title: Physiological differences in insulin-like growth factor binding protein-1 (IGFBP-1) phosphorylation in IGFBP-1 transgenic mice.
abstract: Insulin-like growth factor binding protein (IGFBP)-1 has been shown to alter cellular responses to insulin-like growth factor 1 (IGF-1). Human IGFBP-1 undergoes serine phosphorylation, and this enhances both its affinity for IGF-1 by six- to eightfold and its capacity to inhibit IGF-1 actions. To investigate the physiological role of IGFBP-1 in vivo, transgenic mice have been generated using either the human IGFBP-1 or rat IGFBP-1 transgene. Both lines of mice expressed high concentrations of IGFBP-1 in serum and tissues; however, human IGFBP-1 transgenic mice did not show glucose intolerance and exhibited no significant intrauterine growth retardation, whereas rat IGFBP-1 transgenic mice showed fasting hyperglycemia and intrauterine growth restriction. The aim of this study was to investigate the physiological differences in the phosphorylation state of human IGFBP-1 and rat IGFBP-1 in these transgenic mice. The phosphorylation status of IGFBP-1 in transgenic mouse serum was analyzed by nondenaturing PAGE. Almost all of the IGFBP-1 in serum from the human IGFBP-1 transgenic mice was present as a nonphosphorylated form. Most of the rat IGFBP-1 in the serum of the mice expressing the rat IGFBP-1 was phosphorylated. Immunoprecipitation showed that mouse hepatoma (Hepa 1-6) cells (exposed to [32P]H3PO4) secrete 32P-labeled IGFBP-1. When the human IGFBP-1 transgene was transfected into Hepa 1-6 cells, all of the IGFBP-1 was secreted in the nonphosphorylated form. However, when the rat IGFBP-1 transgene was transfected into these cells, phosphorylated forms of IGFBP-1 were secreted. To confirm this result, the mouse hepatoma cell protein kinase was partially purified. This kinase activity phosphorylated mouse and rat IGFBP-1 in vitro, but it did not phosphorylate human IGFBP-1. Scatchard analysis showed that the affinity of phosphorylated rat IGFBP-1 for IGF-1 was 3.9-fold higher than that of nonphosphorylated human IGFBP-1. We conclude that the mouse IGFBP-1 kinase activity cannot phosphorylate human IGFBP-1, whereas it can phosphorylate rat IGFBP-1. The phosphorylation state of human IGFBP-1 may account for part of the phenotypic differences noted in the two studies of transgenic mice, and it is an important determinant of the capacity of human IGFBP-1 to inhibit IGF-1 actions in vivo.
authors.affiliation: Department of Medicine, University of North Carolina School of Medicine, Chapel Hill 27599-7170, USA.
authors.forename: K
authors.initials: K
authors.lastname: Sakai
authors.forename: A J
authors.initials: AJ
authors.lastname: D'Ercole
authors.forename: L J
authors.initials: LJ
authors.lastname: Murphy
authors.forename: D R
authors.initials: DR
authors.lastname: Clemmons
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D017077:Culture Media, Conditioned / Q000494:pharmacology; D006801:Humans; D018970:Insulin-Like Growth Factor Binding Protein 1 / Q000235:genetics / Q000378:metabolism*; D007334:Insulin-Like Growth Factor I / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic / Q000235:genetics; D010766:Phosphorylation; D020033:Protein Isoforms / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism; D014407:Tumor Cells, Cultured
chemical_list: D017077:Culture Media, Conditioned; D018970:Insulin-Like Growth Factor Binding Protein 1; D020033:Protein Isoforms; D007334:Insulin-Like Growth Factor I; D011494:Protein Kinases