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Identification of Novel in vivo MAP Kinase Substrates in Arabidopsis thaliana Through Use of Tandem Metal Oxide Affinity Chromatography

Open AccessPublished:November 20, 2012DOI:https://doi.org/10.1074/mcp.M112.020560
      Mitogen-activated protein kinase (MPK) cascades are important for eukaryotic signal transduction. They convert extracellular stimuli (e.g. some hormones, growth factors, cytokines, microbe- or damage-associated molecular patterns) into intracellular responses while at the same time amplifying the transmitting signal. By doing so, they ensure proper performance, and eventually survival, of a given organism, for example in times of stress. MPK cascades function via reversible phosphorylation of cascade components MEKKs, MEKs, and MPKs. In plants the identity of most MPK substrates remained elusive until now. Here, we provide a robust and powerful approach to identify and quantify, with high selectivity, site-specific phosphorylation of MPK substrate candidates in the model plant Arabidopsis thaliana. Our approach represents a two-step chromatography combining phosphoprotein enrichment using Al(OH)3-based metal oxide affinity chromatography, tryptic digest of enriched phosphoproteins, and TiO2-based metal oxide affinity chromatography to enrich phosphopeptides from complex protein samples. When applied to transgenic conditional gain-of-function Arabidopsis plants supporting in planta activation of MPKs, the approach allows direct measurement and quantification ex vivo of site-specific phosphorylation of several reported and many yet unknown putative MPK substrates in just a single experiment.
      Mitogen-activated protein kinase (MPK)
      The abbreviations used are:
      GO
      gene ontology
      MPK
      mitogen-activated protein kinase
      MEKK
      MEK-activating kinase
      MEK
      MPK-activating kinase
      MOAC
      metal oxide affinity chromatography
      Nt
      Nicotiana tabacum
      DEX
      dexamethasone
      MQ
      Max Quant
      PD
      Proteome Discoverer
      FDR
      false discovery rate
      PRS
      positive reference set
      RRS
      random reference set
      PCA
      principal component analysis
      ACN
      acetonitrile
      FA
      formic acid
      PEP
      posterior error probability.
      1The abbreviations used are:GO
      gene ontology
      MPK
      mitogen-activated protein kinase
      MEKK
      MEK-activating kinase
      MEK
      MPK-activating kinase
      MOAC
      metal oxide affinity chromatography
      Nt
      Nicotiana tabacum
      DEX
      dexamethasone
      MQ
      Max Quant
      PD
      Proteome Discoverer
      FDR
      false discovery rate
      PRS
      positive reference set
      RRS
      random reference set
      PCA
      principal component analysis
      ACN
      acetonitrile
      FA
      formic acid
      PEP
      posterior error probability.
      cascades are conserved signal amplification modules in eukaryotes (
      • Herskowitz I.
      MAP kinase pathways in yeast: For mating and more.
      ). They transduce extracellular stimuli (such as some hormones, growth factors, cytokines, microbe or damage-associated molecular patterns) into cellular responses. Signaling through MPK cascades requires stimulus-activated MPK kinase (MEK)-activating kinases (MEKKs) that activate their MEK substrates by phosphorylation. MEKs also use phosphorylation to activate their MPK substrates. While MPK cascades have been intensively studied over the past decade, in plants the identity of in vivo MPK substrates that ultimately cause the physiological changes, however, remained largely elusive. This presumably is because of the transient nature of the phosphorylation event and the ephemeral interaction between MPKs and their protein substrates. Compartmentalization and, thus, spatial separation of MPKs and their protein substrates may also impede identification of in vivo MPK substrates.
      Phosphoproteomics is a powerful tool to identify bona-fide MPK substrates. This is because it allows unbiased localization and site-specific quantification of in vivo phosphorylation of hundreds of proteins in a single experiment. In recent years enrichment of phosphorylated peptides using TiO2 and similar metal oxides on proteolytic digestion of the protein extract has become popular and was applied successfully (
