V-1, a protein expressed transiently during murine cerebellar development, regulates actin polymerization via interaction with capping protein
V-1, a protein expressed transiently during murine cerebellar development, regulates actin polymerization via interaction with capping protein
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DOI：
10.1074/jbc.m211509200
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发表时间：
2003-02-21
影响因子：
4.8
通讯作者：
Isobe, T
中科院分区：
文献类型：
--
作者：
Taoka, M;Ichimura, T;Isobe, T
V-1是一个12kda的蛋白，由三个连续的ANK重复序列组成，被认为是蛋白-蛋白相互作用的表面。由于其在小鼠小脑发育期间的时间表达谱，人们认为它在神经发育中起作用，但其确切作用尚不清楚。在这里，我们应用蛋白质组学方法来寻找与V-1相互作用的蛋白质靶点。在培养的293T细胞中表达串联亲和纯化标记的V-1 cDNA，并捕获细胞内形成的蛋白复合物，并用质谱法对其进行表征。我们检测到两个与V-1特异性相关的多肽，它们被鉴定为冠蛋白（CP，也称为CapZ或β -肌动蛋白）的a和13个亚基。CP通过覆盖肌动蛋白丝的倒钩端来调节肌动蛋白聚合。V-1- cp复合物不仅在转染V-1 cDNA的培养细胞中检测到，而且在内源性细胞和小鼠小脑提取物中也检测到。表面等离子体共振光谱分析表明，V-1与CP异二聚体形成稳定的配合物，解离常数为1.2 x 10(-7) m，分子化学计量接近1:1。此外，V-1在体外以剂量依赖的方式抑制cp调节的肌动蛋白聚合。因此，我们的研究结果表明，V-1是一种通过与CP相互作用来调节肌动蛋白聚合动力学的新成分，从而参与各种细胞过程，如肌动蛋白驱动的细胞运动和神经元发育过程中的运动。
V-1 is a 12-kDa protein consisting of three consecutive ANK repeats, which are believed to serve as the surface for protein-protein interactions. It is thought to have a role in neural development for its temporal profile of expression during murine cerebellar development, but its precise role remains unknown. Here we applied the proteomic approach to search for protein targets that interact with V-1. The V-1 cDNA attached with a tandem affinity purification tag was expressed in the cultured 293T cells, and the protein complex formed within the cells were captured and characterized by mass spectrometry. We detected two polypeptides specifically associated with V-1, which were identified as the a and 13 subunits of the capping protein (CP, alternatively called CapZ or beta-actinin). CP regulates actin polymerization by capping the barbed end of the actin filament. The V-1-CP complex was detected not only in cultured cells transfected with the V-1 cDNA but also endogenously in cells as well as in murine cerebellar extracts. An analysis of the V-1/CP interaction by surface plasmon resonance spectroscopy showed that V-1 formed a stable complex with the CP heterodimer with a dissociation constant of 1.2 x 10(-7) m and a molecular stoichiometry of similar to1:1. In addition, V-1 inhibited the CP-regulated actin polymerization in vitro in a dose-dependent manner. Thus, our results suggest that V-1 is a novel component that regulates the dynamics of actin polymerization by interacting with CP and thereby participates in a variety of cellular processes such as actin-driven cell movements and motility during neuronal development.