The C/EBP family of bZIP proteins plays a significant role in the regulation of a variety of biological processes. A member of this family, liver specific transcription factor CCAAT/IEnhancer Binding Protein a (C/EBPa), is highly expressed in differentiated cells and is a strong inhibitor of cell proliferation. Levels of C/EBPa are reduced when liver starts to proliferate after partial hepatectomy or during development of hepatocarcinomas. Elucidation of molecular mechanisms of C/EBPa mediated growth arrest is important for the understanding of liver pathology. Our data clearly demonstrate that C/EBPa mediated growth arrest does not require transcriptional activity of C/EBPa and occurs on the level of protein:protein interactions. We recently found that CEBPa is able to interact with two key kinases that drive the cell cycle: cdk2 and cdk4. This interaction leads to inhibition of cdk2 and cdk4 kinase activity. In addition, C/EBPa interacts with p107 and disrupts S-phase specific p107-E2F-cdk2-cyclin A complex. It has been recently shown that another transcription factor, muscle specific protein MyoD, causes growth arrest via direct inhibition of cdk4. Therefore, the major hypothesis of this application is that tissue specific transcription factors (such as C/EBPat) bring about growth arrest through direct interaction with cell cycle proteins. This hypothesis will be examined by investigating C/EBPa mediated growth arrest. In this application, we will a)- examine the role of C/EBPa interaction with cdk4 and cdk2 in growth arrest, b) - define the role of C/EBPa-p 107 interaction in regulation of E2F transcription and c) determine the mechanisms that regulate interaction of C/EBPa with cell cycle proteins. A variety of biological systems will be used for these studies including animal models and stable clones containing C/EBPa and its mutant forms under inducible promoter. In addition, we found that C/EBPa forms a high molecular weight complex with Rb in old, but not in young animals. Using this biological model, we will test the hypothesis that post-translational modifications of C/EBPa regulate the interaction of C/EBPa with cell cycle proteins.
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