A Highly Sensitive Detection Method, Phos-tag<sup>TM</sup> Affinity SDS-PAGE, Used to Analyze a Possible Substrate of CDPK-Related Protein Kinase5 in <i>Arabidopsis</i> — Oak Academic Publishing
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A Highly Sensitive Detection Method, Phos-tag<sup>TM</sup> Affinity SDS-PAGE, Used to Analyze a Possible Substrate of CDPK-Related Protein Kinase5 in <i>Arabidopsis</i>
State Key Laboratory of Hybrid Rice, Wuhan University, Wuhan, China
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State Key Laboratory of Hybrid Rice, Wuhan University, Wuhan, China
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State Key Laboratory of Hybrid Rice, Wuhan University, Wuhan, China
1 State Key Laboratory of Hybrid Rice, Wuhan University, Wuhan, China
2 State Key Laboratory of Hybrid Rice, Wuhan University, Wuhan, China
3 State Key Laboratory of Hybrid Rice, Wuhan University, Wuhan, China
Phosphorylation of proteins is an important post-translational modification. Me thods to determine the phosphorylation state of proteins are very important to evaluate diverse biological processes. CRK5 is the CDPK-related protein kinase in <i> Arabidopsis </i> , WD-repeat protein (WDRP) might be CRK5-interact-protein based on Y2H results. Here, we used bimolecular fluorescence complementation (BiFC) further to study and visualize the interaction between CRK5 and WDRP in living cells. Then, we combined Phos-tag <sup> TM </sup> SDS-PAGE with western blot (WB) analysis, using WDRP antibody and the anti-6×His antibody, to detect phosphorylated WDRP. This approach confirmed that WDRP might be phosphorylated by CRK5 <i> in vitro </i> . Site mutation analysis suggested that serine-70 might be the amino acid phosphorylated by CRK5 in WDRP. Cell extracts isolated from WT, OERK5, and <i> crk </i> 5 used to analyze the kinase reaction using recombinant WDRP as substrate. These results demonstrated that WDRP was phosphorylated by cell extracts and that there may be additional kinases that phosphorylate WDRP in <i> Arabidopsis </i> . Phos-tag <sup> TM </sup> SDS-PAGE thus provides a suitable and convenient method for analysis of phosphorylation in plants.
KeywordsCalcium-Dependent Protein Kinase (CDPK)CDPK-Related Protein Kinase (CRK)WD-Repeat Protein (WDRP)Protein PhosphorylationPhos-tag<sup>TM</sup>
Ubersax, J.A. and Ferrell Jr., J.E. (2007) Mechanisms of Specificity in Protein Phosphorylation. Nature Reviews Molecular Cell Biology, 8, 530-541. https://doi.org/10.1038/nrm2203
Takeda, H., Kawasaki, A., Takahashi, M., Yamada, A. and Koike, T. (2003) Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry of Phosphorylated Compounds Using a Novel Phosphate Capture Molecule. Rapid Communications of Mass Spectrometry, 17, 2075-2081. https://doi.org/10.1002/rcm.1154
Kinoshita, E., Kinoshita-Kikuta, E. and Koike, T. (2009) Separation and Detection of Large Phosphoproteins Using Phos-tag SDS-PAGE. Nature Protocols, 4, 1513-1521. https://doi.org/10.1038/nprot.2009.154
Kinoshita, E., Kinoshita-Kikuta, E. and Koike, T. (2015) Advances in Phos-tag-Based Methodologies for Separation and Detection of the Phosphoproteome. Biochimica et Biophysica Acta (BBA)—Proteins and Proteomics, 1854, 601-608. https://doi.org/10.1016/j.bbapap.2014.10.004
Xie, K., Chen, J., Wang, Q. and Yang, Y. (2014) Direct Phosphorylation and Activation of a Mitogen-Activated Protein Kinase by a Calcium-Dependent Protein Kinase in Rice. Plant Cell, 26, 3077-3089. https://doi.org/10.1105/tpc.114.126441
Sugiyama, Y., Katayama, S., Kameshita, I., Morisawa, K., Higuchi, T., Todaka, H., Kinoshita, E., Kinoshita-Kikuta, E., Koike, T., Taniguchi, T. and Sakamoto, S. (2015) Expression and Phosphorylation State Analysis of Intracellular Protein Kinases Using Multi-PK antibody and Phos-tag SDS-PAGE. MethodsX, 2, 469-474. https://doi.org/10.1016/j.mex.2015.11.007
