Characterization of insulin-like growth factor binding protein-1 kinases from human hepatoma cells.
The phosphorylation of insulin-like growth factor binding protein-I (IGFBP-1) alters its binding affinity for insulin-like growth factor I (IGF-I) and thus regulates the bioavailability of IGF-I for binding to the IGF-I receptor. The kinase(s) responsible for the phosphorylation of IGFBP-1 has not been identified. This study was designed to characterize the IGFBP-1 kinase activity in HepG2 human hepatoma cells, a cell line that secretes IGFBP-1 primarily as phosphorylated isoforms. IGFBP-1 kinase activity was partially purified from detergent extracts of the cells by phosphocellulose chromatography and gel filtration. Two kinases of approximate M(r) 150,000 (peak I kinase) and M(r) 50,000 (peak II kinase) were identified. Each kinase phosphorylated IGFBP-1 at serine residues that were phosphorylated by intact HepG2 cells. The kinases were distinct based on their differential sensitivity to inhibition by heparin (IC50 = 2.5 and 16.5 micrograms/ml, peak I and II kinase, respectively) and inhibition by the isoquinoline sulfonamide CKI-7 (IC50 = 50 microM and 100 microM, peak I and II kinase, respectively). In addition, a tenfold molar excess of nonradioactive GTP relative to [gamma-32P]ATP lowered the incorporation of 32P into IGFBP-1 by 80% when the reaction was catalyzed by the peak I kinase, whereas GTP had no effect on the reaction catalyzed by the peak II kinase. In the presence of polylysine, IGFBP-1 was radiolabeled by the partially purified kinase activity when [gamma-32P]GTP served as the phosphate donor indicating the presence of casein kinase II activity. Furthermore, IGFBP-1 was phosphorylated by purified casein kinase I and casein kinase II at sites phosphorylated by the peak I and II kinases. Our data suggest that at least two kinases could be responsible for the phosphorylation of IGFBP-1 in intact HepG2 cells and that the kinases are related to the casein kinase family of protein kinases.
Ankrapp
D P
DP
Department of Medicine, University of North Carolina, Chapel Hill 27599, USA.
Jones
J I
JI
Clemmons
D R
DR
D000595:Amino Acid Sequence; D006528:Carcinoma, Hepatocellular / Q000201:enzymology*; D047390:Casein Kinase II; D047388:Casein Kinases; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000378:metabolism; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D018970:Insulin-Like Growth Factor Binding Protein 1 / Q000378:metabolism*; D007546:Isoquinolines / Q000494:pharmacology; D008113:Liver Neoplasms / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010449:Peptide Mapping; D010766:Phosphorylation; D010768:Phosphoserine / Q000032:analysis; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D004791:Enzyme Inhibitors; D018970:Insulin-Like Growth Factor Binding Protein 1; D007546:Isoquinolines; C058815:N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide; D010768:Phosphoserine; D006160:Guanosine Triphosphate; D006493:Heparin; D011494:Protein Kinases; D047390:Casein Kinase II; D047388:Casein Kinases; D017346:Protein Serine-Threonine Kinases