Vinculin Phosphorylation at Tyr 1065 Regulates Vinculin Conformation and Tension Development in Airway Smooth Muscle Tissues [Cell Biology]

December 13th, 2013 by Huang, Y., Day, R. N., Gunst, S. J.

Vinculin localizes to membrane adhesion junctions in smooth muscle tissues where its head domain binds to talin and its tail domain binds to filamentous actin, thus linking actin filaments to the extracellular matrix. Vinculin can assume a closed conformation in which the head and tail domains bind to each other and mask the binding sites for actin and talin, and an open activated conformation that exposes the binding sites for talin and actin. Acetylcholine (ACh) stimulation of tracheal smooth muscle tissues induces the recruitment of vinculin to the cell membrane and its interaction with talin and actin, which is required for active tension development. Vinculin phosphorylation at Tyr 1065 on its C-terminal increases concurrently with tension development in tracheal smooth muscle tissues. In the present study, the role of vinculin phosphorylation at Tyr 1065 in regulating the conformation and function of vinculin during airway smooth muscle contraction was evaluated. Vinculin constructs with point mutations at Tyr 1065 (vinculin Y1065F and vinculin Y1065E) and vinculin conformation-sensitive FRET probes were expressed in smooth muscle tissues to determine how Tyr 1065 phosphorylation affects smooth muscle contraction and the conformation and cellular functions of vinculin. The results show that vinculin phosphorylation at tyrosine 1065 is required for normal tension generation in airway smooth muscle during contractile stimulation, and that Tyr 1065 phosphorylation regulates the conformation and scaffolding activity of the vinculin molecule. We conclude that the phosphorylation of vinculin at tyrosine 1065 provides a mechanism for regulating the function of vinculin in airway smooth muscle in response to contractile stimulation.