Smékalová, V., Luptovciak, I., Komis, G., Samajová, O., Ovecka, M., Doskocilová, A., Takác, T., Vadovic, P., Novák, O., Pechan, T., Ziemann, A., Kosútová, P. and Samaj, J. (2014) Involvement of YODA and Mitogen Activated Protein Kinase 6 in Arabidopsis Post-Embryogenic Rootdevelopment through Auxin Up-Egulation and Cell Division Plane Orientation. New Phytologyist, 203, 1175-1193. https://doi.org/10.1111/nph.12880
Kinoshita, E., Kinoshita-Kikuta, E., Shiba, A., Edahiro, K., Inoue, Y., Yamamoto, K., Yoshida, M. and Koike, T. (2014) Profiling of Protein Thiophosphorylation by Phos-tag Affinity Electrophoresis: Evaluation of Adenosine 5’ -O-(3-thiotriphosphate) as a Phosphoryl Donor in Protein Kinase Reactions. Proteomics, 14, 668-679. https://doi.org/10.1002/pmic.201300533
Zheng, W., Li, J., Wang, S., Cao, S., Jiang, J., Chen, C., Ding, C., Qin, C., Ye, X., Gao, G.F. and Liu, W. (2015) Phosphorylation Controls the Nuclear-Cytoplasmic Shuttling of Influenza A Virus Nucleoprotein. Journal of Virology, 89, 5822-5834. https://doi.org/10.1128/JVI.00015-15
Chakrabandhu, K., Huault, S., Durivault, J., Lang, K., Ta Ngoc, L., Bole, A., Doma, E., Dérijard, B., Gérard, J.P., Pierres, M. and Hueber, A.O. (2016) An Evolution-Guided Analysis Reveals a Multi-Signaling Regulation of Fas by Tyrosine Phosphorylation and Its Implication in Human Cancers. PLOS Biology, 14, e1002401. http://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.1002401 https://doi.org/10.1371/journal.pbio.1002401
Schwamborn, R., Brown, E. and Haase, J. (2016) Elevation of Cortical Serotonin Transporter Activity upon Peripheral Immune Challenge Is Regulated Independently of p38 Mitogen-Activated Protein Kinase Activation and Transporter Phosphorylation. Journal of Neurochemistry, 137, 423-435. https://doi.org/10.1111/jnc.13596
Sutherland, C., MacDonald, J.A. and Walsh, M.P. (2016) Analysis of Phosphorylation of the Myosin-Targeting Subunit of Myosin Light Chain Phosphatase by Phos-tag SDS-PAGE. American Journal of Physiology-Cell Physiology, 310: C681-691. https://doi.org/10.1152/ajpcell.00327.2015
Kimura, T., Sharma, G., Ishiguro, K. and Hisanaga, S.I. (2018) Phospho-Tau Bar Code: Analysis of Phosphoisotypes of Tau and Its Application to Tauopathy. Frontiers in Neuroscience, 12, 44. https://doi.org/10.3389/fnins.2018.00044
Zhang, F., Xiang, L., Yu, Q., Zhang, H., Zhang, T., Zeng, J., Geng, C., Li, L., Fu, X., Shen, Q., Yang, C., Lan, X., Chen, M., Tang, K. and Liao, Z. (2017) Artemisinin Biosynthesis Promoting Kinase 1 Positively Regulates Artemisinin Biosynthesis through Phosphorylating AabZIP1. Journal of Experimental Botany, 69, 1109-1123. https://doi.org/10.1093/jxb/erx444
Chappellaz, M., Segboer, H., Ulke-Lemée, A., Sutherland, C., Chen, H.M. and MacDonald, J.A. (2018) Quantitation of Myosin Regulatory Light Chain Phosphorylation in Biological Samples with Multiple Reaction Monitoring Mass Spectrometry. Biochimica et Biophysica Acta (BBA)—Proteins and Proteomics, 1866, 608-616. https://doi.org/10.1016/j.bbapap.2018.03.008
Fujimoto, T., Kuwahara, T., Eguchi, T., Sakurai, M., Komori, T. and Iwatsubo, T. (2018) Parkinson’s Disease-Associated Mutant LRRK2 Phosphorylates Rab7L1 and Modifies Trans-Golgi Morphology. Biochemical and Biophysical Research Communications, 495, 1708-1715. https://doi.org/10.1016/j.bbrc.2017.12.024
van de Poel, S., Dreer, M., Velic, A., Macek, B., Baskaran, P., Iftner, T. and Stubenrauch, F. (2018) Identification and Functional Characterization of Phosphorylation Sites of the Human Papillomavirus 31 E8^E2 Protein. Journal of Virology, 92, e01743.
