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The mechanisms controlling growth and cell cycle are essential for the mammalian cell. Perturbations like DNA damage result in cell cycle arrest, senescence, or apoptosis. Dysfunction of these mechanisms often give rise cancer. Involved in these processes on different levels is a protein called EAPP (E2F Associated PhosphoProtein) that we have identified and characterized. Tumor cells often overexpress EAPP, indicating that it confers a selective advantage to these cells. EAPP levels increase upon DNA damage and higher EAPP levels protect cells from apoptosis. Scrutinizing the function of EAPP is currently our main goal.
To investigate the role of EAPP we alter its level and intracellular localization in different cell types. The downregulation of EAPP is conducted by RNAi to lower its expression in a dose-dependent manner, the knock out by CRISPR-Cas9. Effects of elevated EAPP levels can be studied by creating cells allowing its inducible overexpression. EAPP is primarily nuclear but also exists in the cytoplasm. Expression of an ER-EAPP fusion protein allows the hormone-dependent control of its localization. The consequences of these interventions are studied by comparative RNA-seq, analysis of protein levels, growth characteristics and response to cellular stress.
Johann Rotheneder studied Biochemistry at the University of Vienna, Austria and obtained a PhD at the Institute of Molecular Biology in Vienna where he continued as a postdoctoral fellow. In 2002 he became tenured as an Associate Professor at the Department of Medical Biochemistry of the Medical University of Vienna.
We identified EAPP in a yeast two-hybrid screen for E2F1-interacting proteins. It also interacts with E2F2 and E2F3a, the other activating members of the E2F family of transcription factors. These interactions result in elevated E2F-dependent transcription.
Overexpression of EAPP results in cell cycle arrest. This is mediated by the cdk inhibitor p21. We could show that EAPP associates with the p21 promoter and is required for the assembly of the transcription pre-initiation complex. EAPP levels increase upon cellular stress resulting in elevated p21 which is anti-apoptotic. Lower EAPP thus facilitates, higher EAPP prevents apoptosis.
DNA double strand breaks result in rapid activation of Chk2. This kinase phosphorylates and inactivates cdc25C, a phosphatase required for entry of mitosis. After repair of the breaks Chk2 has to be inactivated to allow continuation of the cell cycle. We showed that EAPP specifically binds phosphorylated Chk2 and this results in its inactivation by the recruitment of specific phosphatases.
EAPP: gatekeeper at the crossroad of apoptosis and p21-mediated cell-cycle arrest.
Andorfer, P; Rotheneder, H
Regulation of the E2F-associated phosphoprotein promoter by GC-box binding proteins.
Schwarzmayr, Ludwig; Andorfer, Peter; Novy, Michael; Rotheneder, Hans
EAPP modulates the activity of p21 and Chk2.
Andorfer, Peter; Schwarzmayr, Ludwig; Rotheneder, Hans