Insulin-Stimulated Phosphorylation of Recombinant pp120/HA4, an Endogenous Substrate of the Insulin Receptor Tyrosine Kinase

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...

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Bibliographic Details
Published in:	Biochemistry (Easton) Vol. 34; no. 29; pp. 9341 - 9349
Main Authors:	Najjar, Sonia M, Philippe, Neuberet, Suzuki, Yoshifumi, Ignacio, Gladys A, Formisano, Pietro, Accili, Domenico, Taylor, Simeon I
Format:	Journal Article
Language:	English
Published:	United States American Chemical Society 25-07-1995
Subjects:	3T3 Cells Adenosine Triphosphatases - metabolism Alanine Amino Acid Sequence Animals Base Sequence Cell Membrane - metabolism DNA Primers Focal Adhesion Kinase 1 Focal Adhesion Protein-Tyrosine Kinases Humans Insulin - pharmacology Liver - metabolism Macromolecular Substances Membrane Glycoproteins - metabolism Mice Molecular Sequence Data Mutagenesis, Site-Directed Phenylalanine Phosphorylation Point Mutation Polymerase Chain Reaction Protein-Tyrosine Kinases - isolation & purification Protein-Tyrosine Kinases - metabolism Receptor, Insulin - biosynthesis Receptor, Insulin - metabolism Recombinant Fusion Proteins - biosynthesis Recombinant Proteins - biosynthesis Recombinant Proteins - metabolism Substrate Specificity Transfection
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Summary:	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.
Bibliography:	ark:/67375/TPS-FQ8N1FBJ-7 istex:4A15398B2E15AB9316CA0A6A921C7EF4A06CCD5D
ISSN:	0006-2960 1520-4995
DOI:	10.1021/bi00029a009