Wernimont, A.K., Artz, J.D., Finerty Jr., P., Lin, Y.H., Amani, M., Allali-Hassani, A., Senisterra, G., Vedadi, M., Tempel, W., Mackenzie, F., Chau, I., Lourido, S., Sibley, L.D. and Hui, R. (2010) Structures of Apicomplexan Calcium-Dependent Protein Kinases Reveal Mechanism of Activation by Calcium. Nature Structural & Molecular Biology, 17, 596-601. https://doi.org/10.1038/nsmb.1795
Ingram, J.R., Knockenhauer, K.E., Markus, B.M., Mandelbaum, J., Ramek, A., Shan, Y., Shaw, D.E., Schwartz, T.U., Ploegh, H.L. and Lourido, S. (2015) Allosteric Activation of Apicomplexan Calcium-Dependent Protein Kinases. Proceedings of the National Academy of Sciences of the United States of America, 112, E4975-E4984. https://doi.org/10.1073/pnas.1505914112
Hrabak, E.M., Chan, C.W.M., Gribskov, M., Harper, J.F., Choi, J.H., Halford, N., Kudla, J., Luan, S., Nimmo, H.G., Sussman, M.R., Thomas, M., Walker-Simmons, K., Zhu, J.-K. and Harmon, A.C. (2003) The Arabidopsis CDPK-SnRK Superfamily of Protein Kinases. Plant Physiology, 132, 666-680. https://doi.org/10.1104/pp.102.011999
Zhang, L. and Lu, Y.T. (2003) Calmodulin-Binding Protein Kinases in Plants. Trends in Plant Science, 8, 123-127. https://doi.org/10.1016/S1360-1385(03)00013-X
Du, W., Wang, Y., Liang, S.P. and Lu, Y.-T. (2005) Biochemical and Expression Analysis of an Arabidopsis Calcium-Dependent Protein Kinase-Related Kinase. Plant Science, 168, 1181-1192. https://doi.org/10.1016/j.plantsci.2004.12.019
Wang, Y., Liang, S., Xie, Q.-G. and Lu, Y.-T. (2004) Characterization of a Calmodulin-Regulated Ca2+-Dependent-Protein-Kinase-Related Protein Kinase, AtCRK1, from Arabidopsis. Biochemical Journal, 383, 73-81. https://doi.org/10.1042/BJ20031907
Rigó, G., Ayaydin, F., Tietz, O., Zsigmond, L., Kovács, H., Páy, A., Salchert, K., Darula, Z., Medzihradszky, K.F., Szabados, L., Palme, K., Koncz, C. and Cséplo, á. (2013) Inactivation of Plasma Membrane-Localized CDPK-RELATED KINASE5 Decelerates PIN2 Exocytosis and Root Gravitropic Response in Arabidopsis. Plant Cell, 25, 1592-1608. https://doi.org/10.1105/tpc.113.110452
Nemoto, K., Takemori, N., Seki, M., Shinozaki, K. and Sawasaki, T. (2015) Members of the Plant CRK Superfamily Are Capable of Trans- and Autophosphorylation of Tyrosine Residues. Journal of Biological Chemistry, 290, 16665-16677. https://doi.org/10.1074/jbc.M114.617274
Liese, A. and Romeis, T. (2013) Biochemical Regulation of in Vivo Function of Plant Calcium-Dependent Protein Kinases (CDPK). Biochimica et Biophysica Acta (BBA)—Molecular Cell Research, 1833, 1582-1589. https://doi.org/10.1016/j.bbamcr.2012.10.024
Ito, T., Nakata, M., Fukazawa, J., Ishida, S. and Takahashi, Y. (2014) Scaffold Function of Ca2+-Dependent Protein Kinase: Tobacco Ca2+-Dependent Protein KINASE1 Transfers 14-3-3 to the Substrate Repression of Shoot Growth after Phosphorylation. Plant Physiology, 165, 1737-1750. https://doi.org/10.1104/pp.114.236448
