PHOSPHORYLATION OF INSULIN-LIKE GROWTH-FACTOR (IGF)-BINDING PROTEIN-1 IN CELL-CULTURE AND INVIVO - EFFECTS ON AFFINITY FOR IGF-I
PHOSPHORYLATION OF INSULIN-LIKE GROWTH-FACTOR (IGF)-BINDING PROTEIN-1 IN CELL-CULTURE AND INVIVO - EFFECTS ON AFFINITY FOR IGF-I
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DOI：
10.1073/pnas.88.17.7481
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发表时间：
1991-09-01
影响因子：
11.1
通讯作者：
CLEMMONS, DR
中科院分区：
文献类型：
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作者：
JONES, JI;DERCOLE, AJ;CLEMMONS, DR
胰岛素样生长因子（IGF-I和IGF-II）存在于细胞外液中，与特异性IGF结合蛋白（IGFBPs）结合。我们和其他人已经报道了具有相同氨基酸序列和分子大小的IGFBP-1的不同制剂的不同生物活性。这一观察结果促使我们确定IGFBP-1是否经历翻译后修饰。免疫沉淀法用于显示中国仓鼠卵巢细胞（用人IGFBP-1 cDNA构建体转染）和人肝癌（HepG 2）细胞在与[P-32]正磷酸盐孵育后分泌P-32标记的IGFBP-1。P-32标记的IGFBP-1的磷酸化氨基酸分析仅显示磷酸丝氨酸残基。开发了一种可以将非磷酸化IGFBP-1与四种或五种磷酸化异构体分离的方法。使用这种技术，我们证明，人羊水和人胎血清含有大比例的非磷酸化IGFBP-1，以及磷酸化形式。相反，HepG 2细胞和人蜕膜细胞主要分泌磷酸化亚型。这些观察结果表明IGFBP-1作为磷蛋白分泌，随后在体内被去磷酸化。结合研究表明，HepG 2细胞分泌的磷酸化IGFBP-1对IGF-I的亲和力比去磷酸化后高6倍。我们的结论是IGFBP-1是磷酸化的，这种磷酸化是一种生理上重要的翻译后修饰。
The insulin-like growth factors (IGF-I and IGF-II) are present in extracellular fluids bound to specific IGF-binding proteins (IGFBPs). We and others have reported varying biologic activity of different preparations of IGFBP-1 that appeared to have identical amino acid sequences and molecular sizes. This observation prompted us to determine whether IGFBP-1 undergoes posttranslational modifications. Immunoprecipitation was used to show that Chinese hamster ovary cells (transfected with a human IGFBP-1 cDNA construct) and human hepatoma (HepG2) cells secrete P-32-labeled IGFBP-1 following incubation with [P-32]orthophosphate. Phospho amino acid analysis of P-32-labeled IGFBP-1 revealed only phosphoserine residues. A method was developed that could separate nonphosphorylated IGFBP-1 from four or five phosphorylated isoforms. Using this technique we demonstrated that human amniotic fluid and human fetal serum contain a large proportion of nonphosphorylated IGFBP-1, as well as phosphorylated forms. In contrast, HepG2 cells and human decidual cells secrete predominantly the phosphorylated isoforms. These observations suggest that IGFBP-1 is secreted as a phosphoprotein and is subsequently dephosphorylated in vivo. Binding studies showed that the phosphorylated IGFBP-1 secreted by HepG2 cells has a 6-fold higher affinity for IGF-I than it does after dephosphorylation. We conclude that IGFBP-1 is phosphorylated and that this phosphorylation is a physiologically important posttranslational modification.