Wood C K, Turner C E, Jackson P, Critchley D R
Department of Biochemistry, University of Leicester, UK.
J Cell Sci. 1994 Feb;107 ( Pt 2):709-17.
Paxillin and vinculin are cytoskeletal proteins that colocalise to focal adhesions, specialised regions of the cell involved in attachment to the extracellular matrix. These two molecules form part of a complex of proteins that link the actin network to the plasma membrane. Paxillin has been shown to bind directly in vitro to the C-terminal region of vinculin (Turner et al. (1990). J. Cell Biol. 111, 1059-1068), which also contains a focal adhesion targeting sequence (Bendori et al. (1989). J. Cell Biol. 108, 2383-2393). In the present study, we have used a series of vinculin deletion mutants to map more precisely the sites in vinculin responsible for paxillin binding and focal adhesion localisation. A glutathione-S-transferase fusion protein spanning vinculin residues 881-1000 was sufficient to support 125I-paxillin binding in a gel-blot assay while no detectable binding was observed to a fusion protein spanning residues 881-978. Transfection experiments using cDNAs encoding chick vinculin residues 398-1066 and 398-1028 demonstrated that amino acids C-terminal to residue 1028 were not necessary for targeting to focal adhesions. In contrast, a vinculin polypeptide expressed from a cDNA encoding residues 398-1000 failed to localise to focal adhesions in stably transfected NIH3T3 cells. We have therefore identified a region of 50 amino acids (residues 979-1028) within the C-terminal region of vinculin that contains both the paxillin-binding site and the focal adhesion targeting sequence. This region is highly conserved in human and chicken vinculin and is likely to be important in regulation of the assembly of focal adhesions.
桩蛋白和纽蛋白是细胞骨架蛋白，它们共定位于粘着斑，即细胞中参与附着到细胞外基质的特殊区域。这两种分子是将肌动蛋白网络与质膜连接起来的蛋白质复合物的一部分。已表明桩蛋白在体外可直接与纽蛋白的C末端区域结合（特纳等人，《细胞生物学杂志》，1990年，第111卷，第1059 - 1068页），该区域也包含一个粘着斑靶向序列（本多里等人，《细胞生物学杂志》，1989年，第108卷，第2383 - 2393页）。在本研究中，我们使用了一系列纽蛋白缺失突变体来更精确地定位纽蛋白中负责与桩蛋白结合及粘着斑定位的位点。在凝胶印迹分析中，一个跨越纽蛋白881 - 1000位残基的谷胱甘肽 - S - 转移酶融合蛋白足以支持125I - 桩蛋白的结合，而对于一个跨越881 - 978位残基的融合蛋白则未观察到可检测到的结合。使用编码鸡纽蛋白398 - 1066位残基和398 - 1028位残基的cDNA进行的转染实验表明，1028位残基C末端的氨基酸对于靶向粘着斑并非必需。相反，从编码398 - 1000位残基的cDNA表达的纽蛋白多肽在稳定转染的NIH3T3细胞中未能定位于粘着斑。因此，我们在纽蛋白的C末端区域鉴定出了一个由50个氨基酸组成的区域（979 - 1028位残基），该区域同时包含桩蛋白结合位点和粘着斑靶向序列。该区域在人和鸡的纽蛋白中高度保守，可能在粘着斑组装的调控中起重要作用。
