Role of Mastermind in Notch Transcriptional Activation

描述（由申请人提供）：Notch信号转导通路控制对神经发生和造血重要的细胞命运决定，并参与各种癌症，包括白血病。信号传导导致Notch受体的细胞内结构域（ICD）的蛋白水解释放，Notch受体是CSL增强子结合蛋白的专用转录共激活因子。我们的实验室最近开发了一种无细胞系统，在一个简单的模型增强子上重现Notch转录。这些研究表明，ICD仅与第二个Notch共激活因子Mastermind（MAM）一起激活转录。我们发现MAM招募CBP/p300组蛋白乙酰转移酶以及介体复合物的其他组分，包括激酶CDK 8。最后，我们发现与MAM共表达也促进了体内ICD的磷酸化和降解。这些结果表明，MAM可能会招募一个核激酶耦合激活与营业额的ICD。在这里，我建议调查的MAM相关的共激活蛋白在Notch转录的作用和协调激活与ICD的蛋白水解降解通过以下具体目标：I。定义Notch共激活子的Mastermind家族的保守性质，II。评估CDK 8的蛋白激酶活性是否有助于Notch转录和/或ICD在体内和体外的周转，和III.检查c-ski相互作用蛋白（SKIP）在Notch体外转录中的作用。通过这些研究，我们希望更好地了解Notch反式激活机制及其在ICD修饰和破坏中的作用，这些研究将促进我们对Notch增强子复合物在转化过程中诱导内源性靶基因的机制的理解。

DESCRIPTION (provided by applicant): The Notch signal transduction pathway controls cell fate decisions important for neurogenesis and hematopoiesis and is involved in various cancers including leukemia. Signaling results in the proteolytic release of the intracellular domain (ICD) of the Notch receptor, a dedicated transcriptional co-activator of CSL enhancer-binding proteins. Our lab recently developed a cell-free system that recapitulates Notch transcription on a simple model enhancer. These studies revealed that the ICD activates transcription only in conjunction with a second Notch co-activator, Mastermind (MAM). We showed that MAM recruits the CBP/p300 histone acetyltransferase as well as other components of the mediator complex including the kinase, CDK8. Finally, we showed that co-expression with MAM also promotes the phosphorylation and degradation of the ICD in vivo. These findings suggest that MAM may recruit a nuclear kinase to couple activation with turnover of the ICD. Here I propose to investigate the role of MAM-associated co-activator proteins in Notch transcription and the co-ordination of activation with proteolytic degradation of the ICD through the following specific aims: I. Define the conserved properties of the Mastermind family of Notch co-activators, II. Assess whether the protein kinase activity of CDK8 contributes to Notch transcription and/or the turnover of the ICD in vivo and in vitro, and III. Examine the role of the c-ski-interacting protein (SKIP) in Notch transcription in vitro. Through these studies we hope to better understand the Notch transactivation mechanism and its role in the modification and destruction of the ICD and taken together, these studies will advance our understanding of the mechanism by which the Notch enhancer complex induces endogenous target genes during transformation.