title: Angiotensin II activates at least two tyrosine kinases in rat liver epithelial cells. Separation of the major calcium-regulated tyrosine kinase from p125FAK.
abstract: In rat liver epithelial cell lines (WB or GN4), angiotensin II (Ang II) stimulates cytosolic tyrosine kinase activity, in part, through a calcium-dependent mechanism. In other cell types, selected hormones that activate Gi- or Gq-coupled receptors stimulate the soluble tyrosine kinase, p125FAK. Immunoprecipitation of p125FAK from Ang II-activated GN4 cells demonstrated a doubling of p125FAK kinase activity. However, an additional Ang II-activated tyrosine kinase (or kinases) representing the majority of the total activity was detected when the remaining cell lysate, immunodepleted of p125FAK, was reimmunoprecipitated with an anti-phosphotyrosine antibody. Cytochalasin D pretreatment blocks G-protein receptor-dependent tyrosine phosphorylation in Swiss 3T3 cells. While cytochalasin D decreased the Tyr(P) content of 65-75-kDa substrates in Ang II-treated GN4 cells, it did not diminish tyrosine phosphorylation of 115-130-kDa substrates, again suggesting activation of at least two tyrosine kinase pathways in GN4 cells. To search for additional Ang II-activated enzymes, we used molecular techniques to identify 20 tyrosine kinase sequences in these cell lines. None was the major cytosolic enzyme activated by Ang II. Specifically, JAK2, which had been shown by others to be stimulated by Ang II in smooth muscle cells, was not activated by Ang II in GN4 cells. Finally, we purified Tyr(P)-containing tyrosine kinases from Ang II-treated cells, using anti-Tyr(P) and ATP affinity resins; 80% of the tyrosine kinase activity migrated as a single 115-120-kDa tyrosine-phosphorylated protein immunologically distinct from p125FAK. In summary, Ang II activates at least two separate tyrosine kinases in rat liver epithelial cells; p125FAK and a presumably novel, cytosolic 115-120-kDa protein referred to as the calcium-dependent tyrosine kinase.
mesh_terms: D000595:Amino Acid Sequence; D000804:Angiotensin II / Q000494:pharmacology*; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D015815:Cell Adhesion Molecules / Q000302:isolation & purification / Q000378:metabolism*; D002460:Cell Line; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D017124:Conserved Sequence; D003600:Cytosol / Q000378:metabolism; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004848:Epithelium / Q000201:enzymology; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D019204:GTP-Binding Proteins / Q000378:metabolism; D015723:Gene Library; D006801:Humans; D015151:Immunoblotting; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011505:Protein-Tyrosine Kinases / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D015815:Cell Adhesion Molecules; D017931:DNA Primers; D010750:Phosphoproteins; D011994:Recombinant Proteins; D000804:Angiotensin II; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; C496052:Ptk2 protein, rat; D019204:GTP-Binding Proteins; D002118:Calcium