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首页> 外文期刊>Molecular and Cellular Biology >Conditionally oncogenic forms of the A-Raf and B-Raf protein kinases display different biological and biochemical properties in NIH 3T3 cells.
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Conditionally oncogenic forms of the A-Raf and B-Raf protein kinases display different biological and biochemical properties in NIH 3T3 cells.

机译：A-Raf和B-Raf蛋白激酶的有条件致癌形式在NIH 3T3细胞中显示出不同的生物学和生化特性。

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The protein kinase domains of mouse A-Raf and B-Raf were expressed as fusion proteins with the hormone binding domain of the human estrogen receptor in mammalian cells. In the absence of estradiol, 3T3 and rat1a cells expressing delta A-Raf:ER and delta B-Raf:ER were nontransformed, but upon the addition of estradiol the cells became oncogenically transformed. Morphological oncogenic transformation was more rapid and distinctive in cells expressing delta B-Raf:ER compared with cells expressing delta A-Raf:ER. Biochemical analysis of cells transformed by delta A-Raf:ER and delta B-Raf:ER revealed several interesting differences. The activation of delta B-Raf:ER consistently led to the rapid and robust activation of both MEK and p42/p44 MAP kinases. By contrast, the activation of delta A-Raf:ER led to a weak activation of MEK and the p42/p44 MAP kinases. The extent of activation of MEK in cells correlated with the ability of the different Raf kinases to phosphorylate and activate MEK1 in vitro. delta B-Raf:ER phosphorylated MEK1 approximately 10 times more efficiently than delta Raf-1:ER and at least 500 times more efficiently than delta A-Raf:ER under the conditions of the immune-complex kinase assays. These results were confirmed with epitope-tagged versions of the Raf kinase domains expressed in insect cells. The activation of all three delta Raf:ER proteins in 3T3 cells led to the hyperphosphorylation of the resident p74raf-1 and mSOS1 proteins, suggesting the possibility of "cross-talk" between the different Raf kinases and feedback regulation of intracellular signaling pathways. The activation of either delta B-Raf:ER or delta Raf-1:ER in quiescent 3T3 cells was insufficient to promote the entry of the cells into DNA synthesis. By contrast, the activation of delta A-Raf:ER in quiescent 3T3 cells was sufficient to promote the entry of the cells into S phase after prolonged exposure to beta-estradiol. The delta Raf:ER system has allowed us to reveal significant differences between the biological and biochemical properties of oncogenic forms of the Raf family of protein kinases. We anticipate that cells expressing these proteins and other estradiol-regulated protein kinases will be useful tools in future attempts to unravel the complex web of interactions involved in intracellular signal transduction pathways.
机译：小鼠A-Raf和B-Raf的蛋白激酶结构域表达为与哺乳动物细胞中人雌激素受体的激素结合结构域的融合蛋白。在没有雌二醇的情况下，表达δA-Raf：ER和δB-Raf：ER的3T3和rat1a细胞未转化，但添加雌二醇后，细胞发生了癌变。与表达δA-Raf：ER的细胞相比，在表达δB-Raf：ER的细胞中形态致癌转化更为迅速和独特。通过δA-Raf：ER和δB-Raf：ER转化的细胞的生化分析显示出一些有趣的差异。 δB-Raf：ER的激活始终导致MEK和p42 / p44 MAP激酶的快速而强劲的激活。相比之下，δA-Raf：ER的激活导致MEK和p42 / p44 MAP激酶的激活较弱。细胞中MEK的激活程度与不同Raf激酶在体外磷酸化和激活MEK1的能力有关。在免疫复合激酶测定的条件下，δB-Raf：ER磷酸化MEK1的效率比δRaf-1：ER大约高10倍，比δA-Raf：ER的效率至少高500倍。用昆虫细胞中表达的Raf激酶结构域的表位标记形式证实了这些结果。 3T3细胞中所有三个delta Raf：ER蛋白的激活导致驻留的p74raf-1和mSOS1蛋白过度磷酸化，这提示了不同Raf激酶之间“串扰”和细胞内信号通路反馈调节的可能性。在静止的3T3细胞中，δB-Raf：ER或del Raf-1：ER的激活不足以促进细胞进入DNA合成。相反，在静止的3T3细胞中，δA-Raf：ER的激活足以促进细胞长时间暴露于β-雌二醇后进入S期。 ΔRaf：ER系统使我们能够揭示蛋白激酶Raf家族致癌形式的生物学和生化特性之间的显着差异。我们预期表达这些蛋白质和其他雌二醇调节的蛋白激酶的细胞将在未来的尝试中揭示细胞内信号转导途径中复杂的相互作用网时将是有用的工具。

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