Insulin-like growth factor-binding protein-1 is phosphorylated by cultured human endometrial stromal cells and multiple protein kinases in vitro.
The insulin-like growth factor-binding protein IGF-BP1 is a major secretory protein of human endometrial stromal cells decidualized in culture. Anion exchange chromatography and nondenaturing gel electrophoresis showed IGF-BP1 to exist in five electrophoretically and chromatographically distinct isoforms. IGF-BP1 variants migrated as a quintet on nondenaturing polyacrylamide gels and as a single band (28 kDa) on sodium dodecyl sulfate-polyacrylamide gels. Alkaline phosphatase treatment reduced the IGF-BP1 variants to a single band. Cells incubated with [32P]orthophosphate for 12 h secreted four 32P-labeled IGF-BP1 phosphovariants, and their migration coincided with those bands that were eliminated by alkaline phosphatase treatment. In cells treated with medroxyprogesterone acetate and relaxin, the concentration of phosphorylated IGF-BP1 was increased dramatically as compared with controls. All the phosphovariants were confirmed to be IGF-BP1 by their ability to be supershifted on nondenaturing polyacrylamide gels after binding a monoclonal antibody to IGF-BP1. Thin layer electrophoresis of IGF-BP1 acid hydrolysates showed IGF-BP1 to be phosphorylated exclusively on serine. Non-phosphorylated IGF-BP1 was phosphorylated by the catalytic subunit of the cAMP-dependent protein kinase and casein kinase II in vitro. This suggests that IGF-BP1 may be a substrate of multiple protein kinases in vivo.
Frost
R A
RA
Department of Obstetrics and Gynecology, State University of New York, Stony Brook 11794.
Tseng
L
L
D000328:Adult; D000469:Alkaline Phosphatase / Q000378:metabolism; D047388:Casein Kinases; D002478:Cells, Cultured; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004717:Endometrium / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D005260:Female; D006801:Humans; D008875:Middle Aged; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism*; D017451:Receptors, Somatomedin; D013379:Substrate Specificity
D011956:Receptors, Cell Surface; D017451:Receptors, Somatomedin; D011494:Protein Kinases; D047388:Casein Kinases; D000469:Alkaline Phosphatase