title: Molecular dissection of actopaxin-integrin-linked kinase-Paxillin interactions and their role in subcellular localization.
abstract: Paxillin is a focal adhesion adapter protein involved in integrin signaling. We have recently reported that the paxillin LD1 motif acts as a binding interface for both the actin-binding protein actopaxin and the serine/threonine integrin-linked kinase (ILK). In this report we demonstrate the direct association between actopaxin and ILK and dissect the role of the respective interactions in their subcellular localization. Co-immunoprecipitation experiments were employed to map the binding sites on ILK and actopaxin. ILK binds to the CH2 domain of actopaxin. However, an actopaxin CH2 domain mutant defective for paxillin binding (paxillin binding subdomain mutant) retains the capacity to bind ILK, indicating that paxillin and ILK binding sites on actopaxin are distinct. Actopaxin binds to the C terminus of ILK. Despite the direct binding between actopaxin and ILK, mutation analysis confirmed a primary role for paxillin in their localization to focal adhesions. Interestingly, an ILK mutant (E359K) that was previously reported to act as dominant negative for ILK function was unable to bind actopaxin or paxillin and failed to localize to focal adhesions. This mutant also exhibited in vitro kinase activity comparable with wild-type ILK. Taken together, these data suggest that normal ILK signaling is dependent on efficient localization involving multiple protein interactions.
authors.affiliation: Department of Cell and Developmental Biology, State University of New York Upstate Medical University, Syracuse, New York 13210, USA.
authors.forename: Sotiris N
authors.initials: SN
authors.lastname: Nikolopoulos
authors.forename: Christopher E
authors.initials: CE
authors.lastname: Turner
mesh_terms: D000185:Actinin; D000818:Animals; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D022001:Focal Adhesions / Q000378:metabolism; D017930:Genes, Reporter; D006801:Humans; D007150:Immunohistochemistry; D008840:Microfilament Proteins / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism; D051419:Paxillin; D010750:Phosphoproteins / Q000378:metabolism*; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection; D000099200:Scaffold Protein ILK; D000096985:Calponins
chemical_list: D000185:Actinin; D003598:Cytoskeletal Proteins; D008840:Microfilament Proteins; D051419:Paxillin; D010750:Phosphoproteins; D017346:Protein Serine-Threonine Kinases; D011993:Recombinant Fusion Proteins; D000099200:Scaffold Protein ILK; C486590:PARVA protein, human; D000096985:Calponins; C496061:PXN protein, human; C496063:Pxn protein, rat