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. 2019 Nov 1;10:4996. doi: 10.1038/s41467-019-12847-6

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 5 — Flg22 induces BAK1-mediated NIK1 phosphorylation, which enhances NIK1’s affinity for receptors. a Flg22 perception triggers NIK1 rapid mobility shift. Protoplasts were transfected with NIK1-HA and treated with 100 nM flg22 for the indicated time points. Total input proteins were stained with Coomassie brilliant blue staining (CBB). b Verification of NIK1 in vivo phosphorylation by λPP treatment. Protein extracts from protoplasts transfected with NIK1-HA were treated with λPP following the standard protocol. c The kinase inhibitor K252a blocks flg22-induced NIK1 mobility shift. K252a was applied 1 h before flg22 treatment. Controls were solvent (DMSO) treatment. d Flg22-induced NIK1 phosphorylation requires FLS2 and its kinase activity. Protoplasts isolated from fls2 mutants were transfected with NIK1-HA and empty vector control, FLAG-tagged FLS2 or FLS2 kinase mutant (FLS2km). e Flg22-mediated NIK1 phosphorylation requires BAK1. NIK1-HA was expressed in protoplasts of Col-0 or bak1-4 mutants and flg22 was applied 10 min before samples collection. f Flg22-induced in vivo phosphorylation of NIK1 detected by different phospho-antibodies. Seedlings of NIK1-HA-overexpressing lines were treated with 100 nM flg22 for the indicated time points. NIK1-HA was immunoprecipitated from total protein extracts, fractionated by SDS-PAGE and immunoblotted with α-phosphoserine (α-pSer), α-phosphothreonine (α-Thr), α-phosphotyrosine (α-Tyr) and α-HA antibodies. g BAK1, but not FLS2, directly phosphorylates the NIK1 cytosolic domain. An in vitro kinase assay was performed using MBP-FLS2JK or MBP-BAK1JK as a kinase and GST-NIK1JKKm as the substrate. Phosphorylation was analysed by autoradiography (Upper), and the protein loading was shown by CBB (Lower). h NIK1 Thr474 is phosphorylated by BAK1 in vitro as shown by MS analysis. i Phosphorylation of NIK1 promotes its interaction with FLS2. FLS2-FLAG was co-expressed with NIK1-HA, NIK1Km-HA or NIK1-T474D-HA in protoplasts. Co-IP was performed with α-FLAG Agarose (IP: α-FLAG), and the proteins were immunoblotted with an α-HA antibody (IB: α-HA). j Quantitative data (n = 30 for h. k A NIK1 phosphomimetic form shows stronger interaction with BAK1. Co-IP assay was performed with the sample co-expressing BAK1-FLAG and T474A-HA or T474D-HA using α-FLAG Agarose. l Quantitative data from three biological replicates for j. Source data are provided as a Source Data file