Calcium-dependent protein kinases from Arabidopsis show substrate specificity differences in an analysis of 103 substrates.
Calcium-dependent protein kinases from Arabidopsis show substrate specificity differences in an analysis of 103 substrates.
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DOI：
10.3389/fpls.2011.00036
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发表时间：
2011
影响因子：
5.6
通讯作者：
Harper JF
中科院分区：
文献类型：
--
作者：
Curran A;Chang IF;Chang CL;Garg S;Miguel RM;Barron YD;Li Y;Romanowsky S;Cushman JC;Gribskov M;Harmon AC;Harper JF
底物的鉴定对于理解任何基于蛋白激酶的信号转导途径都是一项关键挑战。在拟南芥中，有1000多种不同的蛋白激酶，其中34种属于钙依赖蛋白激酶（CPK）家族。虽然CPK参与调节植物生物学的多个方面，从离子运输到转录，但对于底物特异性的异构体特异性差异或磷酸化靶点的数量知之甚少。在此，利用体外激酶测定法比较了拟南芥的四种CPK（CPK1、10、16和34）的磷酸化靶点。通过使用103种不同底物进行的测定，揭示了每种激酶在底物特异性方面的显著差异。例如，CPK16能使应激调节蛋白Di19 - 2的一个肽段中的丝氨酸109磷酸化，Km约为70μM，但该位点不会被CPK1、10或34显著磷酸化。相反，CPK1、10和34能使93种其他不被CPK16识别的肽底物磷酸化。通过动力学分析验证了所有四种CPK之间底物特异性差异的实例。为了测试体内磷酸化事件与体外激酶活性之间的相关性，用274种合成肽进行了测定，这些肽包含先前在从植物中分离的蛋白质中定位的磷酸化位点（体内定位位点）。其中，74个（27%）被发现可被所测试的四种CPK中的至少一种磷酸化。这27%的成功率验证了一种将特定激酶（如CPK）的活性与新兴技术所揭示的植物体内数千个磷酸化位点联系起来的有效策略。
The identification of substrates represents a critical challenge for understanding any protein kinase-based signal transduction pathway. In Arabidopsis, there are more than 1000 different protein kinases, 34 of which belong to a family of Ca2+-dependent protein kinases (CPKs). While CPKs are implicated in regulating diverse aspects of plant biology, from ion transport to transcription, relatively little is known about isoform-specific differences in substrate specificity, or the number of phosphorylation targets. Here, in vitro kinase assays were used to compare phosphorylation targets of four CPKs from Arabidopsis (CPK1, 10, 16, and 34). Significant differences in substrate specificity for each kinase were revealed by assays using 103 different substrates. For example CPK16 phosphorylated Serine 109 in a peptide from the stress-regulated protein, Di19-2 with KM ∼70 μM, but this site was not phosphorylated significantly by CPKs 1, 10, or 34. In contrast, CPKs 1, 10, and 34 phosphorylated 93 other peptide substrates not recognized by CPK16. Examples of substrate specificity differences among all four CPKs were verified by kinetic analyses. To test the correlation between in vivo phosphorylation events and in vitro kinase activities, assays were performed with 274 synthetic peptides that contained phosphorylation sites previously mapped in proteins isolated from plants (in vivo-mapped sites). Of these, 74 (27%) were found to be phosphorylated by at least one of the four CPKs tested. This 27% success rate validates a robust strategy for linking the activities of specific kinases, such as CPKs, to the thousands of in planta phosphorylation sites that are being uncovered by emerging technologies.
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拟南芥中串联亲和纯化的 14-3-3 蛋白复合物的蛋白质组学分析。
影响因子：
3.4
作者：
Chang, Ing-Feng;Curran, Amy;Woolsey, Rebekah;Quilici, David;Cushman, John C.;Mittler, Ron;Harmon, Alice;Harper, Jeffrey F.
通讯作者：
Harper, Jeffrey F.
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Schulze WX
影响因子：
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作者：
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通讯作者：
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影响因子：
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影响因子：
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作者：
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通讯作者：
Sibley LD