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This version published online on May 15, 2003
Endocrinology, doi:10.1210/en.2003-0089
A more recent version of this article appeared on September 1, 2003
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Submitted on January 17, 2003
Accepted on May 7, 2003

Phosphorylation of Insulin-like Growth Factor Binding Protein-3 by Breast Cancer Cells Membranes Enhances IGF-I Binding

Suresh Mishra1 and Liam J. Murphy1*

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

* To whom correspondence should be addressed. E-mail: ljmurph{at}cc.umanitoba.ca.

Cross-linking of non-glycosylated biotinylated IGFBP-3 to T-47D cell membranes identifies complexes with Mr of 32, 50, 70 and 100 kDa. Non-biotinylated glycosylated IGFBP-3 competed for binding to each of these sites. The 32 kDa band, approximated the size of intact non-glycosylated IGFBP-3, but its abundance was enhanced by cross-linking and it had a more acidic pI on isoelectric focusing suggesting that it had undergone phosphorylation. Immobilized IGFBP-3 was phosphorylated in the presence of 32P-{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 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 non-phosphorylated 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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