PHOSPHORYLATION OF INSULIN-LIKE GROWTH-FACTOR (IGF)-BINDING PROTEIN-1 IN CELL-CULTURE AND INVIVO - EFFECTS ON AFFINITY FOR IGF-I

被引:293
作者
JONES, JI
DERCOLE, AJ
CAMACHOHUBNER, C
CLEMMONS, DR
机构
[1] UNIV N CAROLINA,SCH MED,DEPT PEDIAT,CHAPEL HILL,NC 27599
[2] UNIV N CAROLINA,SCH MED,DEPT MED,CHAPEL HILL,NC 27599
关键词
CHINESE HAMSTER OVARY CELLS; HEPATOMA CELLS; DECIDUAL CELLS; AMNIOTIC FLUID; NON-SDS POLYACRYLAMIDE GEL ELECTROPHORESIS;
D O I
10.1073/pnas.88.17.7481
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
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.
引用
收藏
页码:7481 / 7485
页数:5
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