title: Insulin-stimulated phosphorylation of recombinant pp120/HA4, an endogenous substrate of the insulin receptor tyrosine kinase.
abstract: Insulin binding to the alpha-subunit of its receptor stimulates the receptor tyrosine kinase to phosphorylate the beta-subunit and several endogenous protein substrates, including pp120/HA4, a liver-specific plasma membrane glycoprotein of M(r) 20,000. Analysis of the deduced amino acid sequence of rat liver pp120/HA4 revealed two potential sites for tyrosine phosphorylation in the cytoplasmic domain (Tyr488 and Tyr513), as well as a potential cAMP-dependent protein kinase phosphorylation site (Ser503). To determine which of these sites is phosphorylated in response to insulin, each of these amino acid residues was altered by site-directed mutagenesis. Mutant cDNAs were then expressed by stable transfection in NIH 3T3 cells. Two mutations (Phe488 and Ala503) impaired insulin-induced phosphorylation of pp120/HA4, suggesting that pp120/HA4 undergoes multisite phosphorylation. It seems likely that Tyr488 is phosphorylated by the insulin receptor kinase, and phosphorylation of Ser513 may contribute to the regulation of tyrosine phosphorylation. Since pp120/HA4 is believed to be associated with a Ca2+/Mg(2+)-dependent ecto-ATPase activity, we determined the effects of insulin-induced phosphorylation on this enzymatic activity. In NIH 3T3 cells co-expressing the insulin receptor and pp120/HA4, insulin caused a 2-fold increase in ecto-ATPase activity. Moreover, elimination of the phosphorylation sites of pp120/HA4 impaired the ability of insulin to stimulate the ecto-ATPase activity. These data suggest that tyrosine phosphorylation of pp120/HA4 may regulate Ca2+/Mg(2+)-dependent ecto-ATPase activity.
authors.affiliation: Diabetes Branch, National Institute of Diabetes and Digestive and Kidney Disease, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: S M
authors.initials: SM
authors.lastname: Najjar
authors.forename: N
authors.initials: N
authors.lastname: Philippe
authors.forename: Y
authors.initials: Y
authors.lastname: Suzuki
authors.forename: G A
authors.initials: GA
authors.lastname: Ignacio
authors.forename: P
authors.initials: P
authors.lastname: Formisano
authors.forename: D
authors.initials: D
authors.lastname: Accili
authors.forename: S I
authors.initials: SI
authors.lastname: Taylor
mesh_terms: D016475:3T3 Cells; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000409:Alanine; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism; D017931:DNA Primers; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D008099:Liver / Q000378:metabolism*; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine; D010766:Phosphorylation; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011505:Protein-Tyrosine Kinases / Q000302:isolation & purification / Q000378:metabolism*; D011972:Receptor, Insulin / Q000096:biosynthesis / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D013379:Substrate Specificity; D014162:Transfection
chemical_list: D017931:DNA Primers; D007328:Insulin; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D010649:Phenylalanine; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; C496051:Ptk2 protein, mouse; D000251:Adenosine Triphosphatases; C027201:ectoATPase; D000409:Alanine