Cyclin-dependent Kinases and Cardiac Growth

描述(由申请人提供)：这是申请人对细胞周期蛋白依赖性蛋白激酶(CDK)及其在心脏生长中的功能作用的研究的竞争性更新。 肥大的生长是心脏病死亡率的风险因素。仍然缺乏信息来解释每个细胞的RNA和蛋白质的全球增长。肥大的信号导致RNA聚合酶II(RNAPII)羧基末端结构域(CTD)的磷酸化，这在其他生物环境中是转录延长所必需的。CTD激酶包括两个基础转录因子TFIIH(细胞周期蛋白HCdk7)和正转录延伸因子b(P-TEFb，细胞周期蛋白T-CDK9)。在过去的支持期间，我们对心脏CDK的兴趣导致我们发现在肥厚中CDK7和CDK9的激活是由信号蛋白Gaq和钙调神经磷酸酶或慢性机械应激触发的。只有CDK9能被急性负荷或在培养中被肥大激动剂内皮素(ET-1)激活。在ET-1诱导的CTD磷酸化和生长过程中，CDK9通过药物抑制和显性负性蛋白发挥优先作用。ET-1不增加细胞周期蛋白T-CDK9的表达或组装，而是引起内源性CDK9抑制因子7SK-SnRNA的解离。CDK9的活性通过抑制其抑制物(7SK)和阻止其激活物(Cyclin T1)在小鼠心肌中的下调而被证明是限制心脏生长的。 基于这些发现和额外的临时工作，我们建议研究心肌中细胞周期蛋白T-CDK9-RNAPII级联的功能和基本机制：(1)研究转基因小鼠中细胞周期蛋白T1和细胞周期蛋白CDK9的功能获得突变。(2)利用条件性显性负性突变和功能缺失突变，确定CDK9的基本功能。(3)验证肥大信号通过细胞周期蛋白T-CDK9通过RNAPII诱导启动子逃逸的预测。

DESCRIPTION (provided by applicant): This is a competing renewal of the Applicant's investigation of cyclin-dependent protein kinases (Cdks) and their functional role in cardiac growth. Hypertrophic growth is a risk factor for mortality in heart diseases. Information is still lacking to explain this global increase in RNA and protein per cell. Hypertrophic signals cause phosphorylation of the RNA polymerase II (RNAPII) carboxyl-terminal domain (CTD), which is required in other biological settings for transcript elongation. CTD kinases include components of two basal transcription factors, TFIIH (cyclin HCdk7) and positive transcription elongation factor b (P-TEFb, cyclin T-Cdk9). During the past period of support, our interest in cardiac Cdks led us to discover the activation of Cdk7 and Cdk9 in hypertrophy triggered by the signaling proteins Gaq and calcineurin or chronic mechanical stress. Only Cdk9 was activated by acute load or, in culture, by the hypertrophic agonist endothelin (ET-1). A preferential role for Cdk9 was shown in CTD phosphorylation and growth induced by ET-1, using pharmacological inhibition and dominant-negative proteins. ET-1 did not increase expression or assembly of cyclin T-Cdk9 but, rather, caused dissociation of 7SK snRNA, an endogenous Cdk9 inhibitor. Activity of Cdk9 was proven to be limiting for cardiac growth, by suppressing its inhibitor (7SK) in cultured myocytes and preventing downregulation of its activator (cyclin T1) in mouse myocardium. Based on these findings and additional interim work, we propose to study the function and fundamental mechanisms of the cyclin T-Cdk9-RNAPII cascade in cardiac muscle: (1) To study gain-of-function mutations for cyclin T1 and Cdk9 in transgenic mice, singly and in combination. (2) To define the essential functions of Cdk9, using conditional dominant-negative and loss-of-function mutations. (3) To test the prediction that hypertrophic signals, through cyclin T-Cdk9, induce promoter escape by RNAPII.