      • Grimsrud P.A.
      • Swaney D.L.
      • Wenger C.D.
      • Beauchene N.A.
      • Coon J.J.
      Phosphoproteomics for the masses.
      ). However, despite the high sensitivity of modern mass spectrometers the large dynamic range of protein abundance and the transient nature of protein phosphorylation remained major difficulties in MS-based phosphoproteomics.
      Here, we used a novel approach combining dual metal oxide affinity chromatography (MOAC) of proteins and peptides with LC-MS/MS to identify and quantify site-specific phosphorylation of in vivo MPK substrate candidates in Arabidopsis thaliana. We took advantage of transgenic, conditional gain-of-function Arabidopsis plants harboring a gene for a constitutively active MEKmutant protein of Nicotiana tabacum (NtMEK2DD) under control of the dexamethasone (DEX)-inducible GVG promoter (
      • Ren D.
      • Yang H.
      • Zhang S.
      Cell death mediated by MAPK is associated with hydrogen peroxide production in Arabidopsis.
      ,
      • Liu Y.
      • Zhang S.
      Phosphorylation of 1-aminocyclopropane-1-carboxylic acid synthase by MPK6, a stress-responsive mitogen-activated protein kinase, induces ethylene biosynthesis in Arabidopsis.
      ). In the NtMEK2DD mutant protein amino acids serine (S) and threonine (T) are substituted by aspartic acid (D) within the phosphorylation site motif (S/T-X5-S/T) between kinase subdomains VII and VIII of NtMEK2. In GVG:FLAG-NtMEK2DD transgenic plants, DEX treatment leads to NtMEK2DD accumulation associated with constitutive and specific phosphorylation of the MEK2 target proteins AtMPK3 and AtMPK6 (
      • Liu Y.
      • Zhang S.
      Phosphorylation of 1-aminocyclopropane-1-carboxylic acid synthase by MPK6, a stress-responsive mitogen-activated protein kinase, induces ethylene biosynthesis in Arabidopsis.
      ) and their in vivo substrates. By combined use of GVG::FLAG-NtMEK2DD plants with a powerful consecutive enrichment of phosphoproteins and phosphopeptides followed by LC-MS/MS analysis, we directly measured site-specific phosphorylation of 141 putative and mostly novel MPK substrates. Among the MPK substrate candidates were previously unknown transcription coactivators, kinases other than MPKs, and other proteins with a role in cell signaling. Network analysis of MPK substrate candidates and their interaction partners disclosed significant enrichment of these groups of proteins. The analyses also closely link, for the first time, MPK signaling modules with the central oscillator of the circadian clock, TIME FOR COFFEE (TIC), and gating of red-light signaling to the clock, and to phototropism.

      CONCLUSION

      Dynamics of protein abundance in a given cell or tissue and the transient nature of the phosphorylation event are major challenges in phosphoproteomics. These obstacles limit the identification and site-specific quantification of the phosphorylation of low-abundant proteins, such as many MPK substrates with a crucial role in cell signaling. We introduce the dual MOAC-based enrichment of phosphopeptides from complex protein samples. The approach effectively targets the phosphate moiety of phosphoproteins and phosphopeptides and, thus allows probing of the phosphoproteome to unprecedented depth. Application of tandem MOAC disclosed the identity of numerous novel phosphorylation sites and potential in vivo targets of Arabidopsis MPKs, particularly of MPK3 and MPK6, in just a single experiment. Our findings decipher involvement of MPK substrates in important biological processes such as plant response to stress, circadian clock, phototropism, and morphogenesis.

      Acknowledgments

      We thank Shuqun Zhang for providing seeds of GVG::FLAG-NtMEK2DD transgenic plants. We appreciate financial support of EN by the European Marie-Curie-international training network MERIT. We thank the European Project PATHONET for financial support of WH, and we acknowledge the German Science Foundation (DFG) for supporting this project.

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