PHOSPHORYLATION OF RAT INSULIN-LIKE-GROWTH-FACTOR BINDING PROTEIN-1 DOES NOT AFFECT ITS BIOLOGICAL PROPERTIES

Insulin-like growth factors (IGFs) I and II stimulate growth and expre ssion of specific genes through binding to cell membrane receptors. IG F binding proteins also bind IGF-1 with higher affinity than the recep tor, They are found in the circulation and tissues and can modulate IG F actions. Human IGFBP-1 is phosphorylated on serine residues, which i ncreases its affinity for IGF-1. An acidic, presumably phosphorylated, form of human IGFBP-1 inhibits IGF-1-stimulated DNA synthesis in cult ured cells, while a less acidic, unphosphorylated form potentiates thi s function, Phosphorylation of human IGFBP-3, however, does not affect its affinity for IGF-1. Previously we found that multiple forms of ra t IGFBP-1 are obtained by anion-exchange chromatography, raising the p ossibility that it also is phosphorylated, which led us to examine its properties. Phosphopeptide analysis of P-32-labeled, immunoprecipitat ed rat IGFBP-1 synthesized by H-4-II-EC3 rat hepatoma cells indicated that it is phosphorylated on two sites that were deduced to be ser107 and ser132 in the central nonconserved domain. Dephosphorylation of pu rified phosphorylated rat IGFBP-1 did not affect its affinity for IGF- 1 or its specific binding activity, and the dephosphorylated form inhi bited DNA synthesis in 3T3 cells. Incubation of cells labeled with rad ioactive proline in the presence of monensin and brefeldin A, which in hibit secretion at different sites, led to intracellular accumulation of the least phosphorylated form of rat IGFBP-1, but prevented further phosphorylation. The results suggested that phosphorylation occurs at two sites in cells, the cis-Golgi and the trans-Gels network. In summ ary, these studies have shown that rat IGFBP-1 is phosphorylated on tw o sites by reactions that occur in different secretory organelles and that similar to human IGFBP-3, but unlike human IGFBP-1, phosphorylati on does not affect its affinity for IGF-1. (C) 1998 Academic Press.