Integrin-linked kinase (ILK) binding to paxillin LD1 motif regulates ILK localization to focal adhesions.
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.
Nikolopoulos
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Department of Cell and Developmental Biology, State University of New York Upstate Medical University, Syracuse, New York 13210, USA.
Turner
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D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003598:Cytoskeletal Proteins / Q000737:chemistry* / Q000378:metabolism*; D022001:Focal Adhesions / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000378:metabolism; D009154:Mutation; D051419:Paxillin; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D051381:Rats; D017386:Sequence Homology, Amino Acid; D000099200:Scaffold Protein ILK
D003598:Cytoskeletal Proteins; D051419:Paxillin; D010750:Phosphoproteins; D017346:Protein Serine-Threonine Kinases; D000099200:Scaffold Protein ILK; C496061:PXN protein, human; C496063:Pxn protein, rat