Identification of the sites of phosphorylation in insulin-like growth factor binding protein-1. Regulation of its affinity by phosphorylation of serine 101.
Serine phosphorylation of insulin-like growth factor binding protein-1 (IGFBP-1) has been shown to alter its affinity for the insulin-like growth factors (IGF-I and IGF-II) and to modify its capacity to modulate cellular responses to the IGFs. Because of this, we determined the sites of serine phosphorylation. Purification of 32P-labeled IGFBP-1 was followed by digestion with trypsin and endoproteinase Glu-C and radiosequencing of labeled peptides. Three serines were found to be phosphorylated, with Ser101, Ser119, and Ser169 containing 70%, 5%, and 25% of the incorporated 32P, respectively. A mutated IGFBP-1, substituting alanine for serine at positions 98 and 101, was expressed in CHO cells. On nondenaturing gels, the wild type protein migrated as five isoforms (one non-phosphorylated and four phosphorylated). However, in the mutated protein, the most rapidly migrating band (a phosphorylated form) was not present. The cells containing the mutated cDNA incorporated 60% less 32P into immunoprecipitable IGFBP-1. The mutated protein had a 3-fold reduction in affinity for IGF-I compared to the wild type protein. We conclude that Ser101 represents the major site of phosphorylation containing 63% of the total 32P incorporated and that phosphorylation of Ser101 is important for maintenance of high affinity binding for this growth factor.
Jones
J I
JI
Department of Medicine, University of North Carolina, Chapel Hill 27599.
Busby
W H
WH
Wright
G
G
Smith
C E
CE
Kimack
N M
NM
Clemmons
D R
DR
D000409:Alanine; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D013006:Growth Hormone / Q000235:genetics; D006801:Humans; D018970:Insulin-Like Growth Factor Binding Protein 1; D007334:Insulin-Like Growth Factor I / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010710:Phosphates / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012694:Serine; D014162:Transfection
D002352:Carrier Proteins; D018970:Insulin-Like Growth Factor Binding Protein 1; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D010710:Phosphates; D010761:Phosphorus Radioisotopes; D011994:Recombinant Proteins; D012694:Serine; D007334:Insulin-Like Growth Factor I; D013006:Growth Hormone; D000409:Alanine