title: The cytoplasmic domain of tissue factor is phosphorylated by a protein kinase C-dependent mechanism.
abstract: Tissue factor (TF) is an integral membrane glycoprotein that serves as a cellular receptor and cofactor for the activation of the plasma protease factor VII. TF activity in both monocytes and endothelial cells is regulated by various cytokines and mitogens, including the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA). Three TF constructs (full-length human, a cytoplasmic domain deletion mutant, and a human-rat TF chimera), expressed in a human kidney cell line, were used to examine the in vivo phosphorylation state of TF after PMA treatment. The cytoplasmic domains of both rat and human TF were rapidly phosphorylated after cells were treated with 10-100 nM PMA. This response was completely abolished by preincubating cells with staurosporine, the potent PKC inhibitor, prior to PMA treatment. Localization of the phosphorylation site(s) to the cytoplasmic domain was demonstrated using a deletion mutant of TF and by CNBr digestion at the single methionine residue (Met-210) in the TF sequence. The rat TF cytoplasmic domain was phosphorylated to a higher specific activity than the human TF cytoplasmic domain. Phosphoamino acid analysis of the chimeric TF revealed both phosphothreonine and phosphoserine, whereas human TF contained only phosphoserine. Thus both potential phosphoacceptor sites are phosphorylated in the rat TF cytoplasmic domain. Alignment of TF cDNA sequences of mouse, rat, rabbit, and man revealed that the phosphoacceptor site (X-S*/T*-P-X, where asterisk indicates the phosphorylated residue) in the cytoplasmic domain has been conserved through evolution.
authors.affiliation: Genentech, Inc., South San Francisco, California 94080.
authors.forename: T F
authors.initials: TF
authors.lastname: Zioncheck
authors.forename: S
authors.initials: S
authors.lastname: Roy
authors.forename: G A
authors.initials: GA
authors.lastname: Vehar
mesh_terms: D000470:Alkaloids / Q000494:pharmacology; D000595:Amino Acid Sequence; D002460:Cell Line; D003593:Cytoplasm / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011233:Precipitin Tests; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D016415:Sequence Alignment; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013925:Thromboplastin / Q000235:genetics / Q000378:metabolism*
chemical_list: D000470:Alkaloids; D013925:Thromboplastin; D011493:Protein Kinase C; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate