ag-213 and Liver-Neoplasms

Regulated endocytosis by growth factor receptors requires intact receptor-associated tyrosine kinase activity. To determine whether a similar requirement exists for the asialoglycoprotein (ASGP) receptor which lacks intrinsic tyrosine kinase activity and participates in constitutive endocytosis, we examined the effect of three tyrosine kinase inhibitors, tyrphostin, genistein, and staurosporine, on receptor-mediated endocytosis in the human hepatoma line HepG2. These compounds inhibited early receptor internalization from the plasma membrane to internal protease-resistant sites in a concentration-dependent manner. This effect correlated with their inhibition of tyrosine phosphorylation of the ASGP receptor in vitro. Receptor trafficking subsequent to receptor internalization was unaffected. Endocytosis of another constitutively internalized protein, the transferrin receptor, was also inhibited by these compounds. In contrast, pinocytosis of the fluid-phase marker Lucifer yellow was not inhibited. The tyrosine kinase inhibitors also decreased the endocytic rate of transfected ASGP receptor H1 subunit in SK-Hep-1 cells. Therefore an intact ASGP receptor heterooligomeric complex is not required for this effect. Mutation of the single cytoplasmic tyrosine at position 5 of the H1 subunit to phenylalanine produced an ASGP receptor which was endocytosed regardless of treatment with the tyrosine kinase inhibitors. We conclude that tyrosine kinase activity modulates the rate of receptor endocytosis at a point early in the internalization process.

Topics: Adenosine Triphosphate; Alkaloids; Asialoglycoprotein Receptor; Carcinoma, Hepatocellular; Catechols; Cell Line; Endocytosis; Genistein; Humans; Isoflavones; Kinetics; Liver Neoplasms; Nitriles; Phosphorylation; Phosphotyrosine; Protein-Tyrosine Kinases; Receptor Protein-Tyrosine Kinases; Receptors, Cell Surface; Signal Transduction; Staurosporine; Transferrin; Tumor Cells, Cultured; Tyrosine; Tyrphostins

Interleukin-6 (IL-6), leukemia inhibitory factor, oncostatin M, IL-11, and ciliary neurotropic factor are a family of cytokines and neuronal differentiation factors which bind to composite plasma membrane receptors sharing the signal transducing subunit gp130. We have shown recently that IL-6 and leukemia inhibitory factor rapidly activate a latent cytoplasmic transcription factor, acute-phase response factor (APRF), by tyrosine phosphorylation, which then binds to IL-6 response elements of various IL-6 target genes. Here we demonstrate that APRF is activated by all cytokines acting through gp130 and is detected in a wide variety of cell types, indicating a central role of this transcription factor in gp130-mediated signaling. APRF activation is also observed in vitro upon addition of IL-6 to cell homogenates. Protein tyrosine kinase inhibitors block both the tyrosine phosphorylation and DNA binding of APRF. The factor was purified to homogeneity from rat liver and shown to consist of a single 87-kDa polypeptide, while two forms (89 and 87 kDa) are isolated from human hepatoma cells. As reported earlier, the binding sequence specificity of APRF is shared by gamma interferon (IFN-gamma) activation factor, which is formed by the Stat91 protein. Partial amino acid sequence obtained from purified rat APRF demonstrated that it is likely to be related to Stat91. In fact, an antiserum raised against the amino-terminal portion of Stat91 cross-reacted with APRF, suggesting the relatedness of APRF and Stat91. Altogether, these data indicate that APRF belongs to a growing family of Stat-related proteins and that IFN-gamma and IL-6 use similar signaling pathways to activate IFN-gamma activation factor and APRF, respectively.

Topics: 3T3 Cells; Alkaloids; Amino Acid Sequence; Animals; Base Sequence; Carcinoma, Hepatocellular; Catechols; Cell Line; Cytokines; DNA-Binding Proteins; Genistein; Growth Inhibitors; Humans; Immune Sera; Interferon-gamma; Interleukin-11; Interleukin-6; Isoflavones; Kinetics; Leukemia Inhibitory Factor; Liver Neoplasms; Lymphokines; Mice; Molecular Sequence Data; Nerve Growth Factors; Nerve Tissue Proteins; Nitriles; Oligodeoxyribonucleotides; Oncostatin M; Peptides; Protein Kinase Inhibitors; Rats; Recombinant Proteins; Signal Transduction; Sphingosine; STAT3 Transcription Factor; Staurosporine; Trans-Activators; Transcription Factors; Tumor Cells, Cultured; Tyrphostins