title: Mitogen-induced, FAK-dependent tyrosine phosphorylation of the SSeCKS scaffolding protein.
abstract: The ability of mitogens to rapidly induce tyrosine phosphorylation of cellular proteins has been taken as evidence of participation in subsequent signaling pathways. SSeCKS, a major protein kinase C (PKC) substrate with protein scaffolding and tumor suppressive properties, becomes tyrosine phosphorylated in NIH3T3 and rodent embryo fibroblasts after short-term treatment with epidermal growth factor (EGF), platelet-derived growth factor (PDGF), or fetal calf serum in the presence of pervanadate, but not by treatment with insulin or insulin-like growth factor-1. The relative phosphotyrosine level on SSeCKS was higher in actively dividing cells than in confluent cultures. Tyrosine phosphorylation of SSeCKS was apparent in cells deficient in Src, Fyn, Yes, or Abl tyrosine kinases or in NIH3T3 cells expressing a temperature-sensitive v-Src allele, but not in FAK-deficient embryo fibroblasts. Purified FAK or Src enzyme failed to directly phosphorylate SSeCKS in vitro. EGF failed to induce SSeCKS tyrosine phosphorylation in FAK-/- fibroblasts, indicating that the EGF receptor is probably not the direct kinase of SSeCKS. Phosphorylation under these conditions was rescued by the transient reexpression of wt-FAK but not FAK mutated at Y397, a major autophosphorylation and SH2-based docking site. Adhesion of FAK+/+ cells to fibronectin failed to significantly induce SSeCKS tyrosine phosphorylation although FAK was activated, suggesting that SSeCKS phosphorylation is mediated through a growth factor receptor-FAK rather than an integrin-FAK pathway. Moreover, PDGF could induce SSeCKS tyrosine phosphorylation in the absence of FAK activation, suggesting a role for FAK SH2-based docking rather than kinase activity. Immunofluorescence analysis showed that in FAK-/- cells, SSeCKS costains along F-actin stress fibers, in contrast to FAK+/+ cells, where most SSeCKS stains at the cell edge and along a cortical cytoskeletal matrix. This correlated with increased coprecipitation of SSeCKS with biotin-phalloidin-bound F-actin from FAK-/- compared to FAK+/+ cell lysates. Similarly, bacterially expressed, unphosphorylated SSeCKS cosedimented with F-actin in ultracentrifugation assays. These data suggest that mitogen-induced, FAK-dependent tyrosine phosphorylation of SSeCKS modulates its binding to the actin-based cytoskeleton, suggesting a role for SSeCKS in mitogen-induced cytoskeletal reorganization.
authors.affiliation: Department of Medicine and the Ruttenberg Cancer Center, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, New York 10029-6574, USA.
authors.forename: Wei
authors.initials: W
authors.lastname: Xia
authors.forename: Irwin H
authors.initials: IH
authors.lastname: Gelman
mesh_terms: D016475:3T3 Cells; D054758:A Kinase Anchor Proteins; D000199:Actins / Q000378:metabolism; D000818:Animals; D018797:Cell Cycle Proteins; D002460:Cell Line; D004815:Epidermal Growth Factor / Q000494:pharmacology; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D051379:Mice; D018345:Mice, Knockout; D008934:Mitogens / Q000378:metabolism*; D010766:Phosphorylation; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D011233:Precipitin Tests; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D014443:Tyrosine / Q000378:metabolism*
chemical_list: D054758:A Kinase Anchor Proteins; D000199:Actins; C491161:Akap12 protein, mouse; C102361:Akap12 protein, rat; D018797:Cell Cycle Proteins; D008934:Mitogens; D010982:Platelet-Derived Growth Factor; D014443:Tyrosine; D004815:Epidermal Growth Factor; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496051:Ptk2 protein, mouse; C496052:Ptk2 protein, rat