植物蛋白磷酸化的检测方法

doi:10.11983/CBB19208

摘要/Abstract

摘要： 蛋白磷酸化是一种重要的蛋白质翻译后修饰方式, 几乎参与植物所有生命过程的调节。蛋白磷酸化过程主要指在蛋白激酶的催化作用下, 将三磷酸腺苷(ATP)上的γ位磷酸基团转移到底物蛋白特定氨基酸残基上的过程。底物蛋白上被磷酸化的常见氨基酸有丝氨酸、苏氨酸及酪氨酸, 磷酸基团与氨基酸中的羟基通过酯键连接。该文详细描述了几种常用的蛋白质体外及体内磷酸化的检测方法及注意事项。

关键词: 蛋白磷酸化, 蛋白激酶, 植物

Abstract: Protein phosphorylation is one of the important protein posttranslational modifications that is involved in the regulation of most cellular processes in plants. Protein kinases catalyze the phosphorylation by transferring the phosphate group in ATP to the substrate proteins. The phosphate is usually covalently linked to the hydroxyl group of specific amino acid residues in the substrates by an ester bond. The mostly studied phosphorylation sites are serine, threonine, and tyrosine residues. Here, we present protocols and related tips for the in vitro and in vivo protein phosphorylation assays.

Key words: protein phosphorylation, protein kinase, plant

朱丹,曹汉威,李媛,任东涛. 植物蛋白磷酸化的检测方法. 植物学报, 2020, 55(1): 76-82.

Dan Zhu,Hanwei Cao,Yuan Li,Dongtao Ren. Protocols for Analyzing Plant Phospho-proteins. Chinese Bulletin of Botany, 2020, 55(1): 76-82.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB19208

https://www.chinbullbotany.com/CN/Y2020/V55/I1/76

图/表 3

图1 原核表达的拟南芥激酶MPK6对3个推测底物的体外磷酸分析激酶(MPK6)连接有6×His标签、推测的底物蛋白(A、B和C)连接有Flag标签。体外磷酸化反应完成后, 反应体系中的蛋白用SDS-PAGE gel分离。然后进行磷酸化底物的放射自显影分析(上图)。底物蛋白(中图)和激酶(下图)的免疫印迹分析显示底物、激酶各自在磷酸化反应中的蛋白使用量基本一致。

Figure 1 An in vitro phosphorylation assay of 3 putative substrate proteins by MPK6 in Arabidopsis thaliana MPK6 was fused with the 6×His tag and the 3 putative substrates were fused with the Flag tag. After phosphorylation reaction, the proteins in the mixture were separated on a SDS- PAGE gel. The gel was dried and exposed to X-ray film (top). Immunoblotting with anti-His and anti-Flag antibodies were used to show the levels of the substrate (middle) and kinase (bottom) proteins in the reactions.

图2 Phos-tag gel和SDS-PAGE gel电泳后, 免疫印迹检测磷酸化蛋白的对比分析磷酸酶(如Calf intestine alkaline phosphatase, CIAP)可去除磷酸化蛋白上的磷酸基团。同一样品在CIAP处理前(-)、后(+), 磷酸化和非磷酸化形式的蛋白经Phos-tag gel (上图)和SDS- PAGE gel (下图)电泳后的免疫印迹分析结果。经Phos-tag gel电泳, 未加CIAP的样品中特定蛋白的磷酸化(P-form)和非磷酸化形式(None-P-form)被清晰地分开(上图); 加CIAP后磷酸化形式消失而非磷酸化形式条带增强。经SDS-PAGE gel电泳, 未加和加CIAP处理的样品中, 特定蛋白的磷酸化和非磷酸化形式分开不明显(下图)。

Figure 2 Immunoblotting detection of phospho-proteins in samples after Phos-tag gel and SDS-PAGE gel separation Phospho-proteins can be dephosphorylated by phosphatases (e.g. Calf intestine alkaline phosphatase, CIAP). Protein samples treated with (+) or without (-) CIAP were separated by Phos-tag (top) and SDS-PAGE (bottom) gels, and the specific protein was further detected by immunoblotting. The P-form and None-P-form of the protein were separated clearly by a Phos-tag gel (top), while the two forms were not separated by a SDS-PAGE gel (bottom). The missing of the upper band and the increasing of the bottom band after Phos-tag gel separation indicated that P-form protein was completely dephosphorylated by CIAP treatment.

图3 拟南芥幼苗总蛋白双向凝胶电泳后的磷酸化蛋白Pro-Q Diamond染色检测分析 2周龄拟南芥幼苗总蛋白提取液经双向凝胶电泳分离后, 用Pro-Q Diamond试剂对磷酸化蛋白进行染色(左图)。荧光扫描后的凝胶再进行考马斯亮蓝染色(blue-silver staining) (右图)。

Figure 3 Pro-Q staining assay of the phospho-proteins in total proteins extracted from Arabidopsis thaliana seedlings and separated by a 2D gel Two-week-old Arabidopsis thaliana seedlings were used for total protein extraction. The total proteins were separated by a 2D gel and the phospho-proteins were stained by Pro-Q (left). The gel was scanned with a Typhoon 9410 fluorescence scanner, and then stained with blue-silver to visualize the proteins (right).

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