The in vivo phosphorylation and glycosylation of human insulin-like growth factor-binding protein-5.
Mass spectrometry is often used to determine post-translational modifications by analysis of tryptic digests of proteins. Here we demonstrate that the analysis of tryptic peptides together with analysis of the full-length protein provided optimal characterization of insulin-like growth factor-binding protein-5 (IGFBP-5) phosphorylation and glycosylation. IGFBP-5 binds insulin-like growth factors with high affinity and has important roles in cell survival, differentiation, and apoptosis. Until now, the primary structure of IGFBP-5 has been incompletely defined. We analyzed human IGFBP-5 from T47D cells by mass spectrometry to determine all of the in vivo post-translational modifications. In full-length IGFBP-5, 31% of the protein was unmodified, 37% was monophosphorylated, and 4% was diphosphorylated with no other modification. The remaining 27% was glycosylated, more than half of which was also monophosphorylated. The major phosphorylation site was Ser(96) in the central domain, and a minor phosphorylation site was Ser(248) near the C terminus. Neither site was phosphorylated in vitro by casein kinase 2, ruling it out as the in vivo kinase. An in vivo phosphorylation site was also found in IGFBP-2 at an analogous position, Ser(106). IGFBP-5 was heterogeneously O-glycosylated mainly by sialylated core 1 type glycans. The most abundant structure contained N-acetylhexosamine, hexose, and two N-acetylneuraminic acid carbohydrates. A small amount of sialylated core 2 type glycan was also present. Phosphorylation and O-glycosylation both affected IGFBP-5 binding to heparin but not insulin-like growth factor binding or ternary complex formation with the acid-labile subunit. The results reveal the first description of the in vivo phosphorylation of IGFBP-5 and its glycan composition.
Graham
Mark E
ME
Cell Signalling Unit, Children's Medical Research Institute, Locked Bag 23, Wentworthville, New South Wales 2145, Australia.
Kilby
Dean M
DM
Firth
Sue M
SM
Robinson
Phillip J
PJ
Baxter
Robert C
RC
D000595:Amino Acid Sequence; D047390:Casein Kinase II / Q000378:metabolism; D045744:Cell Line, Tumor; D006031:Glycosylation; D006493:Heparin / Q000378:metabolism; D006801:Humans; D018975:Insulin-Like Growth Factor Binding Protein 5 / Q000378:metabolism*; D007334:Insulin-Like Growth Factor I / Q000378:metabolism; D007335:Insulin-Like Growth Factor II / Q000378:metabolism; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011134:Polysaccharides / Q000378:metabolism*; D011499:Protein Processing, Post-Translational
D018975:Insulin-Like Growth Factor Binding Protein 5; D011134:Polysaccharides; D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II; D006493:Heparin; D047390:Casein Kinase II