Isolation of Intrinsically Active (MEK-independent) Variants of the ERK Family of Mitogen-activated Protein (MAP) Kinases
Isolation of Intrinsically Active (MEK-independent) Variants of the ERK Family of Mitogen-activated Protein (MAP) Kinases
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DOI：
10.1074/jbc.m806443200
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发表时间：
2008-12-12
影响因子：
4.8
通讯作者：
Engelberg, David
中科院分区：
文献类型：
--
作者：
Levin-Salomon, Vered;Kogan, Konstantin;Engelberg, David
MAPK是细胞信号传导途径的关键组分，具有独特的激活机制：即相邻苏氨酸和酪氨酸残基的双重磷酸化。ERK酶形成参与增殖、分化、发育、学习和记忆的MAPK亚家族。每个Erk分子在这些过程中的确切作用尚不清楚。解决这个问题的有效策略是单独激活每个分子，例如，通过表达它们的固有活性变体。然而，这样的分子到目前为止还没有产生。在这里，我们报告的隔离，通过一个专门设计的遗传筛选，酵母MAPK Mpk 1/Erk的六个变体（每个携带一个点突变）是活跃的，独立于上游磷酸化。其中一个激活突变R68 S发生在哺乳动物Erk 1（Arg-84）和Erk 2（Arg-65）以及果蝇ERK Rolled（Arg-80）中保守的残基上。当作为重组蛋白在体外测试时，用Ser替换这种保守的Arg使得这些MAPK固有地具有非常高的活性水平。Arg至Ser突变与七标记突变（产生Erk 2（R65 S + D319 N）和Rolled（R80 S + D334 N））的组合导致甚至更高的活性（分别为45%和70%，相对于完全活性的双重磷酸化Erk 2或Rolled）。发现Erk 2（R65 S）和Erk 2（R65 S + D319 N）在人HEK 293细胞中表达时也具有自发活性。我们进一步揭示了突变体的作用机制，并表明它涉及获得自磷酸化活性。因此，已经获得了在体外和体内具有自发活性的第一代Erk分子。
MAPKs are key components of cell signaling pathways with a unique activation mechanism: i.e. dual phosphorylation of neighboring threonine and tyrosine residues. The ERK enzymes form a subfamily of MAPKs involved in proliferation, differentiation, development, learning, and memory. The exact role of each Erk molecule in these processes is not clear. An efficient strategy for addressing this question is to activate individually each molecule, for example, by expressing intrinsically active variants of them. However, such molecules were not produced so far. Here, we report on the isolation, via a specifically designed genetic screen, of six variants ( each carries a point mutation) of the yeast MAPK Mpk1/Erk that are active, independent of upstream phosphorylation. One of the activating mutations, R68S, occurred in a residue conserved in the mammalian Erk1 (Arg-84) and Erk2 (Arg-65) and in the Drosophila ERK Rolled (Arg-80). Replacing this conserved Arg with Ser rendered these MAPKs intrinsically active to very high levels when tested in vitro as recombinant proteins. Combination of the Arg to Ser mutation with the sevenmaker mutation ( producing Erk2(R65S+D319N) and Rolled(R80S+D334N)) resulted in even higher activity ( 45 and 70%, respectively, in reference to fully active dually phosphorylated Erk2 or Rolled). Erk2(R65S) and Erk2(R65S+D319N) were found to be spontaneously active also when expressed in human HEK293 cells. We further revealed the mechanism of action of the mutants and show that it involves acquisition of autophosphorylation activity. Thus, a first generation of Erk molecules that are spontaneously active in vitro and in vivo has been obtained.