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

Departments of Internal Medicine and Physiology, University of Manitoba, Winnipeg, Canada, R3E 0W3

Address all correspondence and requests for reprints to: Liam J. Murphy, Room 843, John Buhler Research Centre, University of Manitoba 715 McDermot Avenue, Winnipeg, Canada, MB R3E 3P4. E-mail: ljmurph{at}cc.umanitoba.ca.

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 ³²P-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 ¹²⁵I-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 ¹²⁵I-IGF-I than nonphosphorylated IGFBP-3. Treatment with alkaline phosphatase significantly reduced ¹²⁵I-IGF-I binding to phosphorylated immobilized IGFBP-3 and also reduced ¹²⁵I-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.

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