Phosphorylation of insulin-like growth factor (IGF)-binding protein 1 in cell culture and in vivo: effects on affinity for IGF-I.
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 32P-labeled IGFBP-1 following incubation with [32P]orthophosphate. Phospho amino acid analysis of 32P-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.
Jones
J I
JI
Department of Medicine, University of North Carolina School of Medicine, Chapel Hill 27599-7170.
D'Ercole
A J
AJ
Camacho-Hubner
C
C
Clemmons
D R
DR
D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D015151:Immunoblotting; D018969:Insulin-Like Growth Factor Binding Proteins; D007334:Insulin-Like Growth Factor I / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D010766:Phosphorylation; D011485:Protein Binding; D014162:Transfection
D002352:Carrier Proteins; D018969:Insulin-Like Growth Factor Binding Proteins; D007334:Insulin-Like Growth Factor I