Nikolopoulos S N, Turner C E
Department of Cell and Developmental Biology, State University of New York Upstate Medical University, Syracuse, New York 13210, USA.
J Biol Chem. 2001 Jun 29;276(26):23499-505. doi: 10.1074/jbc.M102163200. Epub 2001 Apr 13.
Paxillin is a focal adhesion adapter protein involved in integrin signaling. Paxillin LD motifs bind several focal adhesion proteins including the focal adhesion kinase, vinculin, the Arf-GTPase-activating protein paxillin-kinase linker, and the newly identified actin-binding protein actopaxin. Microsequencing of peptides derived from a 50-kDa paxillin LD1 motif-binding protein revealed 100% identity with integrin-linked kinase (ILK)-1, a serine/threonine kinase that has been implicated in integrin, growth factor, and Wnt signaling pathways. Cloning of ILK from rat smooth muscle cells generated a cDNA that exhibited 99.6% identity at the amino acid level with human ILK-1. A monoclonal antibody raised against a region of the carboxyl terminus of ILK, which is identical in rat and human ILK-1 protein, recognized a 50-kDa protein in all cultured cells and tissues examined. Binding experiments showed that ILK binds directly to the paxillin LD1 motif in vitro. Co-immunoprecipitation from fibroblasts confirmed that the association between paxillin and ILK occurs in vivo in both adherent cells and cells in suspension. Immunofluorescence microscopy of fibroblasts demonstrated that endogenous ILK as well as transfected green fluorescent protein-ILK co-localizes with paxillin in focal adhesions. Analysis of the deduced amino acid sequence of ILK identified a paxillin-binding subdomain in the carboxyl terminus of ILK. In contrast to wild-type ILK, paxillin-binding subdomain mutants of ILK were unable to bind to the paxillin LD1 motif in vitro and failed to localize to focal adhesions. Thus, paxillin binding is necessary for efficient focal adhesion targeting of ILK and may therefore impact the role of ILK in integrin-mediated signal transduction events.
桩蛋白是一种参与整合素信号传导的粘着斑衔接蛋白。桩蛋白的LD基序结合多种粘着斑蛋白，包括粘着斑激酶、纽蛋白、Arf - GTP酶激活蛋白桩蛋白激酶连接体，以及新鉴定出的肌动蛋白结合蛋白肌动桩蛋白。对源自一种50 kDa桩蛋白LD1基序结合蛋白的肽段进行微量测序，结果显示其与整合素连接激酶（ILK）-1具有100%的同一性，ILK -1是一种丝氨酸/苏氨酸激酶，已被证明参与整合素、生长因子和Wnt信号通路。从大鼠平滑肌细胞中克隆ILK得到一个cDNA，该cDNA在氨基酸水平上与人ILK -1具有99.6%的同一性。针对ILK羧基末端区域产生的单克隆抗体（该区域在大鼠和人ILK -1蛋白中是相同的），在所有检测的培养细胞和组织中识别出一种50 kDa的蛋白。结合实验表明，ILK在体外直接与桩蛋白的LD1基序结合。从成纤维细胞进行的共免疫沉淀证实，桩蛋白和ILK之间的结合在体内的贴壁细胞和悬浮细胞中均会发生。对成纤维细胞进行免疫荧光显微镜检查表明，内源性ILK以及转染的绿色荧光蛋白 - ILK与桩蛋白在粘着斑中共定位。对ILK推导的氨基酸序列分析确定了ILK羧基末端的一个桩蛋白结合亚结构域。与野生型ILK相反，ILK的桩蛋白结合亚结构域突变体在体外无法与桩蛋白的LD1基序结合，并且无法定位到粘着斑。因此，桩蛋白结合对于ILK有效地靶向粘着斑是必要的，因此可能会影响ILK在整合素介导的信号转导事件中的作用。
