INDUCED EXPRESSION FROM AP-1/ETS-DRIVEN PROMOTERS REQUIRES RAF-1 KINASE

Raf-1丝氨酸/苏氨酸蛋白激酶具有关键的 连接细胞生长因子受体激活的开关 细胞核中的转录事件。 我们先前已经 通过使用Raf-1-显性负突变体显示Raf-1是必需的 对于血清和TPA诱导的癌基因反应元件表达， 多瘤病毒增强子 Raf-1的最小区域显示， 这种显性阴性表型（Raf-C4）含有半胱氨酸指 母题 为了确保Raf-C4蛋白阻断AP-1/Ets依赖性 转录，并确定是否富含半胱氨酸的区域内 保守区1是这种活性所必需的，我们测试了这种能力， 的Raf-C4 pm 17阻断AP-1/Ets驱动的HepG 2细胞中的表达。 拉夫 C4 pm 17含有单个氨基酸取代（Cys至Ser，在 168)在Raf-C4富含半胱氨酸的区域内。 这种替代强烈 降低了Raf-C4的显性负效应。 因此， Raf-1中的半胱氨酸指基序可能是与 上游调控因素。 v-Ras还激活了转录从癌基因反应元件， 多瘤病毒增强子 为了确定Raf-1是否介导反式激活， 通过Ras，我们观察了Raf-C4阻断Ras诱导的激活的能力， 通过癌基因反应元件。 v-Ha-Ras高效 反式激活pB 4X CAT表达，Raf-C4完全阻断Ras- 诱导表达。 这些结果表明，Raf-1是必需的， Ras的反式激活。 Raf-C4似乎是通过滴定 Raf-1激活因子，在血清或TPA后由Ras诱导 NIH 3 T3细胞。 此外，我们发现Raf-1和Ras 通过癌基因反应元件协同反式激活， 富含半胱氨酸的区域是这种效应所必需的。 无法 Raf-1半胱氨酸指突变体（Raf-pm 17）与Ras 表明它不能结合Ras诱导的Raf-1激活剂。 目前的工作旨在确定和表征这种 Ras诱导的Raf-1激活剂。

Raf-1 serine/threonine protein kinase has the hallmarks of a critical switch that connects growth factor receptor activation at the cell membrane with transcriptional events in the nucleus. We have previously shown by the use of Raf-1-dominant negative mutants that Raf-1 is required for serum- and TPA-induced expression from the oncogene-responsive element in the polyomavirus enhancer. The minimal region of Raf-1 that displayed this dominant negative phenotype (Raf-C4) contains a cysteine finger motif. To insure that the Raf-C4 protein blocks AP-1/Ets-dependent transcription and to determine if the cysteine-rich region within conserved region 1 is necessary for this activity, we tested the ability of Raf-C4pm17 to block AP-1/Ets-driven expression in HepG2 cells. Raf- C4pm17 contains a single amino acid substitution (Cys to Ser at position 168) within the Raf-C4 cysteine-rich region. This substitution strongly decreases the dominant negative effect of Raf-C4. Thus, the conserved cysteine finger motif in Raf-1 is probably necessary for interactions with upstream regulatory factors. v-Ras also activates transcription from the oncogene-responsive element in the polyomavirus enhancer. To determine if Raf-1 mediates transactivation by Ras, we looked at the ability of Raf-C4 to block Ras-induced activation through the oncogene-responsive element. v-Ha-Ras efficiently transactivated pB4X CAT expression, and Raf-C4 completely blocked Ras- induced expression. These results demonstrate that Raf-1 is necessary for transactivation by Ras. Raf-C4 appears to function by titrating out a Raf-1 activating factor that is induced by Ras following serum or TPA treatment of NIH 3T3 cells. In addition, we show that Raf-1 and Ras cooperate in transactivation through the oncogene-responsive element and that the cysteine-rich region is necessary for this effect. The inability of the Raf-1 cysteine finger mutant (Raf-pm17) to cooperate with Ras suggests that it is incapable of binding the Ras-induced Raf-1 activator. Current work is aimed at the identification and characterization of this Ras-induced Raf-1 activator.