Abstract
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 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 terminus 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.
Highlights
Vinculin assumes a closed inactive or an open talin/actin-binding conformation
Expression of the Nonphosphorylatable Vinculin Mutant Y1065F Suppresses Endogenous Vinculin Phosphorylation at Tyr1065 (Fig. 2)—Tracheal smooth muscle tissues were transfected with EGFP plasmids encoding WT vinculin, the nonphosphorylatable vinculin mutant Y1065F, or the phosphorylation mimetic Y1065E, or they were sham-treated
We found that conformation-sensitive vinculin FRET probes expressed in smooth muscle tissues undergo conversion from an autoinhibited “closed” conformation to an open “activated” conformation at the mem
Summary
Vinculin assumes a closed inactive or an open talin/actin-binding conformation. During airway smooth muscle contraction, vinculin undergoes Tyr1065 phosphorylation. The results show that vinculin phosphorylation at tyrosine 1065 is required for normal tension generation in airway smooth muscle during contractile stimulation and that Tyr1065 phosphorylation regulates the conformation and scaffolding activity of the vinculin molecule. Our studies have shown that vinculin plays an active role in tension generation in airway smooth muscle tissues during active contraction [10, 23] In these tissues, contractile stimulation triggers the recruitment of vinculin to the membrane and its assembly into membrane adhesomes that regulate actin polymerization [10, 23, 24]. In smooth muscle tissues, the recruitment of vinculin to adhesion complexes and its conversion to an open conformation in which it can bind to talin and actin are critical for contraction and tension development. We conclude that the phosphorylation of vinculin at Tyr1065 provides a mechanism for regulating the function of vinculin in airway smooth muscle in response to external stimulation
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