Abstract
Phosphorylation of a substrate by protein kinases leads to the activation or inactivation of numerous signaling pathways and metabolic processes. The assessment of kinase activity by using a specific or generic substrate plays a crucial role in characterization of kinase specificity and activity. Here we describe a protocol using either a synthetic peptide as a specific substrate or using myelin basic protein (MBP) as a generic substrate for the kinase activity assay. The kinase of interest is fused with a GFP (green fluorescent protein) tag and can be purified by GFP magnetic beads. Kinase–GFP complexes are then incubated with ATP, substrate, and coordinated reaction reagent for the kinase reaction. The assay is then quantified through mass spectrometry or enzymatic luminescence.
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References
Zulawski M, Schulze G, Braginets R, Hartmann S, Schulze WX (2014) The Arabidopsis Kinome: phylogeny and evolutionary insights into functional diversification. BMC Genomics 15:548
van Wijk KJ, Friso G, Walther D, Schulze WX (2014) Meta-analysis of Arabidopsis thaliana phospho-proteomics data reveals compartmentalization of phosphorylation motifs. Plant Cell 26(6):2367–2389
Ardito F, Giuliani M, Perrone D, Troiano G, Lo Muzio L (2017) The crucial role of protein phosphorylation in cell signaling and its use as targeted therapy (Review). Int J Mol Med 40(2):271–280
Trentini DB, Fuhrmann J, Mechtler K, Clausen T (2014) Chasing phosphoarginine proteins: development of a selective enrichment method using a phosphatase trap. Mol Cell Proteomics 13(8):1953–1964
Elkhadragy L, Long W (2019) A radioactive in vitro ERK3 kinase assay. Bio Protoc 9(16):e3332
Kim HS, Bian X, Lee CJ, Kim SE, Park SC, Xie Y, Guo X, Kwak SS (2019) IbMPK3/IbMPK6-mediated IbSPF1 phosphorylation promotes tolerance to bacterial pathogen in sweetpotato. Plant Cell Rep 38(11):1403–1415
Lee HY, Yoon GM (2017) Kinase assay for CONSTITUTIVE TRIPLE RESPONSE 1 (CTR1) in Arabidopsis thaliana. Methods Mol Biol 1573:133–140
Tan S, Abas M, Verstraeten I, Glanc M, Molnar G, Hajny J, Lasak P, Petrik I, Russinova E, Petrasek J, Novak O, Pospisil J, Friml J (2020) Salicylic acid targets protein phosphatase 2A to attenuate growth in plants. Curr Biol 30(3):381–395.e388
Kersten B, Agrawal GK, Iwahashi H, Rakwal R (2006) Plant phosphoproteomics: a long road ahead. Proteomics 6(20):5517–5528
Willmann R, Haischer DJ, Gust AA (2014) Analysis of MAPK activities using MAPK-specific antibodies. Methods Mol Biol 1171:27–37
Wurzinger B, Mair A, Fischer-Schrader K, Nukarinen E, Roustan V, Weckwerth W, Teige M (2017) Redox state-dependent modulation of plant SnRK1 kinase activity differs from AMPK regulation in animals. FEBS Lett 591(21):3625–3636
Zhang M, Su J, Zhang Y, Xu J, Zhang S (2018) Conveying endogenous and exogenous signals: MAPK cascades in plant growth and defense. Curr Opin Plant Biol 45(Pt A):1–10
Hemmila II (1999) LANCEtrade mark: homogeneous assay platform for HTS. J Biomol Screen 4(6):303–308
Mathis G (1995) Probing molecular interactions with homogeneous techniques based on rare earth cryptates and fluorescence energy transfer. Clin Chem 41(9):1391–1397
Seethala R, Menzel R (1997) A homogeneous, fluorescence polarization assay for src-family tyrosine kinases. Anal Biochem 253(2):210–218
Wu JJ (2002) Comparison of SPA, FRET, and FP for kinase assays. Methods Mol Biol 190:65–85
Jia Y, Gu XJ, Brinker A, Warmuth M (2008) Measuring the tyrosine kinase activity: a review of biochemical and cellular assay technologies. Expert Opin Drug Discovery 3(8):959–978
