Cardiogenic Gene Switch, Role of SRF Phosphorylation

描述（由申请方提供）：血清反应因子（SRF）作为主要调控平台，由于其对中胚层形成的强制性要求以及其与其他早期心脏富集转录辅因子形成特异性蛋白质-蛋白质结合的能力，可能在心脏祖细胞的定型中发挥核心作用。SRF无效突变体的分析支持这样的想法，即SRF是哺乳动物心脏中胚层形成的重要调节剂，并将SRF置于心脏细胞定型和分化的调节层次中的非常高的位置。我们发现两个保守的SRF MADS盒残基Thr 159和Ser 162的调节磷酸化构成了一种新型基因开关，用于激活和抑制SRF依赖性心源性基因程序。蛋白激酶C（PKC）对SRF具有深刻的抑制活性，主要将磷酸化引导至Ser 162，其次是Thr 159。VEGF通过Flkl受体的信号传导和驱动PKC活性可能部分地通过阻断心源性收缩蛋白基因活性的表达来抑制SRF肌源性活性。此外，VEGF可能发挥抑制活性，形成心脏形成区域的后边缘，并可能在从形成心脏垫到出现非收缩性内皮细胞的过渡期间稍后发出信号。磷酸化的SRF基因开关还可以允许以心源性基因活性为代价激活内皮血管生成程序。因此，该提案的中心主题将是确定SRF的磷酸化如何在MADS盒的α 1螺旋中仅相隔三个氨基酸的两个位点联网以在心脏中胚层的出现以及胚胎小鼠心脏的发育和形成期间调节下游基因靶点。通过以下四个目的：目的一：是否磷酸化SRF MADS盒发生在心脏发生和小鼠心脏的制作？目的II SRF MADS盒的磷酸化是否作为基因调控开关？目的III SRF MADS盒的磷酸化是否在指定早期心脏和/或内皮/血管生成程序中具有直接作用？目的IV VEGF信号通路是否抑制SRF依赖的心源性基因活性？

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?