TRANSCRIPTIONAL REGULATION OF C-FOS AND C-JUN

本课题旨在研究生长因子诱导c-fos和c-jun原癌基因的机制。它们受不同的通路和序列元件的调控。对这些基因调控的研究将有助于理解这些基因是如何开启和关闭的，并有助于识别刺激细胞生长的关键信号通路的其他组成部分。c-fos启动子受生长因子通过SRE位点调控。通往该位点的途径导致SRF相关的TCF蛋白磷酸化，但血清和其他因素也以一种未知的机制独立于TCF激活SRE。最有可能的调节机制涉及额外的络合蛋白。最近发现的一种srf结合蛋白是bZIP因子ATF6。抑制ATF6水平可降低血清对SRE的诱导。此外，GALA-ATF6可以通过p38 MAPK通路调控，从而参与调控。ATF6的DNA结合、异源二聚化和调控将被表征。ATF6和/或一个假定的伴侣将被测试在c-fos基因的生长因子调节中的作用。由于已知的srf结合蛋白似乎不是SRE的血清调节所必需的，我们将鉴定新的srf络合蛋白。在凝胶迁移转移试验中鉴定的一个因子将被纯化和克隆，一个新的哺乳动物双杂交筛选将用于鉴定参与血清c-fos调节的其他srf络合蛋白。c-jun启动子的EGF诱导受MEF2和ATF位点控制。这些位点由ras - rac - MEKK通路调控。该通路如何通过MEF2和ATF位点直接调控c-jun启动子将被研究。在HeLa细胞中，MEF2D与MEF2位点结合，GALA-MEF2D受MEKK调控。初步结果表明，EGF诱导MEF2D磷酸化。我们将绘制MEF2D调控和磷酸化所需的区域。如果需要磷酸化，我们将确定mef2d蛋白激酶。在HeLa细胞中，ATF1结合c-jun ATF位点，EGF诱导ATF1磷酸化。我们将测试这种磷酸化对c-jun调控的重要性，并确定负责egf诱导的ATF1磷酸化的蛋白激酶。

The aim of this proposal is to study the mechanism of induction of the c-fos and c-jun proto-oncogenes by growth factors. They are regulated by distinct pathways and sequence elements. The study of regulation of these genes should lead to an understanding of how these genes are switched on and off and to the identification of additional components of signaling pathways critical for stimulation of cell growth. The c-fos promoter is regulated by growth factors through the SRE site. Pathways to this site lead to phosphorylation of the SRF- associated TCF proteins but serum and other factors also activate the SRE independently of TCF by an unknown mechanism. The most likely regulatory mechanism involves additional complexing proteins. One recently identified SRF-binding protein is the bZIP factor ATF6. Inhibition of ATF6 levels decreased serum induction of the SRE. In addition, GALA-ATF6 can be regulated by p38 MAPK pathways such that it could participate in regulation. DNA binding, heterodimerization and regulation of ATF6 will be characterized. ATF6 and/or a putative partner will be tested for roles in growth factor regulation of the c-fos gene. Since the known SRF-binding proteins do not appear to be required for serum regulation of the SRE we will identify novel SRF-complexing proteins. A factor identified in gel mobility shift assays will be purified and cloned and a novel mammalian two-hybrid screen will be used to identify additional SRF-complexing proteins involved in serum regulation of c-fos. EGF induction of the c-jun promoter is controlled by MEF2 and ATF sites. These sites are regulated by a ras to rac to MEKK pathway. How this pathway directly regulates the c-jun promoter through the MEF2 and ATF sites will be studied. In HeLa cells MEF2D binds to the MEF2 site and GALA-MEF2D is regulated by MEKK. Preliminary results indicate that EGF induces MEF2D phosphorylation. The regions of MEF2D required for its regulation and phosphorylation will be mapped. If phosphorylation is required, we will identify the MEF2D-protein kinase. ATF1 binds to the c-jun ATF site in HeLa cells and EGF induces ATF1 phosphorylation. The importance of this phosphorylation for c-jun regulation will be tested and the protein kinase responsible for EGF-induced ATF1 phosphorylation will be identified.