CELL CYCLE REGULATION IN CARDIOVASCULAR BIOLOGY

通过刺激心肌细胞重新进入细胞分裂周期来再生心肌组织的努力受到出生后心肌细胞无法进行有丝分裂的限制。在G1期和S期激活的转录因子共表达可诱导GO退出和DNA复制，但这些细胞在G2期被阻断。在低等真核生物中，有丝分裂的进入需要许多基因的协调表达，然而，控制它们转录的机制在很大程度上仍然不清楚。调控G2/M转导的转录激活子的特征将极大地促进我们对细胞分裂的理解，并为受损心肌的再生提供新的治疗策略。我们的长期目标是了解细胞分裂的转录调控，以及这种过程如何被操纵以促进心肌再生。我们目前的方法是专注于hCDC5的生物学，这是我们克隆的一种新的人类磷酸蛋白，并证明了它是第一个在哺乳动物细胞中描述的G2/M转录调节因子。拟议的研究将探讨hCDC5调节细胞分裂周期的机制。我们的具体目标是：1)通过测试核定位信号突变来确定hCDC5在核转位中的作用，通过体外激酶试验和磷酸肽定位来确定特定的磷酸化位点，以及测试磷酸化突变的亚细胞定位、转录活性和对G2/M的影响；2)通过亲和层析和酵母双杂交筛选来确定调节hCDC5活性的相关蛋白；以及3)通过靶标的选择和扩增、酵母结合位点的选择和cDNA消减来鉴定hCDC5的哺乳动物靶标。这些研究将为G2/M调控的转录机制以及在未分裂组织中操纵细胞周期的试剂提供基本的见解。

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.