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. 2013 Sep 5;4(10):771–781. doi: 10.1007/s13238-013-3053-6

Identification and functional analysis of phosphorylation residues of the Arabidopsis BOTRYTIS-INDUCED KINASE1

Jinhua Xu 1,2, Xiaochao Wei 1,2, Limin Yan 3, Dan Liu 1,2, Yuanyuan Ma 3, Yu Guo 2,4, Chune Peng 1,2, Honggang Zhou 2,4, Cheng Yang 2,4, Zhiyong Lou 3,✉, Wenqing Shui 1,2,✉
PMCID: PMC4875430  PMID: 24104392

Abstract

Arabidopsis BOTRYTIS-INDUCED KINASE1 (BIK1) is a receptor-like cytoplasmic kinase acting early in multiple signaling pathways important for plant growth and innate immunity. It is known to form a signaling complex with a cell-surface receptor FLS2 and a co-receptor kinase BAK1 to transduce signals upon perception of pathogen-associated molecular patterns (PAMPs). Although site-specific phosphorylation is speculated to mediate the activation and function of BIK1, few studies have been devoted to complete profiling of BIK1 phosphorylation residues. Here, we identified nineteen in vitro autophosphorylation sites of BIK1 including three phosphotyrosine sites, thereby proving BIK1 is a dual-specificity kinase for the first time. The kinase activity of BIK1 substitution mutants were explicitly assessed using quantitative mass spectrometry (MS). Thr-237, Thr-242 and Tyr-250 were found to most significantly affect BIK1 activity in autophosphorylation and phosphorylation of BAK1 in vitro. A structural model of BIK1 was built to further illustrate the molecular functions of specific phosphorylation residues. We also mapped new sites of FLS2 phosphorylation by BIK1, which are different from those by BAK1. These in vitro results could provide new hypotheses for more in-depth in vivo studies leading to deeper understanding of how phosphorylation contributes to BIK1 activation and mediates downstream signaling specificity.

Electronic Supplementary Material

Supplementary material is available for this article at 10.1007/s13238-013-3053-6 and is accessible for authorized users.

Keywords: phosphorylation, BIK1, receptor-like cytoplasmic kinase, quantitative mass spectrometry

Electronic supplementary material

13238_2013_3053_MOESM1_ESM.pdf (841.7KB, pdf)

Supplementary material, approximately 841 KB.

13238_2013_3053_MOESM2_ESM.pdf (38KB, pdf)

Supplementary material, approximately 38 KB.

Footnotes

These authors contributed equally to the work.

Electronic Supplementary Material

Supplementary material is available for this article at 10.1007/s13238-013-3053-6 and is accessible for authorized users.

Contributor Information

Zhiyong Lou, Email: louzy@xtal.tsinghua.edu.cn.

Wenqing Shui, Email: angelshui@nankai.edu.cn.

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Associated Data

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Supplementary Materials

13238_2013_3053_MOESM1_ESM.pdf (841.7KB, pdf)

Supplementary material, approximately 841 KB.

13238_2013_3053_MOESM2_ESM.pdf (38KB, pdf)

Supplementary material, approximately 38 KB.

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