Engelman J A, Zhang X L, Razani B, Pestell R G, Lisanti M P
Department of Molecular Pharmacology, Albert Einstein Cancer Center, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
J Biol Chem. 1999 Nov 5;274(45):32333-41. doi: 10.1074/jbc.274.45.32333.
Caveolin-1 is a principal component of caveolae membranes in vivo. Caveolin-1 mRNA and protein expression are down-regulated in NIH 3T3 cells in response to transformation by activated oncogenes, such as H-Ras(G12V) and v-Abl. The mechanisms governing this down-regulation event remain unknown. Here, we show that caveolin-1 gene expression is directly regulated by activation of the Ras-p42/44 MAP kinase cascade. Down regulation of caveolin-1 protein expression by Ras is independent of (i) the type of activating mutation (G12V versus Q61L) and (ii) the form of activated Ras transfected (H-Ras versus K-Ras versus N-Ras). Treatment of Ras or Raf-transformed NIH 3T3 cells with a well characterized MEK inhibitor (PD 98059) restores caveolin-1 protein expression. In contrast, treatment of v-Src and v-Abl transformed NIH 3T3 cells with PD 98059 does not restore caveolin-1 expression. Thus, there must be at least two pathways for down-regulating caveolin-1 expression: one that is p42/44 MAP kinase-dependent and another that is p42/44 MAP kinase-independent. We focused our efforts on the p42/44 MAP kinase-dependent pathway. The activity of a panel of caveolin-1 promoter constructs was evaluated using transient expression in H-Ras(G12V) transformed NIH 3T3 cells. We show that caveolin-1 promoter activity is up-regulated approximately 5-fold by inhibition of the p42/44 MAP kinase cascade. Using electrophoretic mobility shift assays we provide evidence that the caveolin-1 promoter (from -156 to -561) is differentially bound by transcription factors in normal and H-Ras(G12V)-transformed cells. We also show that activation of protein kinase A (PKA) signaling is sufficient to down-regulate caveolin-1 protein expression and promoter activity. Thus, we have identified two signaling pathways (Ras-p42/44 MAP kinase and PKA) that transcriptionally down-regulate caveolin-1 gene expression.
小窝蛋白-1是体内小窝膜的主要成分。在NIH 3T3细胞中，小窝蛋白-1的mRNA和蛋白质表达会因被激活的癌基因（如H-Ras(G12V)和v-Abl）转化而被下调。调控这一下调事件的机制尚不清楚。在此，我们表明小窝蛋白-1基因表达直接受Ras-p42/44丝裂原活化蛋白激酶级联反应激活的调控。Ras对小窝蛋白-1蛋白表达的下调与（i）激活突变的类型（G12V与Q61L）和（ii）转染的活化Ras的形式（H-Ras与K-Ras与N-Ras）无关。用一种特性明确的MEK抑制剂（PD 98059）处理Ras或Raf转化的NIH 3T3细胞可恢复小窝蛋白-1蛋白表达。相比之下，用PD 98059处理v-Src和v-Abl转化的NIH 3T3细胞并不能恢复小窝蛋白-1的表达。因此，至少存在两条下调小窝蛋白-1表达的途径：一条是依赖p42/44丝裂原活化蛋白激酶的途径，另一条是不依赖p42/44丝裂原活化蛋白激酶的途径。我们将研究重点放在了依赖p42/44丝裂原活化蛋白激酶的途径上。使用在H-Ras(G12V)转化的NIH 3T3细胞中的瞬时表达来评估一组小窝蛋白-1启动子构建体的活性。我们表明，通过抑制p42/44丝裂原活化蛋白激酶级联反应，小窝蛋白-1启动子活性上调约5倍。使用电泳迁移率变动分析，我们提供证据表明小窝蛋白-1启动子（从-156至-561）在正常细胞和H-Ras(G12V)转化的细胞中与转录因子的结合存在差异。我们还表明蛋白激酶A（PKA）信号的激活足以下调小窝蛋白-1蛋白表达和启动子活性。因此，我们确定了两条转录下调小窝蛋白-1基因表达的信号通路（Ras-p42/44丝裂原活化蛋白激酶和PKA）。
Zotero 插件