Tang Dale D, Turner Christopher E, Gunst Susan J
Department of Cellular and Integrative Physiology, Indiana University School of Medicine, 635 Barnhill Drive, Indianapolis, IN 46202, USA.
J Physiol. 2003 Nov 15;553(Pt 1):21-35. doi: 10.1113/jphysiol.2003.045047. Epub 2003 Aug 29.
The adapter protein paxillin has been implicated in the regulation of cytoskeletal organization and cell motility. Paxillin undergoes tyrosine phosphorylation in response to the contractile stimulation of smooth muscle, and the depletion of paxillin by antisense inhibits smooth muscle contraction. In the present study, acetylcholine (ACh)-stimulation of tracheal smooth muscle tissues increased paxillin phosphorylation at tyr-31 and tyr-118 by three- to fourfold. The role of tyr-31 and tyr-118 phosphorylation of paxillin in smooth muscle was evaluated by introducing plasmids encoding wild type paxillin or paxillin mutants F31, F118 or F31/118 (phenylalanine substitution at tyrosine sites 31, 118) into tracheal smooth muscle strips by reversible permeabilization, and incubating the tissues for 2 days. The expression of recombinant proteins was confirmed by immunoblot and immunofluorescence analysis. Expression of the paxillin mutants F31, F118 or F31/118 inhibited the contractile response to ACh stimulation but did not inhibit the increase in myosin light chain phosphorylation. The expression of wild type paxillin had no significant affect on force or myosin light chain phosphorylation. ACh stimulation reduced G-actin/F-actin ratio in tissues expressing wild type paxillin; whereas the agonist-induced decrease in G-actin/F-actin was inhibited in strips expressing paxillin mutant F31/118. The paxillin mutant F31/118 showed a marked decrease in their interaction with the SH2/SH3 adaptor protein CrkII but not with vinculin or focal adhesion kinase. We conclude that paxillin phosphorylation at tyr-31 and tyr-118 regulates active tension development during contractile stimulation. Paxillin phosphorylation at these two sites may be important in regulating actin filament dynamics and organization during smooth muscle contraction.
衔接蛋白桩蛋白与细胞骨架组织调控及细胞运动有关。在平滑肌收缩刺激下，桩蛋白会发生酪氨酸磷酸化，反义技术使桩蛋白缺失会抑制平滑肌收缩。在本研究中，乙酰胆碱（ACh）刺激气管平滑肌组织使桩蛋白酪氨酸31和酪氨酸118位点的磷酸化增加了三到四倍。通过可逆通透法将编码野生型桩蛋白或桩蛋白突变体F31、F118或F31/118（酪氨酸位点31、118处为苯丙氨酸取代）的质粒导入气管平滑肌条，并将组织孵育2天，以此评估桩蛋白酪氨酸31和酪氨酸118磷酸化在平滑肌中的作用。通过免疫印迹和免疫荧光分析证实了重组蛋白的表达。桩蛋白突变体F31、F118或F31/118的表达抑制了对ACh刺激的收缩反应，但未抑制肌球蛋白轻链磷酸化的增加。野生型桩蛋白的表达对张力或肌球蛋白轻链磷酸化无显著影响。ACh刺激降低了表达野生型桩蛋白组织中的G-肌动蛋白/F-肌动蛋白比值；而在表达桩蛋白突变体F31/118的条带中，激动剂诱导的G-肌动蛋白/F-肌动蛋白降低受到抑制。桩蛋白突变体F31/118与SH2/SH3衔接蛋白CrkII的相互作用显著降低，但与纽蛋白或粘着斑激酶的相互作用未降低。我们得出结论，桩蛋白酪氨酸31和酪氨酸118位点的磷酸化在收缩刺激过程中调节主动张力的发展。这两个位点的桩蛋白磷酸化在调节平滑肌收缩过程中的肌动蛋白丝动力学和组织方面可能很重要。
