title: Characterization of tyrosine phosphorylation of paxillin in vitro by focal adhesion kinase.
abstract: The concomitant tyrosine phosphorylation of the focal adhesion protein, paxillin, and the tyrosine kinase, focal adhesion kinase (FAK), in response to multiple stimuli including integrin-mediated cell adhesion suggests that paxillin phosphorylation is closely coupled to FAK activity. In the present study, we have identified a specific tyrosine residue within paxillin, tyrosine 118 (Tyr-118), that represents the principle site of phosphorylation by FAK in vitro. The identification of this site as a target for FAK phosphorylation was accomplished by immunoprecipitating FAK and performing in vitro kinase assays, using as substrate either glutathione S-transferase (GST)-paxillin fusion proteins containing truncations in paxillin sequence or fusion proteins with phenylalanine substitutions for tyrosine residues. GST-paxillin containing a phenylalanine substitution at Tyr-118 (Y118F) was not phosphorylated by FAK immunoprecipitates; however, this mutant was shown to bind FAK equally as well as the wild type fusion protein. As a first step toward assessing the function of paxillin phosphorylation on Tyr-118, a Y118F paxillin cDNA construct was transiently transfected into NIH 3T3 cells. Similar to wild type paxillin, mutated paxillin localized to focal adhesions, indicating that the phosphorylation of paxillin on Tyr-118 is not essential for the recruitment of paxillin to sites of cell adhesion.
authors.affiliation: Department of Anatomy and Cell Biology, State University of New York Health Science Center, Syracuse 13210, USA.
authors.forename: S L
authors.initials: SL
authors.lastname: Bellis
authors.forename: J T
authors.initials: JT
authors.lastname: Miller
authors.forename: C E
authors.initials: CE
authors.lastname: Turner
mesh_terms: D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D015815:Cell Adhesion Molecules / Q000378:metabolism* / Q000502:physiology*; D002642:Chick Embryo; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D051379:Mice; D008969:Molecular Sequence Data; D051419:Paxillin; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000502:physiology*; D014443:Tyrosine / Q000378:metabolism*
chemical_list: D015815:Cell Adhesion Molecules; D003598:Cytoskeletal Proteins; D051419:Paxillin; D010750:Phosphoproteins; C496062:Pxn protein, mouse; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496051:Ptk2 protein, mouse