Abelson 酪氨酸激酶在 Crk 相关底物/丝切蛋白-1 相互作用和气道平滑肌收缩中的作用和调节。

BACKGROUND: Airway smooth muscle contraction is critical for maintenance of appropriate airway tone, and has been implicated in asthma pathogenesis. Smooth muscle contraction requires an "engine" (myosin activation) and a "transmission system" (actin cytoskeletal remodeling). However, the mechanisms that control actin remodeling in smooth muscle are not fully elucidated. The adapter protein Crk-associated substrate (CAS) regulates actin dynamics and the contraction in smooth muscle. In addition, profilin-1 (Pfn-1) and Abelson tyrosine kinase (c-Abl) are also involved in smooth muscle contraction. The interplays among CAS, Pfn-1 and c-Abl in smooth muscle have not been previously investigated. METHODS: The association of CAS with Pfn-1 in mouse tracheal rings was evaluated by co-immunoprecipitation. Tracheal rings from c-Abl conditional knockout mice were used to assess the roles of c-Abl in the protein-protein interaction and smooth muscle contraction. Decoy peptides were utilized to evaluate the importance of CAS/Pfn-1 coupling in smooth muscle contraction. RESULTS: Stimulation with acetylcholine (ACh) increased the interaction of CAS with Pfn-1 in smooth muscle, which was regulated by CAS tyrosine phosphorylation and c-Abl. The CAS/Pfn-1 coupling was also modified by the phosphorylation of cortactin (a protein implicated in Pfn-1 activation). In addition, ACh activation promoted the spatial redistribution of CAS and Pfn-1 in smooth muscle cells, which was reduced by c-Abl knockdown. Inhibition of CAS/Pfn-1 interaction by a decoy peptide attenuated the ACh-induced actin polymerization and contraction without affecting myosin light chain phosphorylation. Furthermore, treatment with the Src inhibitor PP2 and the actin polymerization inhibitor latrunculin A attenuated the ACh-induced c-Abl tyrosine phosphorylation (an indication of c-Abl activation). CONCLUSIONS: Our results suggest a novel activation loop in airway smooth muscle: c-Abl promotes the CAS/Pfn-1 coupling and actin polymerization, which conversely facilitates c-Abl activation. The positive feedback may render c-Abl in active state after contractile stimulation.

背景：气道平滑肌收缩对于维持适当的气道张力至关重要，并且与哮喘发病机制有关。平滑肌收缩需要一个“发动机”（肌球蛋白激活）和一个“传动系统”（肌动蛋白细胞骨架重塑）。然而，控制平滑肌中肌动蛋白重塑的机制尚未完全阐明。衔接蛋白 Crk 相关底物（CAS）调节平滑肌中的肌动蛋白动力学和收缩。此外，丝状肌动蛋白结合蛋白-1（Pfn-1）和 Abelson 酪氨酸激酶（c-Abl）也参与平滑肌收缩。CAS、Pfn-1 和 c-Abl 之间的相互作用在平滑肌中尚未被研究过。

方法：通过共免疫沉淀评估小鼠气管环中 CAS 与 Pfn-1 的结合。使用 c-Abl 条件性敲除小鼠的气管环来评估 c-Abl 在蛋白-蛋白相互作用和平滑肌收缩中的作用。利用诱饵肽来评估 CAS/Pfn-1 偶联在平滑肌收缩中的重要性。

结果：乙酰胆碱（ACh）刺激增加了平滑肌中 CAS 与 Pfn-1 的相互作用，这受 CAS 酪氨酸磷酸化和 c-Abl 的调节。CAS/Pfn-1 偶联也受到皮层蛋白（一种被认为参与 Pfn-1 激活的蛋白质）磷酸化的调节。此外，ACh 激活促进了 CAS 和 Pfn-1 在平滑肌细胞中的空间重分布，而 c-Abl 敲低则减少了这种重分布。通过诱饵肽抑制 CAS/Pfn-1 相互作用减弱了 ACh 诱导的肌动蛋白聚合和收缩，而不影响肌球蛋白轻链磷酸化。此外，Src 抑制剂 PP2 和肌动蛋白聚合抑制剂 latrunculin A 的处理减弱了 ACh 诱导的 c-Abl 酪氨酸磷酸化（表明 c-Abl 激活）。

结论：我们的结果表明气道平滑肌中存在一种新的激活环：c-Abl 促进 CAS/Pfn-1 偶联和肌动蛋白聚合，这反过来又促进了 c-Abl 的激活。这种正反馈可能使 c-Abl 在收缩刺激后处于激活状态。