Abstract
Phosphorylation/dephosphorylation is a key posttranslational mechanism for signal transduction and amplification. Several techniques exist for assessing protein phosphorylation status, but each has its own drawbacks. The fast, straightforward, and low-tech approach described here uses transient overexpression of peptide-tagged proteins in Arabidopsis leaf mesophyll protoplasts and immunoblotting with Phos-tag™ SDS-PAGE and commercial anti-tag antibodies. We illustrate this with two relevant examples related to the SnRK1 protein kinase, which mediates metabolic stress signaling: Arabidopsis thaliana SnRK1 activation by T-loop (auto-)phosphorylation and SnRK1 phosphorylation of the Arabidopsis RAV1 transcription factor, which is involved in seed germination and early seedling development.
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References
Hasan MM, Liu XD, Waseem M, Guang-Qian Y, Alabdallah NM, Jahan MS, Fang XW (2022) ABA activated SnRK2 kinases: an emerging role in plant growth and physiology. Plant Signal Behav 17:1–6
Liu J, Ishitani M, Halfter U, Kim CS, Zhu JK (2000) The Arabidopsis thaliana SOS2 gene encodes a protein kinase that is required for salt tolerance. Proc Natl Acad Sci U S A 97:3730–3734
Broeckx T, Hulsmans S, Rolland F (2016) The plant energy sensor: evolutionary conservation and divergence of SnRK1 structure, regulation, and function. J Exp Bot 67:6215–6252
Crepin N, Rolland F (2019) SnRK1 activation, signaling, and networking for energy homeostasis. Curr Opin Plant Biol 51:29–36
Nukarinen E, Nagele T, Pedrotti L, Wurzinger B, Mair A, Landgraf R, Bornke F, Hanson J, Teige M, Baena-Gonzalez E, Dröger-Laser W, Weckwerth W (2016) Quantitative phosphoproteomics reveals the role of the AMPK plant ortholog SnRK1 as a metabolic master regulator under energy deprivation. Sci Rep 6:31697
Cho HY, Wen TN, Wang YT, Shih MC (2016) Quantitative phosphoproteomics of protein kinase SnRK1 regulated protein phosphorylation in Arabidopsis under submergence. J Exp Bot 67:2745–2760
Carianopol CS, Chan AL, Dong S et al (2020) An abscisic acid-responsive protein interaction network for sucrose non-fermenting related kinase1 in abiotic stress response. Commun Biol 3:1–15
Van Leene J, Eeckhout D, Gadeyne A, Matthijs HC, De Winne N, Persiau G, Van De Slijke E, Persyn F, Mertens T, Smagghe W, Crepin N, Broucke E, Van Damme D, Pleskot R, Rolland F, De Jaeger G (2022) Mapping of the plant SnRK1 kinase signaling network reveals a key regulatory role for the class II T6P synthase-like proteins. Nat Plants 8(11):1245–1261
Yoo SD, Cho YH, Sheen J (2007) Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Protoc 2:1565–1572
Weekes J, Ball KL, Caudwell FB, Hardie DG (1993) Specificity determinants for the Amp-activated protein-kinase and its plant homolog analyzed using synthetic peptides. FEBS Lett 334:335–339
Halford NG, Hey S, Jhurreea D, Laurie S, McKibbin RS, Paul M, Zhang Y (2003) Metabolic signalling and carbon partitioning: role of Snf1-related (SnRK1) protein kinase. J Exp Bot 54:382–375
Johnson LN, Noble ME, Owen DJ (1996) Active and inactive protein kinases: structural basis for regulation. Cell 85:149–158
Kannan N, Neuwald AF (2005) Did protein kinase regulatory mechanisms evolve through elaboration of a simple structural component? J Mol Biol 351:956–972
Nolen B, Taylor S, Ghosh G (2004) Regulation of protein kinases; controlling activity through activation segment conformation. Mol Cell 15:661–675
Iyer GH, Garrod S, Woods VL Jr, Taylor SS (2005) Catalytic independent functions of a protein kinase as revealed by a kinase-dead mutant: study of the Lys72His mutant of cAMP-dependent kinase. J Mol Biol 351:1110–1122
Baena-Gonzalez E, Rolland F, Thevelein JM, Sheen J (2007) A central integrator of transcription networks in plant stress and energy signaling. Nature 448:938–942
Ramon M, Dang TVT, Broeckx T, Hulsmans S, Crepin N, Sheen J, Rolland F (2019) Default activation and nuclear translocation of the plant cellular energy sensor SnRK1 regulate metabolic stress responses and development. Plant Cell 31:1614–1632
Glab N, Oury C, Guerinier T, Domenichini S, Crozet P, Thomas M, Vidal J, Hodges M (2017) The impact of Arabidopsis thaliana SNF1-related-kinase 1 (SnRK1)-activating kinase 1 (SnAK1) and SnAK2 on SnRK1 phosphorylation status: characterization of a SnAK double mutant. Plant J 89:1031–1041
Wang P, Yan Y, Bai Y, Dong Y, Wei Y, Zeng H, Shi H (2021) Phosphorylation of RAV1/2 by KIN10 is essential for transcriptional activation of CAT6/7, which underlies oxidative stress response in cassava. Cell Rep 37:1–19
Sheen J (1996) Ca2+-dependent protein kinases and stress signal transduction in plants. Science 274:1900–1902
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Broucke, E., Rolland, F., Crepin, N. (2023). Fast Identification of In Vivo Protein Phosphorylation Events Using Transient Expression in Leaf Mesophyll Protoplasts and Phos-tagTM SDS-PAGE. In: Couée, I. (eds) Plant Abiotic Stress Signaling. Methods in Molecular Biology, vol 2642. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3044-0_12
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DOI: https://doi.org/10.1007/978-1-0716-3044-0_12
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