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Phosphorylation of dense-plaque proteins talin and paxillin during tracheal smooth muscle contraction
F M Pavalko1, L P Adam, M F Wu
1Department of Physiology and Biophysics, Indiana University School of Medicine, Indianapolis 46202-5120.
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.
Insights
Contractile stimulation increases phosphorylation of talin and paxillin proteins in airway smooth muscle. This suggests a role for these actin-membrane interactions in regulating muscle contraction.
Area of Science:
- Cell Biology
- Physiology
- Biochemistry
Background:
- Cytoskeletal-membrane interactions are crucial for airway smooth muscle (ASM) contraction.
- Understanding the molecular mechanisms regulating these interactions is key to understanding ASM function.
Purpose of the Study:
- To investigate the effect of contractile stimulation on the phosphorylation of key actin-membrane attachment proteins: talin, vinculin, and paxillin.
- To elucidate the signaling pathways involved in these phosphorylation events.
Main Methods:
- Utilized 32P-labeled canine tracheal smooth muscle strips.
- Stimulated muscle strips with acetylcholine (ACh).
- Analyzed protein phosphorylation using 2D phosphopeptide mapping.
Main Results:
- Acetylcholine stimulation significantly increased serine/threonine phosphorylation of talin (2.6-fold).
- Tyrosine phosphorylation of paxillin increased approximately threefold upon ACh stimulation.
- Vinculin phosphorylation remained undetectable in both resting and stimulated states.
Conclusions:
- Phosphorylation of talin and paxillin occurs during ACh-stimulated ASM contraction.
- Distinct signaling pathways activate serine/threonine and tyrosine kinases, respectively, for talin and paxillin phosphorylation.
- These phosphorylation events may regulate the anchoring of contractile filaments to the membrane in ASM cells.