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 foc al adhesion kinase, vinculin, the Arf-GTPase-activating protein paxillin-ki nase linker, and the newly identified actin-binding protein actopaxin, Micr osequencing of peptides derived from a 50-kDa paxillin LD1 motif-binding pr otein revealed 100% identity with integrin-linked kinase (ILK)-1, a serine/ threonine kinase that has been implicated in integrin, growth factor, and W nt signaling pathways. Cloning of ILK from rat smooth muscle cells generate d a cDNA that exhibited 99.6% identity at the amino acid level with human I LK-1. A monoclonal antibody raised against a region of the carboxyl terminu s of ILK, which is identical in rat and human ILK-1 protein, recognized a 5 0-kDa protein in all cultured cells and tissues examined. Binding experimen ts showed that ILK binds directly to the paxillin LD1 motif in vitro, Coimm unoprecipitation from fibroblasts confirmed that the association between pa xillin and ILK occurs in vivo in both adherent cells and cells in suspensio n. Immunofluorescence microscopy of fibroblasts demonstrated that endogenou s ILK as well as transfected green fluorescent protein-ILK co-localizes wit h 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 IL K were unable to bind to the paxillin LD1 motif in vitro and failed to loca lize 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.