Combination of Two Activating Mutations in One HOG1 Gene Forms Hyperactive Enzymes That Induce Growth Arrest
Combination of Two Activating Mutations in One HOG1 Gene Forms Hyperactive Enzymes That Induce Growth Arrest
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一个 HOG1 基因中的两个激活突变组合形成高活性酶，导致生长停滞
DOI：
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发表时间：
2003
影响因子：
5.3
通讯作者：
D. Engelberg
中科院分区：
文献类型：
--
作者：
Gilad Yaakov;M. Bell;S. Hohmann;D. Engelberg
丝裂原活化蛋白激酶（MAPKs）在细胞分化、生长、增殖和凋亡过程中发挥着关键作用。尽管MAPK已被广泛研究，但每种MAPK的精确功能、特定底物和靶基因尚不清楚。这些问题可以通过单一激活给定的MAPK来解决，例如，通过使用构成活性的MAPK酶。我们最近报道了8个酿酒酵母MAPK Hog1的高活性突变体的分离，每个突变体都有一个独特的单点突变。这些突变体获得了高的内在催化活性，但没有施加Hog1途径的全部生物学潜力。在这里，我们描述了我们的尝试，以获得比以前的突变体更活跃的MAPK催化和生物学。我们在同一基因中结合了两个不同的激活点突变，发现两个产生的双突变体获得了不寻常的特性。这些等位基因HOG1D170A，F318L和HOG1D170A，F318S诱导了严重的生长抑制，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（在没有任何刺激的情况下）催化活性和Hog1靶基因的强诱导相关。此外，对这些活性等位基因磷酸化状态的分析表明，它们获得的内在活性不依赖于phospho-Thr174或phospho-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在本质上被突变激活并诱导强烈生物效应的第一个例子。
ABSTRACT 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.
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影响因子：
56.9
作者：
HAN, J;LEE, JD;ULEVITCH, RJ
通讯作者：
ULEVITCH, RJ
DOI：
10.1016/s0021-9258(18)53507-9
发表时间：
1993-03
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
D. Robbins;E. Zhen;H. Owaki;C. Vanderbilt;D. Ebert;T. Geppert;M. Cobb
通讯作者：
D. Robbins;E. Zhen;H. Owaki;C. Vanderbilt;D. Ebert;T. Geppert;M. Cobb
影响因子：
56.9
作者：
Posas, F;Saito, H
通讯作者：
Saito, H
DOI：
10.1073/pnas.94.6.2327
发表时间：
1997-03-18
影响因子：
11.1
作者：
Wang, ZL;Harkins, PC;Goldsmith, EJ
通讯作者：
Goldsmith, EJ
影响因子：
16
作者：
Chang, CI;Xu, BE;Goldsmith, EJ
通讯作者：
Goldsmith, EJ