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Proteomic characterization of MPK4 signaling network and putative substrates

Plant Molecular Biology volume 101, pages 325–339 (2019)Cite this article

Key message

Combining genetic engineering of MPK4 activity and quantitative proteomics, we established an in planta system that enables rapid study of MPK4 signaling networks and potential substrate proteins.

Abstract

Mitogen activated protein kinase 4 (MPK4) is a multifunctional kinase that regulates various signaling events in plant defense, growth, light response and cytokinesis. The question of how a single protein modulates many distinct processes has spurred extensive research into the physiological outcomes resulting from genetic perturbation of MPK4. However, the mechanism by which MPK4 functions is still poorly understood due to limited data on the MPK4 networks including substrate proteins and downstream pathways. Here we introduce an experimental system that combines genetic engineering of kinase activity and quantitative proteomics to rapidly study the signaling networks of MPK4. First, we transiently expressed a constitutively active (MPK4CA) and an inactive (MPK4IN) version of a Brassica napus MPK4 (BnMPK4) in Nicotiana benthamiana leaves. Proteomics analysis revealed that BnMPK4 activation affects multiple pathways (e.g., metabolism, redox regulation, jasmonic acid biosynthesis and stress responses). Furthermore, BnMPK4 activation also increased protein phosphorylation in the phosphoproteome, from which putative MPK4 substrates were identified. Using protein kinase assay, we validated that a transcription factor TCP8-like (TCP8) and a PP2A regulatory subunit TAP46-like (TAP46) were indeed phosphorylated by BnMPK4. Taken together, we demonstrated the utility of proteomics and phosphoproteomics in elucidating kinase signaling networks and in identification of downstream substrates.

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Acknowledgements

The authors thank Dr. Xiaoen Huang for providing N. benthamiana seeds, Dr. Alice Harmon for helpful discussions, and Dr. Joshua A. Silveira for mass spectrometry data acquisition. This research was supported by Grants from the National Science Foundation (MCB 0818051 and MCB 1412547 to S. Chen).

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  1. Tong Zhang

    Present address: Biological Sciences Division, Pacific Northwest National Lab, Richland, WA, 99354, USA

Authors and Affiliations

  1. Department of Biology, University of Florida, Gainesville, FL, 32611, USA

    Tong Zhang, Shweta Chhajed, Jacqueline D. Schneider & Sixue Chen

  2. Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL, 32610, USA

    Guanqiao Feng, Wen-Yuan Song & Sixue Chen

  3. Department of Plant Pathology, University of Florida, Gainesville, FL, 32611, USA

    Wen-Yuan Song

  4. Genetics Institute, University of Florida, Gainesville, FL, 32610, USA

    Wen-Yuan Song & Sixue Chen

  5. Proteomics and Mass Spectrometry, Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, FL, 32610, USA

    Sixue Chen

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Contributions

T.Z., W.S., and S. Chen developed the experimental plan, and T.Z. performed most of the experiments and data analysis. S. Chhajed conducted protein expression and in-solution kinase assay. J.D.S. performed infiltration and sample collection. G.F. built the Nicotiana database and assisted with bioinformatics analysis. T.Z. drafted the manuscript and S. Chen finalized the paper. All authors reviewed and approved the manuscript.

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Correspondence to Sixue Chen.

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11103_2019_908_MOESM1_ESM.pdf

Supplementary material 1 (PDF 446 kb) Fig. S1. MPK4CA expressed in N. benthamiana shows strong phosphorylation on MKS1. In-gel kinase assay, with MKS1 embedded in the gel as a substrate, was used to determine the kinase activity of MPK4s expressed in N. benthamiana leaves 2 days after infiltrationFig. S2. Quality control metrics of the proteomics data. a Accuracy of measurement in m/z. The Δm/z, the difference between measured and theoretical m/z, was plotted against the number of obtained peptide spectral matches (PSMs). b Distribution of XCorr over PSM charge status. c Distribution of signal to noise level of TMT quantification. d Distribution of delta CnFig. S3. Correlation of TMT reporter ion quantification. The correlation among three biological replicates of MPK4CA and MPK4IN samples in global (a and b) and phosphoproteomics (c and d) were plotted. The Pearson correlation coefficient values were shown on the upper right of each plotFig. S4. Heatmap showing the protein fold changes (log2 transformed) of significant proteins involved in stress response. The protein abundance of MPK4CA is normalized to that of MPKIN (median of 0, thus not shown here)Fig. S5. Heatmap showing the protein fold changes (log2 transformed) of significant proteins involved in oxylipin biosynthetic process. The protein abundance of MPK4CA is normalized to that of MPKIN (median of 0, thus not shown here)Fig. S6. Pathway analysis of significantly changed proteins at the global level. Decreased and increased proteins were enriched separately, and the obtained pathways were shown in blue and red, respectivelyFig. S7. Analysis of the MPK4-perturbed phosphoproteome. Distribution patterns of phosphoproteins over the number of phosphopeptides for all the identified phosphoproteins (a) and statistically significant phosphoproteins (b) were plotted. Correlations, in term of fold change, between 2 phosphopeptides that identify the same proteins were plotted for the 208 proteins from the entire phosphoprotein dataset (c) and 67 proteins from the significant phosphoprotein list (d)Fig. S8. Correlation between protein fold change at the phosphorylation and total (global) levels. x axis: log2 fold change of the total protein level; y axis: log2 fold change of the phosphorylation level; black diagonal line: y = x to indicate same levels of change. Majority (207 out of 212) showed a shift toward the left of the y = x line, indicating that increase at the phosphorylation level is much higher than the increase at the total protein level. Of note, 25 % (54 out of 212) showed a decrease at total protein level (blue dots). Only 4% (9 out of 212) showed increase at both levels (red dots). The remaining showed significant increase at phosphorylation level, but insignificant increase at total protein level (black dots)Fig. S9. Pathway analysis of significantly changed proteins at the phosphorylation level. Decreased and increased phosphoproteins were enriched separately, and the obtained pathways were shown in blue and red, respectively

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Zhang, T., Chhajed, S., Schneider, J.D. et al. Proteomic characterization of MPK4 signaling network and putative substrates. Plant Mol Biol 101, 325–339 (2019). https://doi.org/10.1007/s11103-019-00908-9

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Keywords

  • MPK4
  • Proteomics
  • Phosphoproteomics
  • Kinase network
  • Substrate