Physiological Differences in Insulin-Like Growth Factor Binding Protein-1 (IGFBP-1) Phosphorylation in IGFBP-1 Transgenic Mice

Physiological Differences in Insulin-Like Growth Factor Binding Protein-1 (IGFBP-1) Phosphorylation in IGFBP-1 Transgenic Mice Keiji Sakai , A. Joseph D'Ercole , Liam J. Murphy and David R. Clemmons From the Departments of Medicine (K.S., D.R.C.) and Pediatrics (A.J.D.E.), University of North Carolina School of Medicine, Chapel Hill, NC; and the Department of Medicine (L.J.M.), University of Manitoba, Winnipeg, Manitoba, Canada. Address correspondence and reprint requests to David R. Clemmons, MD, Building 7170, 6111 Thurston Bowles, School of Medicine, Division of Endocrinology, University of North Carolina, Chapel Hill, NC 27599-7170. E-mail: endo{at}med.unc.edu . 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 [ 32 P]H 3 PO 4 ) secrete 32 P-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. Scatc