TRANSCRIPTIONAL REGULATION OF C-FOS AND C-JUN

本研究的目的是研究生长因子诱导c-fos和c-jun原癌基因表达的机制。 它们由不同的途径和序列元件调节。 对这些基因调控的研究应有助于了解这些基因是如何被打开和关闭的，并有助于识别对刺激细胞生长至关重要的信号通路的其他组分。c-fos启动子通过SRE位点受生长因子调控。 通往该位点的途径导致SRF相关TCF蛋白的磷酸化，但血清和其他因子也通过未知机制独立于TCF激活SRE。 最可能的调节机制涉及额外的复合蛋白。 最近鉴定的一种SRF结合蛋白是bZIP因子ATF 6。 抑制ATF 6水平降低了SRE的血清诱导。 此外，GALA-ATF 6还可以通过p38 MAPK途径进行调控，从而参与调控。DNA结合，异源二聚体和调节的ATF 6的特点。 将测试ATF 6和/或推定伴侣在c-fos基因的生长因子调节中的作用。 由于已知的SRF结合蛋白似乎不需要血清调节SRE，我们将确定新的SRF复合蛋白。 凝胶迁移率变动分析中确定的一个因素将被纯化和克隆，一种新的哺乳动物双杂交筛选将被用来确定额外的SRF复合蛋白参与血清调节c-fos。EGF对c-jun启动子的诱导由MEF 2和ATF位点控制。 这些位点由ras至rac至MEKK途径调节。 该途径如何通过MEF 2和ATF位点直接调节c-jun启动子将被研究。 在HeLa细胞中，MEF 2D与MEF 2位点结合，GALA-MEF 2D受MEKK调节。初步结果表明，EGF诱导MEF 2D磷酸化。 将绘制MEF 2D调节和磷酸化所需的区域。 如果需要磷酸化，我们将鉴定MEF 2D蛋白激酶。 在HeLa细胞中，ATF 1与c-jun ATF位点结合，EGF诱导ATF 1磷酸化。 将测试这种磷酸化对c-jun调节的重要性，并鉴定负责EGF诱导的ATF 1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.