RAF-1 PROTEIN-KINASE ACTIVATES THE NF-KAPPA-B TRANSCRIPTION FACTOR BY DISSOCIATING THE CYTOPLASMIC NF-KAPPA-B-I-KAPPA-B COMPLEX
RAF-1 PROTEIN-KINASE ACTIVATES THE NF-KAPPA-B TRANSCRIPTION FACTOR BY DISSOCIATING THE CYTOPLASMIC NF-KAPPA-B-I-KAPPA-B COMPLEX
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DOI：
10.1073/pnas.90.20.9247
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发表时间：
1993-10-15
影响因子：
11.1
通讯作者：
SEDIVY, JM
中科院分区：
文献类型：
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作者：
LI, SF;SEDIVY, JM
向静止细胞添加促有丝分裂生长因子触发复杂的信号转导级联，导致基因表达的重编程和进入细胞周期。我们发现c-Raf-1蛋白激酶的致癌变体刺激含有NF-κ B结合位点的启动子的表达。原位免疫荧光分析显示v-raf转化的NIH 3 T3细胞中NF-κ B p65亚基的核水平升高。HeLa细胞胞质提取物与纯化的重组谷胱甘肽S-转移酶-raf融合蛋白在ATP存在下孵育释放活性NF-κ B，其可通过电泳凝胶迁移率变动测定法检测。纯化的重组IkappaB和谷胱甘肽S-转移酶-raf在ATP的存在下共孵育导致IkappaB的磷酸化。GAL 4（激活结构域）-IkappaB和GAL 4（DNA结合结构域）-Raf融合蛋白在酵母中的共表达导致GAL 4响应报告基因的刺激，表明IkappaB和Raf在体内物理相互作用。这些结果表明，Raf-1激酶在信号转导中的功能部分是通过磷酸化细胞质IkappaB-NF-kappaB复合物中的IkappaB以释放活性NF-kappaB来激活NF-kappaB转录因子。
Addition of mitogenic growth factors to quiescent cells triggers complex signal transduction cascades that result in the reprogramming of gene expression and entry into the cell cycle. We have found that an oncogenic variant of the c-Raf-1 protein kinase stimulated the expression of promoters containing NF-kappaB binding sites. In situ immunofluorescence analysis revealed elevated nuclear levels of the p65 subunit of NF-kappaB in v-raf-transformed NIH 3T3 cells. Incubation of HeLa cell cytoplasmic extracts with a purified recombinant glutathione S-transferase-raf fusion protein in the presence of ATP released active NF-kappaB that could be detected by electrophoretic gel mobility shift assay. Coincubation of purified recombinant IkappaB and glutathione S-transferase-raf in the presence of ATP resulted in the phosphorylation of IkappaB. Coexpression of GAL4 (activation domain)-IkappaB and GAL4 (DNA-binding domain)-raf fusion proteins in yeast resulted in stimulation of a GAL4-responsive reporter gene, indicating that IkappaB and Raf interact physically in vivo. These results indicate that the Raf-1 kinase functions in signal transduction in part by activating the NF-kappaB transcription factor by phosphorylating IkappaB in the cytoplasmic IkappaB-NF-kappaB complex to release active NF-kappaB.