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. Employing receptor-stimulation assay using IGF-IR-overexpressing P6 cells, we found that WT-IGFBP-1 inhibited IGF-IRβ autophosphorylation (~2-fold, P < .001), possibly attributable to sequestration of IGF-I. Relative to WT, S98A and S169A mutants did not inhibit receptor autophosphorylation. S119A, on the other hand, greatly stimulated the receptor (2.3-fold, P < .05). The data with S101A matched S119A. In summary, we show that phosphorylation at S98 and S169 resulted in milder changes in IGF-I action; nonetheless most dramatic inhibitory effects on the biological activity of IGF-I were due to IGFBP-1 phosphorylation at S119. Our results provide novel demonstration that IGFBP-1 phosphorylation at S119 can enhance affinity for IGF-I possibly through stabilization of the IGF-IGFBP-1 complex. These data also propose that the synergistic interaction of distinct phosphorylation sites may be important in eliciting more pronounced effects on IGF-I affinity that needs further investigation.
Children’s Health Research Institute, Munich, Germany.
Majida
M
Abu Shehab
Cristiana
C
Iosef
Robert
R
Wildgruber
Girish
G
Sardana
Madhulika B
MB
Gupta
D019943:Amino Acid Substitution; D000818:Animals; D041702:BALB 3T3 Cells; D001665:Binding Sites / Q000235:genetics; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D018970:Insulin-Like Growth Factor Binding Protein 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D017526:Receptor, IGF Type 1 / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
C032231:IGFBP1 protein, human; D018970:Insulin-Like Growth Factor Binding Protein 1; D011994:Recombinant Proteins; D017526:Receptor, IGF Type 1