Gronborg M, Kristiansen TZ, Stensballe A, Andersen JS, Ohara O, Mann M, Jensen ON, Pandey A (2002) A mass spectrometry-based proteomic approach for identification of serine/threonine-phosphorylated proteins by enrichment with phospho-specific antibodies: identification of a novel protein, Frigg, as a protein kinase A substrate. Mol Cell Proteomics 1(7):517–527
Peck SC (2006) Analysis of protein phosphorylation: methods and strategies for studying kinases and substrates. Plant J 45(4):512–522
Brumlik MJ, Wei S, Finstad K, Nesbit J, Hyman LE, Lacey M, Burow ME, Curiel TJ (2004) Identification of a novel mitogen-activated protein kinase in Toxoplasma gondii. Int J Parasitol 34(11):1245–1254
Ghosh AS, Ray D, Dutta S, Raha S (2010) EhMAPK, the mitogen-activated protein kinase from Entamoeba histolytica is associated with cell survival. PLoS One 5(10):e13291
Jiang L, Anderson JC, Gonzalez Besteiro MA, Peck SC (2017) Phosphorylation of Arabidopsis MAP kinase phosphatase 1 (MKP1) is required for PAMP responses and resistance against bacteria. Plant Physiol 175(4):1839–1852
Patel A, Chojnowski AN, Gaskill K, De Martini W, Goldberg RL, Siekierka JJ (2011) The role of a Brugia malayi p38 MAP kinase ortholog (Bm-MPK1) in parasite anti-oxidative stress responses. Mol Biochem Parasitol 176(2):90–97
Zeng N, D’Souza RF, Figueiredo VC, Markworth JF, Roberts LA, Peake JM, Mitchell CJ, Cameron-Smith D (2017) Acute resistance exercise induces Sestrin2 phosphorylation and p62 dephosphorylation in human skeletal muscle. Physiol Rep 5(24)
Zhang J, Gao J, Zhu Z, Song Y, Wang X, Wang X, Zhou X (2020) MKK4/MKK5-MPK1/MPK2 cascade mediates SA-activated leaf senescence via phosphorylation of NPR1 in Arabidopsis. Plant Mol Biol 102(4–5):463–475
Lu SX, Hrabak EM (2013) The myristoylated amino-terminus of an Arabidopsis calcium-dependent protein kinase mediates plasma membrane localization. Plant Mol Biol 82(3):267–278
Minkoff BB, Makino SI, Haruta M, Beebe ET, Wrobel RL, Fox BG, Sussman MR (2017) A cell-free method for expressing and reconstituting membrane proteins enables functional characterization of the plant receptor-like protein kinase FERONIA. J Biol Chem 292(14):5932–5942
Wu XN, Chu L, Xi L, Pertl-Obermeyer H, Li Z, Sklodowski K, Sanchez-Rodriguez C, Obermeyer G, Schulze WX (2019) Sucrose-induced receptor kinase 1 is modulated by an interacting kinase with short extracellular domain. Mol Cell Proteomics 18(8):1556–1571
Wu XN, Sanchez Rodriguez C, Pertl-Obermeyer H, Obermeyer G, Schulze WX (2013) Sucrose-induced receptor kinase SIRK1 regulates a plasma membrane aquaporin in Arabidopsis. Mol Cell Proteomics 12(10):2856–2873
Rappsilber J, Ishihama Y, Mann M (2003) Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics. Anal Chem 75(3):663–670
ADP-Glo™ kinase assay technical manual (2020) Promega Corporation. https://www.promega.de/-/media/files/resources/protocols/technical-manuals/0/adp-glo-kinase-assay-protocol.pdf?la=en. Accessed 3 Aug 2020
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Wang, J., Yang, X., Xi, L., Wu, X.N. (2021). Kinase Activity Assay Using Unspecific Substrate or Specific Synthetic Peptides. In: Wu, X.N. (eds) Plant Phosphoproteomics. Methods in Molecular Biology, vol 2358. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1625-3_17
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DOI: https://doi.org/10.1007/978-1-0716-1625-3_17
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