Cardiogenic Gene Switch, Role of SRF Phosphorylation

描述(由申请人提供)：血清反应因子(SRF)作为主要的调节平台，可能在心脏祖细胞的承诺中发挥核心作用，因为它对中胚层的形成是必需的，并且它能够与其他早期心脏丰富的转录辅助因子进行特定的蛋白质-蛋白质关联。对SRF缺失突变的分析支持这样的观点，即SRF是哺乳动物心脏中胚层形成的重要调节因子，并将SRF置于心脏细胞承诺和分化调控体系中的非常高的位置。我们发现，两个保守的SRF MADS盒残基Thr159和Ser162的受控磷酸化构成了激活和抑制SRF依赖的心脏基因程序的新的基因开关。蛋白激酶C(PKC)对SRF有很强的抑制活性，它主要将磷酸化导向Ser162，其次导向Thr159。血管内皮生长因子可能通过Flk1受体和驱动PKC活性而失活SRF的生肌活性，部分是通过阻断心肌收缩蛋白基因活性的表达来实现的。此外，血管内皮生长因子可能发挥抑制作用，形成心脏形成区域的后缘，并可能在从形成心垫到出现非收缩心内膜的过渡过程中发出信号。磷酸化的SRF基因开关也可能允许激活内皮血管生成程序，但代价是心脏生成基因的活性。因此，这项建议的中心主题将是确定SRF的磷酸化如何在心脏中胚层的出现和小鼠胚胎心脏的形成过程中调节下游基因靶点。SRF位于MADS盒的α1线圈中仅相距3个氨基酸的两个位置。通过以下四个目标：目的I：SRF MADS box的磷酸化是否发生在小鼠心脏的形成和细化过程中？目的II SRF MADS盒的磷酸化是否起基因调控开关的作用？目的III SRF MADS box的磷酸化是否在指定早期心脏和/或内皮/血管生成程序中起直接作用？目的：血管内皮生长因子信号通路是否抑制SRF依赖的心脏基因活性？

DESCRIPTION (provided by applicant): Serum response factor (SRF), as the master regulatory platform, may play a central role in the commitment of cardiac progenitors by virtue of its obligatory requirement for mesoderm formation and by its ability for making specific protein-protein associations with other early cardiac enriched transcription cofactors. Analysis of SRF null mutants supports the idea that SRF is an essential regulator of mammalian cardiac mesoderm formation and places SRF at a very high point in the regulatory hierarchy for cardiac cell commitment and differentiation. We discovered that the regulated phosphorylation of two conserved SRF MADS box residues Thr159 and Ser162 constitutes a novel gene switch for the activation and repression of SRF dependent cardiogenic gene programs. Protein kinase C (PKC) has a profound inhibitory activity on SRF, directing phosphorylation primarily to Ser162 and secondarily to Thr159. It is likely that VEGF by signaling through the Flkl receptor and driving PKC activity may inactivate SRF myogenic activity in part by blocking the expression of cardiogenic contractile protein gene activity. In addition, VEGF may exert inhibitory activity that forms the posterior border of the heart forming region and may signal later during the transition from forming the cardiac cushion to the appearance of the noncontractile endocardium. The phosphorylated SRF gene switch may also allow for the activation of the endothelial-hemangiogenic program at the expense of cardiogenic gene activity. Thus, the central theme of this proposal will be to determine how the phosphorylation of SRF, at two sites that are only three amino acids apart in the alpha 1 coil of the MADS box, is networked to regulate downstream gene targets during the appearance of cardiac mesoderm and the elaboration and formation of the embryonic mouse heart. By the following four aims: Aim I: Does phosphorylation of SRF MADS box occur during cardiogenesis and the elaboration of the murine heart? Aim II Does phosphorylation of the SRF MADS box act as a gene regulatory switch? Aim III Does phosphorylation of SRF MADS box have a direct role in specifying early cardiac and or endothelial/hemangiogenic programs? Aim IV Does the VEGF signaling pathway repress SRF dependent cardiogenic gene activity?