Children’s Health Research Institute, Munich, Germany.
Endocrinology. 2013 Mar;154(3):1130-43. doi: 10.1210/en.2012-1962. Epub 2013 Jan 25.
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.
我们之前的研究表明，缺氧和亮氨酸缺乏会导致 IGF 结合蛋白-1（IGFBP-1）在离散位点发生过度磷酸化，从而显著增强 IGF-I 的亲和力并抑制 IGF-I 刺激的细胞生长。在这项研究中，我们使用突变来研究这些磷酸化位点的功能作用。我们创建了三个 IGFBP-1 突变体，其中单个丝氨酸（S119/S169/S98）残基被丙氨酸取代，并且重新创建了 S101A 用于比较。野生型（WT）和突变 IGFBP-1 在中华仓鼠卵巢细胞中表达，并使用等电聚焦无流电泳分离细胞培养基中的 IGFBP-1。BIACore 分析表明，S98A 和 S169A 的 IGF-I 亲和力变化适中，而 S119A 则大大降低了 IGFBP-1 对 IGF-I 的亲和力（100 倍，P <.0001）。S101A 也得到了类似的结果。突变体的 IGF-I 亲和力变化反映在它们抑制 IGF-I 诱导的受体自动磷酸化的能力上。使用 IGF-IR 过表达 P6 细胞的受体刺激测定，我们发现 WT-IGFBP-1 抑制 IGF-IRβ 自动磷酸化（~2 倍，P <.001），可能归因于 IGF-I 的隔离。与 WT 相比，S98A 和 S169A 突变体不抑制受体自动磷酸化。另一方面，S119A 则极大地刺激了受体（2.3 倍，P <.05）。S101A 的数据与 S119A 匹配。总之，我们表明 S98 和 S169 的磷酸化导致 IGF-I 作用的变化较小；尽管如此，对 IGF-I 生物学活性的最显著抑制作用归因于 IGFBP-1 在 S119 处的磷酸化。我们的研究结果提供了新的证据表明，IGFBP-1 在 S119 处的磷酸化可以增强其与 IGF-I 的亲和力，可能是通过 IGF-IGFBP-1 复合物的稳定。这些数据还表明，不同磷酸化位点的协同相互作用可能在对 IGF-I 亲和力产生更明显影响方面很重要，需要进一步研究。
