Characterization of insulin-like growth factor binding protein-1 kinases from human hepatoma cells

Abstract

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.

Lithium chloride inhibits the expression and secretion of insulin-like growth factor-binding protein-1

“…Lithium has an effect, albeit less potently, on protein kinases other than GSK3 (Davies et al, 2000), including casein kinase 2, which can phosphorylate IGFBP-1 (Ankrapp et al, 1996). In Figure 3(inset) we show that the pattern of isoforms of IGFBP-1 seen on immunoblotting after native PAGE are not changed by 10 mM lithium chloride.…”

The in Vivo Phosphorylation and Glycosylation of Human Insulin-like Growth Factor-binding Protein-5

“…Because both IGFBP-1 and IGFBP-3 are phosphorylated at central domain residues close to the cysteine-rich N-terminal domain, and the kinases involved were considered likely to be CK1, CK2, or a related enzyme (26,31), we also considered whether CK2 might phosphorylate IGFBP-5. Ser 96 was predicted as a potential CK2 phosphorylation site by the kinase specificity prediction program NetPhosK 1.0, and we observed that DRB, a specific inhibitor of CK2, reduced 32 P incorporation into IGFBP-5 in cellular metabolic labeling experiments.…”

The role and regulation of IGFBP-1 phosphorylation in fetal growth restriction

“…1991;Koistinen et al 1993a, b). CK2 modulates IGF-I activity in vitro based on the finding that it phosphorylates IGFBP-1 in HepG2 (Ankrapp et al 1996) and decidual cells (Frost and Tseng. 1991 LC-MS analysis of IGFBP-1 phosphopepƟde peak intensity raƟos calculated from the relaƟve phosphopepƟde peak intensiƟes between the control and FGR groups N-terminal C-terminal Linker region P P P P Ser101 S119 S169…”