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首页> 外文期刊>BMC Plant Biology >Substrate thiophosphorylation by Arabidopsis mitogen-activated protein kinases
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Substrate thiophosphorylation by Arabidopsis mitogen-activated protein kinases

机译：通过拟南芥促丝裂解蛋白激酶谱系硫代磷酸化

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Mitogen-activated protein kinase (MPK) cascades are important to cellular signaling in eukaryotes. They regulate growth, development and the response to environmental challenges. MPK cascades function via reversible phosphorylation of cascade components, MEKK, MEK, and MPK, but also by MPK substrate phosphorylation. Using mass spectrometry, we previously identified many in vivo MPK3 and MPK6 substrates in Arabidopsis thaliana, and we disclosed their phosphorylation sites. We verified phosphorylation of several of our previously identified MPK3/6 substrates using a nonradioactive in vitro labeling assay. We engineered MPK3, MPK4, and MPK6 to accept bio-orthogonal ATPγS analogs for thiophosphorylating their appropriate substrate proteins. Subsequent alkylation of the thiophosphorylated amino acid residue(s) allows immunodetection using thiophosphate ester-specific antibodies. Site-directed mutagenesis of amino acids confirmed the protein substrates’ site-specific phosphorylation by MPK3 and MPK6. A combined assay with MPK3, MPK6, and MPK4 revealed substrate specificity of the individual kinases. Our work demonstrates that the in vitro-labeling assay represents an effective, specific and highly sensitive test for determining kinase-substrate relationships.
机译：丝裂原激活的蛋白激酶（MPK）级联对真核生物中的细胞信号传导很重要。他们规范增长，发展和对环境挑战的反应。 MPK级联通过可逆磷酸化的级联组件，MEKK，MEK和MPK，而且通过MPK底物磷酸化。使用质谱法，我们先前鉴定了拟南芥中的体内MPK3和MPK6底物中的许多鉴定，我们公开了它们的磷酸化位点。我们使用非酰化体外标记测定验证了我们先前鉴定的几个先前鉴定的MPK3 / 6底物的磷酸化。我们设计了MPK3，MPK4和MPK6以接受生物正交ATPγS类似物，用于硫代磷酸化其合适的底物蛋白。随后的硫代磷酸化氨基酸残基的烷基化允许使用硫代磷酸酯特异性抗体免疫缩进。氨基酸的部位定向诱变通过MPK3和MPK6证实了蛋白质底物的特异性磷酸化。具有MPK3，MPK6和MPK4的组合测定显示单个激酶的底物特异性。我们的作品表明，体外标记测定代表了用于确定激酶底物关系的有效，特异性和高敏感的试验。

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