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危重 连接细胞上生长因子受体激活的开关 核内有转录事件的膜。我们之前已经 Raf-1显性负突变体的使用表明Raf-1是必需的 用于血清和TPA诱导的癌基因反应元件的表达 在多瘤病毒增强剂中。显示的Raf-1的最小区域 这种显性负性表型(Raf-C4)含有一个半胱氨酸指 Motif。确保Raf-C4蛋白阻断AP-1/ETS依赖 转录并确定半胱氨酸富集区是否在 保守区1是这个活动所必需的，我们测试了它的能力 Raf-C4pm17阻断AP-1/ETS在HepG2细胞中的表达英国皇家空军- C4pm17含有单一氨基酸取代位(Cys到Ser 168)在Raf-C4半胱氨酸富集区。这种替代很强烈 减少Raf-C4的主要负面影响。因此，保守的 Raf-1中的半胱氨酸指基序可能是与 上游监管因素。 V-RAS还激活来自癌基因反应元件的转录 多瘤病毒增强剂。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 通过致癌基因反应元件。V-Ha-RAS高效 反式激活的pB4X CAT表达和Raf-C4完全阻断RAS- 诱导表达。这些结果表明Raf-1是细胞周期调控所必需的 RAS激活。Raf-C4的功能似乎是通过滴定a 血清或TPA后RAS诱导的RAF-1激活因子 对NIH3T3细胞的处理。此外，我们还证明了Raf-1和Ras 通过癌基因反应元件协同反式激活 这种效应需要富含半胱氨酸的区域。无能为力 Raf-1半胱氨酸指突变体(Raf-pm17)与RAS的协同作用 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一点 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.