Phosphorylation of Insulin-Like Growth Factor (IGF) Binding Protein-3 by Breast Cancer Cell Membranes Enhances IGF-I Binding

Abstract

Cross-linking of nonglycosylated biotinylated IGF binding protein (IGFBP)-3 to T-47D cell membranes identifies complexes with Mr of 32, 50, 70, and 100 kDa. Nonbiotinylated glycosylated IGFBP-3 competed for binding to each of these sites. The 32-kDa band approximated the size of intact nonglycosylated IGFBP-3, but its abundance was enhanced by cross-linking, and it had a more acidic isoelectric point on isoelectric focusing, suggesting that it had undergone phosphorylation. Immobilized IGFBP-3 was phosphorylated in the presence of (32)P-gamma ATP by both T-47D cell membranes and by intact cells treated with phenylarsine oxide to inhibit internalization. MCF-7 and COS-1 cells were also able to bind and phosphorylated IGFBP-3. IGF-I inhibited both IGFBP-3 binding to membranes and phosphorylation. However, incubation of T-47D cells with IGFBP-3 enhanced binding of (125)I-IGF-I to the cell monolayer indicating that membrane bound IGFBP-3 was able to bind IGF-I. Immobilized IGFBP-3 when phosphorylated by T-47D membranes bound significantly more (125)I-IGF-I than nonphosphorylated IGFBP-3. Treatment with alkaline phosphatase significantly reduced (125)I-IGF-I binding to phosphorylated immobilized IGFBP-3 and also reduced (125)I-IGF-I to T-47D cell monolayers preincubated with IGFBP-3. Phosphorylation of IGFBP-3 by T-47D membranes was partially blocked by inhibitors of both protein kinase A and C. These data demonstrate that binding of IGFBP-3 to breast cancer membranes is accompanied by phosphorylation at the plasma membrane and that both processes are inhibited by IGF-I. However, once phosphorylated the ability of IGFBP-3 to bind IGF-I is enhanced, resulting in increased association of the IGF-I with the cell membrane.

Combination Therapy of Insulin-Like Growth Factor Binding Protein-3 and Retinoid X Receptor Ligands Synergize on Prostate Cancer Cell Apoptosis In vitro and In vivo

“…IGFBP-3 is secreted and reuptaken rapidly to the nucleus via distinct endocytic pathways (7) and nuclear localization is mediated by importin-h pathway via a bipartite basic consensus sequence near its COOH terminus (27,28). Phosphorylation seems to affect its ability to bind IGF-I, but reports conflict on the nature of this effect (29,30).…”

Insulin-Like Growth Factor Binding Protein-3 Interacts with Autocrine Motility Factor/Phosphoglucose Isomerase (AMF/PGI) and Inhibits the AMF/PGI Function

“…T47D and MCF-7 cells were obtained from the American Type Tissue Collection (Manassas, Virginia). Cells were cultured as described previously (14) using culture reagents were Life Technologies, Inc. (Burlington, Ontario, Canada). Glycosylated and nonglycosylated IGFBP-3 were obtained from Upstate Biotechnology Inc. (Lake Placid, NY).…”

“…Non glycosylated Escherichia coli-derived IGFBP-3 was biotinylated as described previously (14). Biotinylated IGFBP-3 retained full biological activity in terms of binding to IGF-I and binding to cell membranes (14).…”

“…Non glycosylated Escherichia coli-derived IGFBP-3 was biotinylated as described previously (14). Biotinylated IGFBP-3 retained full biological activity in terms of binding to IGF-I and binding to cell membranes (14). Rabbit muscle-derived AMF/PGI (SigmaAldrich Canada) was dissolved in 500 l of PBS at a concentration of 1 g/l and was incubated with 7.5 l of p-biotinoyl-aminocaproic acid-N-hydroxysuccinamide ester (Roche Molecular Biochemicals, Mannheim, Germany) for 2 h at room temperature.…”

Understanding the Apoptotic Functions of IGFBP-3 in Prostate Cancer

“…Phosphorylation of these sites may affect the ability of IGFBP-3 to become glycosylated because the S111A/ S113A double mutant showed a strongly reduced glycosylation pattern (14). Phosphorylation of IGFBP-3 at the cell membrane of T-47D cells was reported to enhance IGF binding (16). IGFBP-3 can also be phosphorylated by DNA-dependent protein kinase (DNA-PK) and cyclic AMP-dependent protein kinase A (PKA) after incubation with recombinant enzyme and [g-32 P]ATP (17).…”