Transcriptional regulation of c-fos and immediate early genes

描述（由申请人提供）：本提案的目的是表征生长因子受体激活即时早期基因所利用的信号通路。直接早期基因c-fos受血清反应元件（SRE）调控，SRE与血清反应因子（SRF）结合。SRF通过两种途径激活。一个是SRF辅助因子，三元复合因子（tcf）的MARK磷酸化，第二个是通过小GTPase RhoA信号传导。在本提案中，RhoA激活c-fos启动子的途径将被研究。SRF共激活因子MKL1/2将被研究，因为它们已被证明是SRF靶基因的血清和RhoA激活所必需的。MKL1在细胞的血清处理下迅速磷酸化。诱导磷酸化的位点已经被绘制出来，并将被测试它们对血清诱导靶基因的要求。然后将确定负责的蛋白激酶。MKL1结构域在其调控中的作用也将被确定，关键结构域的络合蛋白将被分离。RhoA向SRF的信号传导涉及肌动蛋白丝的变化。然而，RhoA的突变与这一模型相悖。这表明一种新的RhoA效应被用于向SRF发送信号。参与SRF激活的RhoA靶标将以不与RhoA突变体结合作为选择标准进行纯化。我们已经通过微阵列分析鉴定了许多血清诱导基因，并发现一个子集依赖于MKL1来诱导。目前尚不清楚有多少其他基因被TCF甚至SRF激活。我们将开发在这些因素中有缺陷的细胞系，并使用微阵列来确定哪种机制调节整个类别的直接早期基因。这可能会识别出独立于这些因素而激活的基因。这些基因将被定位为新的调控因子，以确定新的信号通路。我们还在微阵列中发现了许多血清抑制基因。这种抑制的机制尚未被广泛研究，许多被抑制的基因具有抗增殖作用。我们将描述这些基因的调控，以确定在早期反应中新的信号通路。

DESCRIPTION (provided by applicant): The aim of this proposal is to characterize the signaling pathways utilized by growth factor receptors to activate immediate early genes. The proto-type immediate early gene c-fos is regulated by a Serum Response Element (SRE), which binds Serum Response Factor (SRF). SRF is activated by two pathways. One is MARK phosphorylation of the SRF cofactors, the ternary complex factors (TCFs), and the second is signaling through the small GTPase RhoA. In this proposal the pathways used by RhoA to activate the c-fos promoter will be studied. The SRF coactivators MKL1/2 will be studied as they have been shown to be required for serum and RhoA activation of SRF target genes. MKL1 is rapidly phosphorylated in response to serum treatment of cells. The sites of inducible phosphorylation have been mapped and will be tested for their requirement for serum induction of target genes. The responsible protein kinase will then be identified. The role of domains of MKL1 in its regulation will also be determined and complexing proteins to critical domains will be isolated. RhoA signaling to SRF has been proposed to involve changes in actin filaments. However, mutations in RhoA argue against this model. This suggests that a novel RhoA effector is used to signal to SRF. Targets of RhoA involved in SRF activation will be purified using lack of binding to RhoA mutants as selection criteria. We have identified many serum inducible genes by microarray analysis and found that a subset is dependent upon MKL1 for its induction. It is unclear how many of the other genes are activated by TCF or even by SRF. We will develop cell lines defective in these factors and use microarrays to determine which mechanisms regulate the entire class of immediate early genes. This will likely identify genes activated independently of these factors. These genes will be mapped for novel regulatory factors to identify new signaling pathways. We also found in microarrays that there are many serum repressible genes. The mechanisms for this repression have not been extensively studied and many of the repressed genes have anti-proliferative effects. We will characterize regulation of these genes to identify new signaling pathways in the immediate early response.