Paxillin LD4 motif binds PAK and PIX through a novel 95-kD ankyrin repeat, ARF-GAP protein: A role in cytoskeletal remodeling.
Paxillin is a focal adhesion adaptor protein involved in the integration of growth factor- and adhesion-mediated signal transduction pathways. Repeats of a leucine-rich sequence named paxillin LD motifs (Brown M.C., M.S. Curtis, and C.E. Turner. 1998. Nature Struct. Biol. 5:677-678) have been implicated in paxillin binding to focal adhesion kinase (FAK) and vinculin. Here we demonstrate that the individual paxillin LD motifs function as discrete and selective protein binding interfaces. A novel scaffolding function is described for paxillin LD4 in the binding of a complex of proteins containing active p21 GTPase-activated kinase (PAK), Nck, and the guanine nucleotide exchange factor, PIX. The association of this complex with paxillin is mediated by a new 95-kD protein, p95PKL (paxillin-kinase linker), which binds directly to paxillin LD4 and PIX. This protein complex also binds to Hic-5, suggesting a conservation of LD function across the paxillin superfamily. Cloning of p95PKL revealed a multidomain protein containing an NH2-terminal ARF-GAP domain, three ankyrin-like repeats, a potential calcium-binding EF hand, calmodulin-binding IQ motifs, a myosin homology domain, and two paxillin-binding subdomains (PBS). Green fluorescent protein- (GFP-) tagged p95PKL localized to focal adhesions/complexes in CHO.K1 cells. Overexpression in neuroblastoma cells of a paxillin LD4 deletion mutant inhibited lamellipodia formation in response to insulin-like growth fac- tor-1. Microinjection of GST-LD4 into NIH3T3 cells significantly decreased cell migration into a wound. These data implicate paxillin as a mediator of p21 GTPase-regulated actin cytoskeletal reorganization through the recruitment to nascent focal adhesion structures of an active PAK/PIX complex potentially via interactions with p95PKL.
D020727:ADP-Ribosylation Factors; D000595:Amino Acid Sequence; D000818:Animals; D017487:Ankyrins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D016466:CHO Cells; D019556:COS Cells; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D015815:Cell Adhesion Molecules / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000378:metabolism; D002465:Cell Movement; D006224:Cricetinae; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D003599:Cytoskeleton / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000378:metabolism; D051416:Focal Adhesion Protein-Tyrosine Kinases; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins / Q000378:metabolism*; D020690:GTPase-Activating Proteins; D047908:Intracellular Signaling Peptides and Proteins; D060588:LIM Domain Proteins; D008969:Molecular Sequence Data; D051419:Paxillin; D010750:Phosphoproteins / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D012091:Repetitive Sequences, Nucleic Acid; D017422:Sequence Analysis, DNA; D013347:Subcellular Fractions; D016596:Vinculin / Q000378:metabolism; D020764:cdc42 GTP-Binding Protein; D054462:p21-Activated Kinases