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（Cdks）及其在心脏生长中的功能作用的研究的竞争性更新。 肥大性生长是心脏病死亡率的一个危险因素。目前仍缺乏信息来解释每个细胞RNA和蛋白质的全球增加。肥大信号引起RNA聚合酶II（RNAPII）羧基末端结构域（CTD）的磷酸化，这是转录物延伸在其他生物学环境中所需的。CTD激酶包括两种基础转录因子TFIIH（细胞周期蛋白HCdk 7）和正转录延伸因子B（P-TEF B，细胞周期蛋白T-Cdk 9）的组分。在过去的支持期间，我们对心脏Cdks的兴趣使我们发现了由信号蛋白Gaq和钙调神经磷酸酶或慢性机械应力触发的肥大中Cdk 7和Cdk 9的激活。只有Cdk 9被激活的急性负荷，或在文化中，由肥大激动剂内皮素（ET-1）。使用药理学抑制和显性负性蛋白，Cdk 9在ET-1诱导的CTD磷酸化和生长中显示出优先作用。ET-1没有增加细胞周期蛋白T-Cdk 9的表达或组装，而是引起内源性Cdk 9抑制剂7SK snRNA的解离。Cdk 9的活性被证明是限制心脏生长，抑制其抑制剂（7SK）在培养的心肌细胞和防止下调其激活剂（细胞周期蛋白T1）在小鼠心肌。 基于以上研究结果和前期工作，我们拟对cyclin T-Cdk 9-RNAP Ⅱ级联反应在心肌中的功能和基本机制进行研究：（1）研究cyclin T1和Cdk 9在转基因小鼠中的功能获得性突变，包括单独突变和联合突变。(2)使用条件显性负突变和功能丧失突变来定义Cdk 9的基本功能。(3)为了测试肥大信号通过细胞周期蛋白T-Cdk 9诱导RNAPII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.