Ebp1-mediated inhibition of cell growth requires serine 363 phosphorylation

Ebpl is an ErbB3 binding phosphoprotein with pleiotropic effects. Overexpression of Ebpl represses transcription of E2F1 responsive cell cycle regulated genes and inhibits cell growth. However, the effect of phosphorylation on Ebpl-mediated transcriptional repression and cell growth inhibition is currently unknown. In this study, we show that serine 363 (S363) of Ebpl is phosphorylated in vivo. Although total Ebpl is located in the nucleus, organelles and the cytoplasm, Ebpl phosphorylated at S363 (Ebpl pS363) is localized exclusively to the nucleus. Mutation of S363 to alanine did not change the subcellular localization of Ebp1. However, the S363A mutation significantly decreased the ability of Ebpl to repress transcription and abrogated its ability to inhibit cell growth. We have previously shown that Ebp1 can bind the E2F1 promoter in vitro and in vivo as part of a protein complex and that Ebpl-transcriptional repression is mediated via its interaction with the co-repressors HDAC2 and mSin3a present in this complex. Although Ebpl S363A interacted with an E2F1 promoter element, it did not bind HDAC2 and mSin3a. These results indicate the importance of S363 phosphorylation in the function of Ebpl.