Identification and validation of new ERK substrates by phosphoproteomic technologies including Phos-tag SDS-PAGE
Identification and validation of new ERK substrates by phosphoproteomic technologies including Phos-tag SDS-PAGE
复制标题
通过磷酸化蛋白质组学技术（包括 Phos-tag SDS-PAGE）鉴定和验证新的 ERK 底物
DOI：
10.1016/j.jprot.2022.104543
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发表时间：
2022
影响因子：
3.3
通讯作者：
Kosako Hidetaka
中科院分区：
文献类型：
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作者：
Yoshikawa Harunori;Nishino Kohei;Kosako Hidetaka
细胞外信号调节激酶（ERK）是丝裂原活化蛋白（MAP）激酶家族的一员，通过磷酸化大量底物来调控各种细胞过程。虽然许多研究已经扩大了ERK底物的数量，但很可能还有其他底物有待发现。在这里，我们已经采用了定量磷酸蛋白质组学的方法来探索新的ERK底物在NIH 3 T3成纤维细胞稳定表达的融合蛋白之间的B-Raf和雌激素受体。在ERK依赖的磷酸化靶点中，我们重点关注NGFI-A结合蛋白2（Nab 2），叉头盒蛋白K1（Foxk 1）和大细胞相关蛋白5（Dlgap 5/HURP）。Phos标记的SDS-PAGE和Western印迹证实了这三种蛋白在细胞中的ERK依赖性磷酸化。PHOS标记的SDS-PAGE显示这些蛋白质被活性ERK高度磷酸化。此外，凝胶内消化的磷酸化蛋白条带从Phos-tag的SDS-PAGE，然后通过LC-MS/MS表明，活性ERK直接磷酸化相同的位点在体外细胞中观察到的。本研究证明了Phos标记的SDS-PAGE用于验证蛋白激酶的候选底物的有用性。SignificanceLabel-free定量磷酸化蛋白质组学鉴定了来自840种蛋白质的1439种磷酸化肽，这些蛋白质在小鼠成纤维细胞中被ERK激活显着增加。通过基因本体和通路分析，我们选择了三个蛋白质参与转录调控和/或肿瘤发生。这些蛋白的磷酸化位点与ERK共有基序一致，并在体外被活性ERK直接磷酸化。Phos-tag SDS-PAGE可用于检测细胞内和体外ERK介导的这些底物的磷酸化。这些新的ERK底物的进一步表征将需要更好地了解ERK信号通路，我们的磷酸化蛋白质组学数据为研究ERK下游底物提供了有用的信息。
The extracellular signal-regulated kinase (ERK), a member of the mitogen-activated protein (MAP) kinase family, governs various cellular processes by phosphorylating a large set of substrates. Although many studies have expanded the number of ERK substrates, it is likely that additional substrates remain to be discovered. Here we have employed a quantitative phosphoproteomic approach to explore novel ERK substrates in NIH3T3 fibroblasts stably expressing a fusion protein between B-Raf and estrogen receptor. Among ERK-dependent phosphorylation targets, we focused on NGFI-A-binding protein 2 (Nab2), forkhead box protein K1 (Foxk1), and Disks large-associated protein 5 (Dlgap5/HURP). Phos-tag SDS-PAGE followed by Western blotting confirmed ERK-dependent phosphorylation of these three proteins in cells. Phos-tag SDS-PAGE ofin vitrokinase assay samples revealed high degrees of phosphorylation of these proteins by active ERK. Furthermore, in-gel digestion of the phosphorylated protein bands from Phos-tag SDS-PAGE followed by LC-MS/MS indicated that active ERK directly phosphorylates the same sitesin vitroas those observed in cells. This study demonstrates the usefulness of Phos-tag SDS-PAGE for validation of candidate substrates of protein kinases.SignificanceLabel-free quantitative phosphoproteomics identified 1439 phosphopeptides derived from 840 proteins that were significantly increased by ERK activation in mouse fibroblasts. Through gene ontology and pathway analysis, we selected three proteins involved in transcriptional regulation and/or tumorigenesis. The identified phosphorylation sites of these proteins conform to the ERK consensus motif and were directly phosphorylated by active ERKin vitro. Phos-tag SDS-PAGE was useful for detecting ERK-mediated phosphorylation of these substrates both in cells andin vitro. Further characterization of these new ERK substrates will be needed to better understand the ERK signaling pathway, and our phosphoproteomic data provide useful information for studying downstream substrates of ERK.