Phosphorylation of dense-plaque proteins talin and paxillin during tracheal smooth muscle contraction

Abstract

Reorganization of cytoskeletal-membrane interactions during contractile stimulation may contribute to the regulation of airway smooth muscle contraction. We investigated the effect of contractile stimulation on the phosphorylation of the actin-membrane attachment proteins talin, vinculin, and paxillin. Stimulation of 32P-labeled canine tracheal smooth muscle strips with acetylcholine (ACh; 10(-3) M) resulted in a rapid 2.6-fold increase in phosphorylation of serine and/or threonine residues, compared with resting levels of 0.22 mol PO4(3-)/mol talin. After stimulation with ACh, phosphorylation of tyrosine residues on paxillin increased approximately threefold. Two-dimensional phosphopeptide mapping of in vivo labeled talin and paxillin indicated phosphorylation on a limited number of sites. Vinculin phosphorylation was undetectable in either resting or ACh-stimulated muscle. We conclude that phosphorylation of talin and paxillin occurs during ACh-stimulated contraction of tracheal smooth muscle and that distinct signaling pathways activate a serine/threonine kinase that phosphorylates talin and a tyrosine kinase that phosphorylates paxillin. The pharmacological activation of airway smooth muscle cells might involve the anchoring of contractile filaments to the membrane.

Paxillin: Adapting to Change

“…Similarly, LIM2 T396/401 and LIM3 S455/479 are phosphorylated after adhesion and stimulation with angiotensin II (26). Growth factors such as activin A (212), EGF (58, 264), heregulin (286), and interleukin (IL)-3 (221), as well as the tumor promoter PMA (26, 58), muscle contraction (202), and virus infection (288) each induces serine/ threonine phosphorylation of paxillin. The onset of mitosis also stimulates paxillin serine/threonine phosphorylation (313), which may function to regulate various paxillin-protein associations leading to focal adhesion and cytoskeletal disassembly associated with cell rounding before cytokinesis.…”

Actin cytoskeletal dynamics in smooth muscle: a new paradigm for the regulation of smooth muscle contraction

“…Paxillin and vinculin bind together in the cytosol (132,136) and are also recruited to membrane adhesion sites in response to contractile stimulation, where they associate with the ILK/ PINCH/␣-parvin complex (95,144). Paxillin is a scaffolding protein that has been shown to undergo tyrosine phosphorylation in response to contractile stimulation in many smooth muscle cell and tissue types (19,71,78,93,101,104,116,122,124,125,129,134,139). The tyrosine phosphorylation of paxillin at two sites, tyrosine 31 and 118, enables it to couple to the SH 2 /SH 3 adaptor protein CrkII, which binds to N-WASp and contributes to N-WASp and Arp2/3 activation (126 -128, 143).…”

Airway smooth muscle dynamics: a common pathway of airway obstruction in asthma

“…The details on how the attachment of thin filaments to adhesion proteins is regulated, especially in partially activated states, remain to be established. However, there is evidence that contractile stimulation of ASM activates a complex of cytoskeletal proteins that associate with transmembrane integrins at sites of cell adhesion [175,176], and that the activation of these proteins is sensitive to mechanical strain [177,178]. The cytoskeletal protein complex that forms at cell adhesion sites may transduce mechanical signals sensed by transmembrane integrin proteins, and initiate signalling pathways that regulate cytoskeletal events, such as the polymerisation of actin, the anchoring of the actin cytoskeleton to adhesion proteins, and the strain-sensitive activation of contractile proteins [55,[179][180][181].…”