title: Monoclonal antibodies mimic insulin activation of ribosomal protein S6 kinase without activation of insulin receptor tyrosine kinase. Studies in cells transfected with normal and mutant human insulin receptors.
abstract: The effects of species-specific monoclonal antibodies to the human insulin receptor on ribosomal protein S6 phosphorylation were studied in rodent cell lines transfected with human insulin receptors. First, Swiss mouse 3T3 fibroblasts expressing normal human insulin receptors (3T3/HIR cells) were studied. Three monoclonal antibodies, MA-5, MA-20, and MA-51, activated S6 kinase in these cells but had no effects in untransfected 3T3 cells. Both insulin and MA-5, the most potent antibody, activated S6 kinase in a similar time- and dose-dependent manner. To measure S6 phosphorylation in vivo, 3T3/HIR cells were preincubated with [32P]Pi and treated with insulin and MA-5. Both agents increased S6 phosphorylation, and their tryptic phosphopeptide maps were similar. MA-5 and the other monoclonal antibodies, unlike insulin, failed to stimulate insulin receptor tyrosine kinase activity either in vitro or in vivo. Moreover, unlike insulin, they failed to increase the tyrosine phosphorylation of the endogenous cytoplasmic protein, pp 185. Next, HTC rat hepatoma cells, expressing a human insulin receptor mutant that had three key tyrosine autophosphorylation sites in the beta-subunit changed to phenylalanines (HTC-IR-F3 cells), were studied. In this cell line but not in untransfected HTC cells, monoclonal antibodies activated S6 kinase without stimulating either insulin receptor autophosphorylation or the tyrosine phosphorylation of pp 185. These data indicate, therefore, that monoclonal antibodies can activate S6 kinase and then increase S6 phosphorylation. Moreover, they suggest that activation of receptor tyrosine kinase and subsequent tyrosine phosphorylation of cellular proteins may not be crucial for activation of S6 kinase by the insulin receptor.
authors.affiliation: Division of Diabetes and Endocrine Research, Mount Zion Hospital, San Francisco California 94120.
authors.forename: C K
authors.initials: CK
authors.lastname: Sung
authors.forename: B A
authors.initials: BA
authors.lastname: Maddux
authors.forename: D M
authors.initials: DM
authors.lastname: Hawley
authors.forename: I D
authors.initials: ID
authors.lastname: Goldfine
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D015153:Blotting, Western; D002478:Cells, Cultured; D015180:Electrophoresis, Gel, Two-Dimensional; D004789:Enzyme Activation; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D009838:Oligodeoxyribonucleotides; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000302:isolation & purification; D010766:Phosphorylation; D011494:Protein Kinases / Q000276:immunology / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011972:Receptor, Insulin / Q000235:genetics / Q000378:metabolism*; D019893:Ribosomal Protein S6 Kinases; D014162:Transfection
chemical_list: D000911:Antibodies, Monoclonal; D007328:Insulin; D009838:Oligodeoxyribonucleotides; D010748:Phosphopeptides; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin; D019893:Ribosomal Protein S6 Kinases