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首页> 外文期刊>Molecular and Cellular Biology >Combination of Two Activating Mutations in One HOG1 Gene Forms Hyperactive Enzymes That Induce Growth Arrest
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Combination of Two Activating Mutations in One HOG1 Gene Forms Hyperactive Enzymes That Induce Growth Arrest

机译：一个HOG1基因中的两个激活突变的组合形成诱导生长停滞的高活性酶。

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Mitogen-activated protein kinases (MAPKs) play key roles in differentiation, growth, proliferation, and apoptosis. Although MAPKs have been extensively studied, the precise function, specific substrates, and target genes of each MAPK are not known. These issues could be addressed by sole activation of a given MAPK, e.g., through the use of constitutively active MAPK enzymes. We have recently reported the isolation of eight hyperactive mutants of the Saccharomyces cerevisiae MAPK Hog1, each of which bears a distinct single point mutation. These mutants acquired high intrinsic catalytic activity but did not impose the full biological potential of the Hog1 pathway. Here we describe our attempt to obtain a MAPK that is more active than the previous mutants both catalytically and biologically. We combined two different activating point mutations in the same gene and found that two of the resulting double mutants acquired unusual properties. These alleles, HOG1D170A,F318L and HOG1D170A,F318S, induced a severe growth inhibition and had to be studied through an inducible expression system. This growth inhibition correlated with very high spontaneous (in the absence of any stimulation) catalytic activity and strong induction of Hog1 target genes. Furthermore, analysis of the phosphorylation status of these active alleles shows that their acquired intrinsic activity is independent of either phospho-Thr174 or phospho-Tyr176. Through fluorescence-activated cell sorting analysis, we show that the effect on cell growth inhibition is not a result of cell death. This study provides the first example of a MAPK that is intrinsically activated by mutations and induces a strong biological effect.
机译：丝裂原激活的蛋白激酶（MAPK）在分化，生长，增殖和凋亡中起关键作用。尽管已经对MAPK进行了广泛的研究，但是每个MAPK的确切功能，特定底物和靶基因尚不清楚。这些问题可以通过单独激活给定的MAPK来解决，例如通过使用组成型活性MAPK酶。最近，我们报道了8个啤酒酵母MAPK Hog1的高活性突变体的分离，每个突变体都有一个明显的单点突变。这些突变体获得高内在的催化活性，但没有强加Hog1途径的全部生物潜力。在这里，我们描述了我们的尝试，以获取一种比以前的突变体在催化和生物学上都更具活性的MAPK。我们在同一个基因中结合了两个不同的激活点突变，发现其中两个最终的双重突变获得了不同寻常的特性。这些等位基因 HOG1 D170A，F318L 和 HOG1 D170A，F318S 引起严重的生长抑制，必须通过诱导表达系统进行研究。这种生长抑制与非常高的自发性（在没有任何刺激的情况下）催化活性和强烈诱导的Hog1靶基因相关。此外，对这些活性等位基因的磷酸化状态的分析表明，它们获得的固有活性独立于磷酸-Thr174或磷酸-Tyr176。通过荧光激活细胞分选分析，我们表明对细胞生长抑制的影响不是细胞死亡的结果。这项研究提供了MAPK的第一个例子，该MAPK被突变固有地激活并诱导了强大的生物学作用。

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