We have recently cloned a novel protein, Ebp1, which interacts with the cytoplasmic domain of the ErbB-3 receptor. Treatment of cells with the ErbB-3 ligand, heregulin (HRG) induces the translocation of Ebp1 from the cytoplasm to the nucleus. The regulated nuclear accumulation of Ebp1 suggests that it may function as a transcriptional coregulator that is sequestered in the cytoplasm before activation similar to STAT or Smads. Overexpression of Ebp1 inhibits proliferation and induces differentiation of human breast cancer cells. Ebp1 interacts with the tumor suppressor protein, Rb and the corepressor Sin3A, and represses transcription of cell cycle-related genes. The overall aim of the current proposal is to further understand the mechanisms of Ebp l's transcriptional repression and to determine how the ability to repress transcription contributes to Ebp1's biological effects.
In Specific Aim 1 we will confirm the role of Ebp1-induced transcriptional repression in mediating its biological effects. Specific experiments include 1) determining the ability of Ebp1 to modulate the activity of endogenous promoters 2) identifying and mapping Ebp1 transcriptional repression domains 3) delineating the interactions of Ebp1 with components of histone deacetylase (HDAC) complexes 4) assessing the ability of Ebp1 to bind DNA as part of a protein complex 5) examining the contribution of Ebpl induced transcriptional repression to its biological effects 6) indentifying Ebp1 binding partners.
In specific Aim 2, we will continue to study regulation of Ebp1 function by phosphorylation. We will a) determine the regulation and subcellular localization of phosphorylated Ebp1 under different growth conditions b) map Ebp1 phosphorylation sites using SELDI technology and use this knowledge to design phosphospecific antibodies c) test the ability of Ebp1 phosphorylation mutants to regulate transcription, cell growth, and differentiation. Third, we will define the normal physiological functions of Ebp1 in a mammalian system by the creation and study of knockout mice. These experiments should contribute to an understanding of how the ability of Ebp1 to repress transcription contributes to its effects on cell growth and differentiation. The development of rational and specific therapies that target repressor complexes is providing a new direction in cancer treatment. An understanding of the mechanisms by which Ebp1 inhibits cell growth and induces differentiation may contribute to the design of new molecular targets for cancer therapy.
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