxillin tyrosine phosphorylation was also not inhibited by the Rho kinase inhibitor Y-27632, by cytochalasin D, or by the inhibition of MLC phosphorylation. We conclude that, in tracheal smooth muscle, Rho mediates Ca2+-insensitive contraction and MLC phosphorylation but that Rho is not required for Ca2+-insensitive paxillin tyrosine phosphorylation. Paxillin phosphorylation also does not require actomyosin activation, nor is it inhibited by the actin filament capping agent cytochalasin D.
Directional Transport of a Bead Bound to Lamellipodial Surface Is Driven by Actin Polymerization
