title: Phosphorylation of tyrosine 285 of PAK1 facilitates βPIX/GIT1 binding and adhesion turnover.
abstract: The p21-activated serine-threonine kinase (PAK1) regulates cell motility and adhesion. We have previously shown that the prolactin (PRL)-activated tyrosine kinase JAK2 phosphorylates PAK1 in vivo and in vitro and identified tyrosines 153, 201, and 285 in PAK1 as sites of JAK2 tyrosyl phosphorylation. Here, we further investigate the role of the tyrosyl phosphorylated PAK1 (pTyr-PAK1) in regulation of cell adhesion. We use human breast cancer T47D cell lines that stably overexpress PAK1 wild type or PAK1 Y3F mutant in which these 3 JAK2 phosphorylation sites were mutated to phenylalanine. We demonstrate that PRL/JAK2-dependent phosphorylation of these tyrosines promotes a motile phenotype in the cells upon adhesion, participates in regulation of cell adhesion on collagen IV, and is required for maximal PAK1 kinase activity. Down-regulation of PAK1 abolishes the effect of PAK1 on cell adhesion. We show that the tyrosyl phosphorylation of PAK1 promotes PAK1 binding to β-PAK1-interacting guanine-nucleotide exchange factor (βPIX) and G protein-coupled receptor kinase-interacting target 1 (GIT1), phosphorylation of paxillin on Ser273, and formation and distribution of adhesion complexes. Using phosphospecific antibodies (Abs) directed to single phosphorylated tyrosines on PAK1, we identified Tyr285 as a site of PRL-dependent phosphorylation of PAK1 by JAK2. Furthermore, using PAK1 Y285F mutant, we provide evidence for a role of pTyr285 in cell adhesion, enhanced βPIX/GIT1 binding, and adhesion turnover. Our immunohistochemistry analysis demonstrates that pTyr285- PAK1 may modulate PAK1 signaling during tumor progression.
authors.affiliation: Departments of *Biological Sciences and Pathology, University of Toledo, Toledo, Ohio, USA.
authors.forename: Alan
authors.initials: A
authors.lastname: Hammer
authors.affiliation: Departments of *Biological Sciences and Pathology, University of Toledo, Toledo, Ohio, USA.
authors.forename: Peter
authors.initials: P
authors.lastname: Oladimeji
authors.affiliation: Departments of *Biological Sciences and Pathology, University of Toledo, Toledo, Ohio, USA.
authors.forename: Luis E
authors.initials: LE
authors.lastname: De Las Casas
authors.affiliation: Departments of *Biological Sciences and Pathology, University of Toledo, Toledo, Ohio, USA mdiakon@utnet.utoledo.edu.
authors.forename: Maria
authors.initials: M
authors.lastname: Diakonova
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology; D002448:Cell Adhesion / Q000502:physiology*; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002465:Cell Movement; D049109:Cell Proliferation; D005260:Female; D005455:Fluorescent Antibody Technique; D006801:Humans; D007124:Immunoenzyme Techniques; D047468:Immunoprecipitation; D051419:Paxillin / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D064067:Rho Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D015398:Signal Transduction; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000378:metabolism*; D054462:p21-Activated Kinases / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; C485647:GIT1 protein, human; D051419:Paxillin; D064067:Rho Guanine Nucleotide Exchange Factors; D014443:Tyrosine; C515903:PAK1 protein, human; D054462:p21-Activated Kinases
keywords: JAK2 kinase; breast cancer cells; cell spreading; prolactin