Phosphorylation of insulin-like growth factor (IGF) binding protein-3 by breast cancer cell membranes enhances IGF-I binding.
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.
Mishra
Suresh
S
Departments of Internal Medicine and Physiology, University of Manitoba, Winnipeg, Canada, R3E 0W3.
Murphy
Liam J
LJ
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D001943:Breast Neoplasms; D019556:COS Cells; D002462:Cell Membrane / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli; D006031:Glycosylation; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000378:metabolism*; D007334:Insulin-Like Growth Factor I / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism; D014407:Tumor Cells, Cultured
D004791:Enzyme Inhibitors; D018972:Insulin-Like Growth Factor Binding Protein 3; D047428:Protein Kinase Inhibitors; D007334:Insulin-Like Growth Factor I; D011494:Protein Kinases; D000469:Alkaline Phosphatase