Phosphorylation of IGFBP-1 at Discrete Sites Elicits Variable Effects on IGF-I Receptor Autophosphorylation

We previously demonstrated that hypoxia and leucine deprivation cause hyperphosphorylation of IGF-binding protein-1 (IGFBP-1) at discrete sites that markedly enhanced IGF-I affinity and inhibited IGF-I-stimulated cell growth. In this study we investigated the functional role of these phosphorylation sites using mutagenesis. We created three IGFBP-1 mutants in which individual serine (S119/S169/S98) residues were substituted with alanine and S101A was recreated for comparison. The wild-type (WT) and mutant IGFBP-1 were expressed in Chinese hamster ovary cells and IGFBP-1 in cell media was isolated using isoelectric-focusing-free-flow electrophoresis. BIACore analysis indicated that the changes in IGF-I affinity for S98A and S169A were moderate, whereas S119A greatly reduced the affinity of IGFBP-1 for IGF-I (100-fold, P < .0001). Similar results were obtained with S101A. The IGF-I affinity changes of the mutants were reflected in their ability to inhibit IGF-I-induced receptor autophosphorylation. Employin...