Ebp1, an ErbB-3 binding protein, interacts with Rb and affects Rb transcriptional regulation

Ebp1, an ErbB-3 binding protein, inhibits the proliferation and induces the differentiation of human breast cancer cells. The mechanisms of these effects are unknown. Rb, the product of the retinoblastoma gene, is an important modulator of cell cycle progression and cellular differentiation. We report that Rb is a binding target for Ebp1. Ebp1 was localized to both the nucleus and the cytoplasm of logarithmically growing AU565 breast cancer cells and HeLa cells as determined by confocal immunofluorescent microscopy. Ebp1 was present in Rb immunoprecipitates derived from AU565 breast cancer cells. GST-Rb also bound endogenous Ebp1. Using GST-Ebp1 constructs, we determined that the 72 C-terminal amino acids of Ebp1 were sufficient to bind Rb. Dephosphorylation of Ebp1 enhanced the interaction of Ebp1 with Rb. The overexpression of Ebp1 in MCF-7 and AU565 (Rb(+)) cells inhibited the activity of the E2F1 regulated cyclin-E promoter. Ebp1 bound E2F1 indirectly via Rb in lysates of MCF-7 cells. The interaction of Ebp1 with Rb may prove to be an important mechanism of Ebp1 induced changes in cell proliferation and differentiation.