Zhang Wenwu, Wu Yidi, Wu Chuanyue, Gunst Susan J
Department of Cellular and Integrative Physiology, Indiana University School of Medicine, 635 Barnhill Drive, Indianapolis, IN 46202, USA.
J Biol Chem. 2007 Nov 23;282(47):34568-80. doi: 10.1074/jbc.M704966200. Epub 2007 Sep 25.
The contractile stimulation of smooth muscle tissues stimulates the recruitment of proteins to membrane adhesion complexes and the initiation of actin polymerization. We hypothesized that integrin-linked kinase (ILK), a beta-integrin-binding scaffolding protein and serine/threonine kinase, and its binding proteins, PINCH, and alpha-parvin may be recruited to membrane adhesion sites during contractile stimulation of tracheal smooth muscle to mediate cytoskeletal processes required for tension development. Immunoprecipitation analysis indicted that ILK, PINCH, and alpha-parvin form a stable cytosolic complex and that the ILK.PINCH.alpha-parvin complex is recruited to integrin adhesion complexes in response to acetylcholine (ACh) stimulation where it associates with paxillin and vinculin. Green fluorescent protein (GFP)-ILK and GFP-PINCH were expressed in tracheal muscle tissues and both endogenous and recombinant ILK and PINCH were recruited to the membrane in response to ACh stimulation. The N-terminal LIM1 domain of PINCH binds to ILK and is required for the targeting of the ILK-PINCH complex to focal adhesion sites in fibroblasts during cell adhesion. We expressed the GFP-PINCH LIM1-2 fragment, consisting only of LIM1-2 domains, in tracheal smooth muscle tissues to competitively inhibit the interaction of ILK with PINCH. The PINCH LIM1-2 fragment inhibited the recruitment of endogenous ILK and PINCH to integrin adhesion sites and prevented their association of ILK with beta-integrins, paxillin, and vinculin. The PINCH LIM1-2 fragment also inhibited tension development, actin polymerization, and activation of the actin nucleation initiator, N-WASp. We conclude that the recruitment of the ILK.PINCH.alpha-parvin complex to membrane adhesion complexes is required to initiate cytoskeletal processes required for tension development in smooth muscle.
平滑肌组织的收缩刺激会促使蛋白质募集到膜粘附复合物，并引发肌动蛋白聚合。我们推测，整合素连接激酶（ILK），一种β整合素结合支架蛋白和丝氨酸/苏氨酸激酶，及其结合蛋白PINCH和α-帕文，可能在气管平滑肌收缩刺激过程中被募集到膜粘附位点，以介导张力发展所需的细胞骨架过程。免疫沉淀分析表明，ILK、PINCH和α-帕文形成稳定的胞质复合物，并且ILK.PINCH.α-帕文复合物在乙酰胆碱（ACh）刺激下被募集到整合素粘附复合物，在那里它与桩蛋白和纽蛋白结合。绿色荧光蛋白（GFP）-ILK和GFP-PINCH在气管肌肉组织中表达，内源性和重组的ILK和PINCH在ACh刺激下均被募集到细胞膜。PINCH的N端LIM1结构域与ILK结合，是细胞粘附过程中ILK-PINCH复合物靶向成纤维细胞粘着斑位点所必需的。我们在气管平滑肌组织中表达仅由LIM1-2结构域组成的GFP-PINCH LIM1-2片段，以竞争性抑制ILK与PINCH的相互作用。PINCH LIM1-2片段抑制内源性ILK和PINCH募集到整合素粘附位点，并阻止ILK与β整合素、桩蛋白和纽蛋白的结合。PINCH LIM1-2片段还抑制张力发展、肌动蛋白聚合以及肌动蛋白成核启动因子N-WASp的激活。我们得出结论，ILK.PINCH.α-帕文复合物募集到膜粘附复合物是启动平滑肌张力发展所需细胞骨架过程所必需的。
