Identification of LIM3 as the principal determinant of paxillin focal adhesion localization and characterization of a novel motif on paxillin directing vinculin and focal adhesion kinase binding.
Identification of LIM3 as the principal determinant of paxillin focal adhesion localization and characterization of a novel motif on paxillin directing vinculin and focal adhesion kinase binding.
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DOI：
10.1083/jcb.135.4.1109
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发表时间：
1996-11
期刊：
影响因子：
--
通讯作者：
Turner CE
中科院分区：
文献类型：
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作者：
Brown MC;Perrotta JA;Turner CE
桩蛋白是一种68-kD的粘着斑磷蛋白，其与几种蛋白质相互作用，包括酪氨酸激酶的src家族成员、转化蛋白v-crk、细胞骨架蛋白粘着斑蛋白和酪氨酸激酶、粘着斑激酶（FAK）。这表明桩蛋白作为分子适配器的功能，负责募集结构和信号分子到粘着斑。目前的研究定义了黏着斑蛋白和FAK的相互作用域桩蛋白，并确定了主要桩蛋白粘着斑靶向基序。使用截短和缺失诱变，我们已经本地化的长春新碱结合位点桩蛋白的连续延伸的21个氨基酸跨越残基143-164。相比之下，FAK与桩蛋白的最大结合除了跨越氨基酸143-164的桩蛋白区域之外还需要包含残基265-313的羧基末端结构域。这些数据表明存在黏着斑蛋白的单个结合位点，和FAK的至少两个结合位点，其被100个氨基酸的插入段分开。氨基酸143-164内的黏着斑蛋白和FAK结合活性是可分离的，因为氨基酸151从带负电荷的谷氨酸突变为不带电荷的极性残基谷氨酰胺（E151 Q）使黏着斑蛋白与桩蛋白的结合降低> 90%，而对FAK的结合能力没有降低。通过转染CHO.K1成纤维细胞，确定桩蛋白内黏着斑蛋白和FAK结合区域的粘着斑靶向要求。值得注意且令人惊讶的是，发现含有消除FAK和/或黏着斑蛋白结合的缺失和点突变的桩蛋白构建体有效靶向粘着斑。此外，含有完整黏着斑蛋白和FAK结合结构域的桩蛋白的氨基末端313个氨基酸的表达未能靶向粘着斑。这表明桩蛋白的其他区域起着粘着斑定位基序的作用。桩蛋白的羧基末端的一半（氨基酸313-559）含有四个连续的双锌指LIM结构域。四个单独的LIM基序的连续羧基末端截短和LIM结构域1、2和3的定点诱变以及缺失诱变的转染分析揭示，靶向桩蛋白至粘着斑的主要机制是通过LIM 3。这些数据表明，桩蛋白定位于局灶性粘连独立的相互作用与黏着斑蛋白和/或FAK，并代表了第一个明确的证明LIM结构域作为一个主要的决定因素的蛋白质亚细胞定位于局灶性粘连。
Paxillin is a 68-kD focal adhesion phosphoprotein that interacts with several proteins including members of the src family of tyrosine kinases, the transforming protein v-crk, and the cytoskeletal proteins vinculin and the tyrosine kinase, focal adhesion kinase (FAK). This suggests a function for paxillin as a molecular adaptor, responsible for the recruitment of structural and signaling molecules to focal adhesions. The current study defines the vinculin- and FAK-interaction domains on paxillin and identifies the principal paxillin focal adhesion targeting motif. Using truncation and deletion mutagenesis, we have localized the vinculin-binding site on paxillin to a contiguous stretch of 21 amino acids spanning residues 143-164. In contrast, maximal binding of FAK to paxillin requires, in addition to the region of paxillin spanning amino acids 143-164, a carboxyl-terminal domain encompassing residues 265-313. These data demonstrate the presence of a single binding site for vinculin, and at least two binding sites for FAK that are separated by an intervening stretch of 100 amino acids. Vinculin- and FAK-binding activities within amino acids 143-164 were separable since mutation of amino acid 151 from a negatively charged glutamic acid to the uncharged polar residue glutamine (E151Q) reduced binding of vinculin to paxillin by >90%, with no reduction in the binding capacity for FAK. The requirement for focal adhesion targeting of the vinculin- and FAK-binding regions within paxillin was determined by transfection into CHO.K1 fibroblasts. Significantly and surprisingly, paxillin constructs containing both deletion and point mutations that abrogate binding of FAK and/or vinculin were found to target effectively to focal adhesions. Additionally, expression of the amino-terminal 313 amino acids of paxillin containing intact vinculin- and FAK-binding domains failed to target to focal adhesions. This indicated other regions of paxillin were functioning as focal adhesion localization motifs. The carboxyl-terminal half of paxillin (amino acids 313-559) contains four contiguous double zinc finger LIM domains. Transfection analyses of sequential carboxyl-terminal truncations of the four individual LIM motifs and site-directed mutagenesis of LIM domains 1, 2, and 3, as well as deletion mutagenesis, revealed that the principal mechanism of targeting paxillin to focal adhesions is through LIM3. These data demonstrate that paxillin localizes to focal adhesions independent of interactions with vinculin and/or FAK, and represents the first definitive demonstration of LIM domains functioning as a primary determinant of protein subcellular localization to focal adhesions.