Ronzier, E., Corratgé-Faillie, C., Sanchez, F., Prado, K., Brière, C., Leonhardt, N., Thibaud, J.B. and Xiong, T.C. (2014) CPK13, a Noncanonical Ca2+-Dependent Protein Kinase, Specifically Inhibits KAT2 and KAT1 Shaker K+ Channels and Reduces Stomatal Opening. Plant Physiology, 166, 314-326. https://doi.org/10.1104/pp.114.240226
Li, R.J., Hua, W. and Lu, Y.T. (2006) Arabidopsis Cytosolic Glutamine Synthetase AtGLN1;1 Is a Potential Substrate of AtCRK3 Involved in Leaf Senescence. Biochemical and Biophysical Research Communication, 342, 119-126. https://doi.org/10.1016/j.bbrc.2006.01.100
Murashige, T. and Skoog, F. (1962) A Revised Medium for Rapid Growth and Bioassays with Tobacco Tissue Cultures. Physiologia Plantarum, 15, 473-497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
Koncz, C., Németh, K., Rédei, G.P. and Schell, J. (1992) T-DNA Insertional Mutagenesis in Arabidopsis. Plant Molecular Biology, 20, 963-976. https://doi.org/10.1007/BF00027166
Clough, S.J. and Bent, A.F. (1998) Floral Dip: A Simplified Method for Agrobacterium-Mediated Transformation of Arabidopsis thaliana. Plant Journal, 16, 735-743. https://doi.org/10.1046/j.1365-313x.1998.00343.x
Davis, A.M., Hall, A., Millar, A., Darrah, C. and Davis, S.J. (2009) Protocol: Streamlined Sub-Protocols for Floral-Dip Transformation and Selection of Transformants in Arabidopsis thaliana. Plant Methods, 5, 3-10. https://doi.org/10.1186/1746-4811-5-3
Lee, L.Y., Fang, M.J., Kuang, L.Y. and Gelvin, S.B. (2008) Vectors for Multi-Color Bimolecular Fluorescence Complementation to Investigate Protein-Protein Interactions in Living Plant Cells. Plant Methods, 4, 24. https://doi.org/10.1186/1746-4811-4-24
Huang, H.D., Lee, T.Y., Tzeng, S.W. and Horng, J.T. (2005) KinasePhos: A Web Tool for Identifying Protein Kinase-Specific Phosphorylation Sites. Nucleic Acids Research, 33, W226-W229. https://doi.org/10.1093/nar/gki471
Lu, Y.-T., Hidaka, H. and Feldman, L.J. (1996) Characterization of a Calcium/Calmodulin-Dependent Protein Kinase Homolog from Maize Roots Showing Light-Regulated Gravitropism. Planta, 199, 16-24. https://doi.org/10.1007/BF00196876
Li, D.F., Li, J., Ma, L., Zhang, L. and Lu, Y.T. (2006) Calmodulin Isoform Specific Activation of a Rice Calmodulin-Binding Kinase Conferred by Only Three Amino Acids of OsCaM61. FEBS Letters, 580, 4325-4331. https://doi.org/10.1016/j.febslet.2006.06.090
Krauß, S., So, J., Hambrock, M., Köhler, A., Kunath, M., Scharff, C., Wessling, M., Grzeschik, K.H., Schneider, R. and Schweiger, S. (2009) Point Mutations in GLI3 Lead to Misregulation of Its Subcellular Localization. PLoS ONE, 4, e7471. http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0007471 https://doi.org/10.1371/journal.pone.0007471
Yuan, E.T., Ino, Y., Kawaguchi, M., Kimura, Y., Hirano, H., Kinoshita-Kikuta, E., Kinoshita, E. and Koike, T. (2017) A Phos-Tag-Based Micropipette-Tip Method for Rapid and Selective Enrichment of Phosphopeptides. Electrophoresis, 38, 2447-2455. https://doi.org/10.1002/elps.201700175