Phosphoproteomic identification of targets of the Arabidopsis sucrose nonfermenting-like kinase SnRK2.8 reveals a connection to metabolic processes
Phosphoproteomic identification of targets of the Arabidopsis sucrose nonfermenting-like kinase SnRK2.8 reveals a connection to metabolic processes
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DOI：
10.1073/pnas.0610208104
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发表时间：
2007-04
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影响因子：
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通讯作者：
Ryoung Shin;S. Alvarez;Adrien Y. Burch;J. Jez;D. Schachtman
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文献类型：
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作者：
Ryoung Shin;S. Alvarez;Adrien Y. Burch;J. Jez;D. Schachtman
SnRK2.8是蔗糖非发酵相关激酶家族的成员，当植物缺乏营养和生长减少时，该家族的表达下调。当这种激酶在拟南芥中过度表达时，植株就会变得更大。为了了解这种激酶是如何调节生长的，我们鉴定了一些被这种激酶磷酸化的蛋白质。一种新的磷酸蛋白质组学方法被使用，在该方法中，来自高表达该激酶的植物的总蛋白与来自该激酶失活的植物的总蛋白进行比较。用一种可识别磷酸化氨基酸的染料染色后，在二维凝胶上比较蛋白质谱。用从大肠杆菌中纯化的激酶和目的蛋白进行体外磷酸化实验，确认候选目的蛋白。7个目的蛋白被SnRK2.8证实为磷酸化。某些靶标，如14-3-3蛋白，调节尚未确定的蛋白质，而其他靶标，如乙醛酶I和核糖5-磷酸异构酶，分别解毒糖酵解副产物和催化碳固定的最后步骤之一。此外，腺苷激酶和60S核糖体蛋白也被确认为SnRK2.8的靶标。利用质谱仪，我们鉴定了SnRK2.8、14-3-3κ和14-3-3χ中的磷酸化残基。这些数据表明，SnRK2.8的表达与植物的生长有关，这可能部分是由于参与代谢过程的酶的磷酸化。
SnRK2.8 is a member of the sucrose nonfermenting-related kinase family that is down-regulated when plants are deprived of nutrients and growth is reduced. When this kinase is over expressed in Arabidopsis, the plants grow larger. To understand how this kinase modulates growth, we identified some of the proteins that are phosphorylated by this kinase. A new phosphoproteomic method was used in which total protein from plants overexpressing the kinase was compared with total protein from plants in which the kinase was inactivated. Protein profiles were compared on two-dimensional gels following staining by a dye that recognizes phosphorylated amino acids. Candidate target proteins were confirmed with in vitro phosphorylation assays, using the kinase and target proteins that were purified from Escherichia coli. Seven target proteins were confirmed as being phosphorylated by SnRK2.8. Certain targets, such as 14-3-3 proteins, regulate as yet unidentified proteins, whereas other targets, such as glyoxalase I and ribose 5-phosphate isomerase, detoxify byproducts from glycolysis and catalyze one of the final steps in carbon fixation, respectively. Also, adenosine kinase and 60S ribosomal protein were confirmed as targets of SnRK2.8. Using mass spectrometry, we identified phosphorylated residues in the SnRK2.8, the 14-3-3κ, and the 14-3-3χ. These data show that the expression of SnRK2.8 is correlated with plant growth, which may in part be due to the phosphorylation of enzymes involved in metabolic processes.