title: The LD4 motif of paxillin regulates cell spreading and motility through an interaction with paxillin kinase linker (PKL).
abstract: The small GTPases of the Rho family are intimately involved in integrin-mediated changes in the actin cytoskeleton that accompany cell spreading and motility. The exact means by which the Rho family members elicit these changes is unclear. Here, we demonstrate that the interaction of paxillin via its LD4 motif with the putative ARF-GAP paxillin kinase linker (PKL) (Turner et al., 1999), is critically involved in the regulation of Rac-dependent changes in the actin cytoskeleton that accompany cell spreading and motility. Overexpression of a paxillin LD4 deletion mutant (paxillinDeltaLD4) in CHO.K1 fibroblasts caused the generation of multiple broad lamellipodia. These morphological changes were accompanied by an increase in cell protrusiveness and random motility, which correlated with prolonged activation of Rac. In contrast, directional motility was inhibited. These alterations in morphology and motility were dependent on a paxillin-PKL interaction. In cells overexpressing paxillinDeltaLD4 mutants, PKL localization to focal contacts was disrupted, whereas that of focal adhesion kinase (FAK) and vinculin was not. In addition, FAK activity during spreading was not compromised by deletion of the paxillin LD4 motif. Furthermore, overexpression of PKL mutants lacking the paxillin-binding site (PKLDeltaPBS2) induced phenotypic changes reminiscent of paxillinDeltaLD4 mutant cells. These data suggest that the paxillin association with PKL is essential for normal integrin-mediated cell spreading, and locomotion and that this interaction is necessary for the regulation of Rac activity during these events.
authors.affiliation: Department of Cell and Developmental Biology, State University of New York, Upstate Medical University, 750 East Adams St., Syracuse, NY 13210, USA.
authors.forename: K A
authors.initials: KA
authors.lastname: West
authors.forename: H
authors.initials: H
authors.lastname: Zhang
authors.forename: M C
authors.initials: MC
authors.lastname: Brown
authors.forename: S N
authors.initials: SN
authors.lastname: Nikolopoulos
authors.forename: M C
authors.initials: MC
authors.lastname: Riedy
authors.forename: A F
authors.initials: AF
authors.lastname: Horwitz
authors.forename: C E
authors.initials: CE
authors.lastname: Turner
mesh_terms: D020727:ADP-Ribosylation Factors / Q000378:metabolism*; D020816:Amino Acid Motifs; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D016466:CHO Cells; D002465:Cell Movement; D002478:Cells, Cultured; D006224:Cricetinae; D003598:Cytoskeletal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D004789:Enzyme Activation; D005347:Fibroblasts / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000378:metabolism*; D017353:Gene Deletion; D005982:Glutathione Transferase / Q000378:metabolism; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D018715:Microscopy, Video; D008957:Models, Genetic; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D051419:Paxillin; D010641:Phenotype; D010750:Phosphoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011554:Pseudopodia / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
chemical_list: C503237:ARFGAP1 protein, human; C503240:ARFGAP3 protein, human; D003598:Cytoskeletal Proteins; D005353:Fibronectins; C430213:GIT2 protein, human; D020690:GTPase-Activating Proteins; D008164:Luminescent Proteins; D051419:Paxillin; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D005982:Glutathione Transferase; D020727:ADP-Ribosylation Factors