title: Hypophosphorylated and inactive Pyk2 associates with paxillin at the microtubule organizing center in hematopoietic cells.
abstract: Pyk2 is a non-receptor tyrosine kinase that regulates cellular adhesion. We generated antibodies to a peptide corresponding to the N-terminus (NT) of Pyk2 and another to a portion of the C-terminal (CT) domain. Only the CT antiserum recovered paxillin-associated Pyk2. These antibodies recognized overlapping but biochemically distinct molecular species of Pyk2 since the CT antiserum recovered Pyk2 after NT antibody immunodepletion. Furthermore, the CT antibody could not immunoblot NT antibody-captured Pyk2. Phosphorylation partially accounts for the differential binding of these antibodies as dephosphorylation of Pyk2 recovered with the NT antibodies allows for recognition by the CT antibody. Additionally, Pyk2 recovered with the NT antibody displays increased serine/threonine phosphorylation. We suggest that the NT epitope is inaccessible to the antibody because Pyk2 is in a closed confirmation in association with paxillin. Upon induction of serine and/or threonine phosphorylation of Pyk2, it opens to a confirmation that allows for antibody binding to the NT epitope but at the same time no longer binds paxillin or the CT antiserum. These antibodies also display differential staining of Pyk2 in both T cells and macrophages. Pyk2 recognized by the CT antibody, but not the NT antibody, colocalized with paxillin at the microtubule-organizing center (MTOC). The MTOC-bound Pyk2 was not tyrosine phosphorylated upon T cell activation. We hypothesize that a reservoir of primarily inactive Pyk2 associates with paxillin at the MTOC, which may allow for rapid delivery of Pyk2 to specific sites of adhesion.
authors.affiliation: Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, AB, Canada.
authors.forename: Joëlle
authors.initials: J
authors.lastname: St-Pierre
authors.forename: Tara L
authors.initials: TL
authors.lastname: Lysechko
authors.forename: Hanne L
authors.initials: HL
authors.lastname: Ostergaard
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D051418:Focal Adhesion Kinase 2 / Q000276:immunology / Q000378:metabolism*; D007106:Immune Sera; D047468:Immunoprecipitation; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D022101:Microtubule-Organizing Center / Q000378:metabolism*; D051419:Paxillin / Q000378:metabolism*; D010766:Phosphorylation; D054730:Protein Interaction Domains and Motifs; D021381:Protein Transport; D011994:Recombinant Proteins / Q000378:metabolism; D013602:T-Lymphocytes, Cytotoxic / Q000378:metabolism*
chemical_list: D007106:Immune Sera; D051419:Paxillin; D011994:Recombinant Proteins; D051418:Focal Adhesion Kinase 2; C496053:Ptk2b protein, mouse