Efforts to regenerate cardiac tissue by stimulating myocyte re- entry into the cell division cycle have been limited by the inability of postnatal cardiac myocytes to undergo mitosis. Co- expression of transcription factors active during G1 and S phase can induce exit from GO and DNA replication, however, these cells are blocked in G2. In lower eukaryotes, mitotic entry requires the coordinated expression of many genes, however, mechanisms controlling their transcription remain largely unknown. The characterization of transcriptional activators regulating G2/M transit would significantly advance our understanding of cell division and provide new therapeutic strategies for the regeneration of damaged myocardium. Our long-term goal is to understand the transcriptional regulation of cell division, and how such processes can be manipulated toward myocardial regeneration. Our current approach is to focus on the biology of hCdc5, a novel human phosphoprotein we cloned and demonstrated to be the first transcriptional regulator of G2/M described in mammalian cells. The proposed studies will address the mechanisms by which hCdc5 regulates the cell division cycle.
Our specific aims are to: 1) determine the role of phosphorylation and nuclear import in regulating hCdc5 function by testing nuclear localization signal mutants for nuclear translocation, identifying specific phosphorylation sites by in vitro kinase assay and phosphopeptide mapping, and testing phosphorylation mutants for subcellular localization, transcriptional activity, and effects on G2/M; 2) identify associated proteins which regulate hCdc5 activity using affinity chromatography and a yeast two-hybrid screen; and 3) identify mammalian targets of hCdc5 by selection and amplification of targets, binding site selection in yeast, and cDNA subtraction. These studies will provide basic insights into transcriptional mechanisms for G2/M control as well as reagents for manipulating the cell cycle in non-dividing tissues.
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