利用激酶客户测定法鉴定拟南芥中的丝裂原活化蛋白激酶底物。

Plant signaling & behavior Pub Date : 2024-12-31 Epub Date: 2024-03-17 DOI:10.1080/15592324.2024.2326238
Sunghwa Bahk, Nagib Ahsan, Jonguk An, Sun Ho Kim, Zakiyah Ramadany, Jong Chan Hong, Jay J Thelen, Woo Sik Chung
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引用次数: 0

摘要

丝裂原活化蛋白激酶(MPK)级联是控制所有真核生物各种细胞反应的重要信号转导元件。MPK 通过下游底物的磷酸化将细胞外刺激转化为细胞反应。虽然 MPK 级联被认为非常复杂,但在植物中只发现了有限数量的 MPK 底物。在这里,我们使用激酶客户(KiC)测定法来鉴定 MPK3 和 MPK6 的新型底物。用代表体内磷酸化位点的大型合成肽库对重组 MPK3 或 MPK6 进行测试,并通过高分辨率串联质谱鉴定磷酸化肽。通过这一筛选,我们分别鉴定出了 23 个和 21 个 MPK3 和 MPK6 的假定客户肽。为了验证推定客户肽的磷酸化情况,我们用分离出的客户肽的重组融合蛋白进行了体外激酶试验。我们发现,分别有 13 个和 9 个重组蛋白被 MPK3 和 MPK6 磷酸化。其中,11 个蛋白被证明是两种 MPK 的新型底物。这项研究表明,KiC测定是一种鉴别MPK新底物的有效方法。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Identification of mitogen-activated protein kinases substrates in Arabidopsis using kinase client assay.

Mitogen-activated protein kinase (MPK) cascades are essential signal transduction components that control a variety of cellular responses in all eukaryotes. MPKs convert extracellular stimuli into cellular responses by the phosphorylation of downstream substrates. Although MPK cascades are predicted to be very complex, only limited numbers of MPK substrates have been identified in plants. Here, we used the kinase client (KiC) assay to identify novel substrates of MPK3 and MPK6. Recombinant MPK3 or MPK6 were tested against a large synthetic peptide library representing in vivo phosphorylation sites, and phosphorylated peptides were identified by high-resolution tandem mass spectrometry. From this screen, we identified 23 and 21 putative client peptides of MPK3 and MPK6, respectively. To verify the phosphorylation of putative client peptides, we performed in vitro kinase assay with recombinant fusion proteins of isolated client peptides. We found that 13 and 9 recombinant proteins were phosphorylated by MPK3 and MPK6. Among them, 11 proteins were proven to be the novel substrates of two MPKs. This study suggests that the KiC assay is a useful method to identify new substrates of MPKs.

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