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A chemical-genetic approach to elucidate protein kinase function in planta

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Abstract

The major objective in protein kinase research is the identification of the biological process, in which an individual enzyme is integrated. Protein kinase-mediated signalling is thereby often addressed by single knock-out mutation- or co-suppression-based reverse genetics approaches. If a protein kinase of interest is a member of a multi gene family, however, no obvious phenotypic alteration in the morphology or in biochemical parameters may become evident because mutant phenotypes may be compensated by functional redundancy or homeostasis. Here we establish a chemical-genetic screen combining ATP-analogue sensitive (as) kinase variants and molecular fingerprinting techniques to study members of the plant calcium-dependent protein kinase (CDPK) family in vivo. CDPKs have been implicated in fast signalling responses upon external abiotic and biotic stress stimuli. CDPKs carrying the as-mutation did not show altered phosphorylation kinetics with ATP as substrate, but were able to use ATP analogues as phosphate donors or as kinase inhibitors. For functional characterization in planta, we have substituted an Arabidopsis thaliana mutant line of AtCPK1 with the respective as-variant under the native CPK1 promoter. Seedlings of Arabidopsis wild type and AtCPK1 as-lines were treated with the ATP analogue inhibitor 1-NA-PP1 and exposed to cold stress conditions. Rapid cold-induced changes in the phosphoproteome were analysed by 2D-gel-electrophoresis and phosphoprotein staining. The comparison between wild type and AtCPK1 as-plants before and after inhibitor treatment revealed differential CPK1-dependent and cold-stress-induced phosphoprotein signals. In this study, we established the chemical-genetic approach as a tool, which allows the investigation of plant-specific classes of protein kinases in planta and which facilitates the identification of rapid changes of molecular biomarkers in kinase-mediated signalling networks.

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References

  • Aboelsaad M, Wu R (1995) A rice membrane calcium-dependent protein-kinase is induced by gibberellin. Plant Physiol 108:787–793

    Article  CAS  Google Scholar 

  • Allen J, Lazerwith S, Shokat K (2005) Bio-orthogonal affinity purification of direct kinase substrates. J Am Chem Soc 127:5288–5289

    Article  PubMed  CAS  Google Scholar 

  • Asano T, Tanaka N, Yang G, Hayashi N, Komatsu S (2005) Genome-wide identification of the rice calcium-dependent protein kinase and its closely related kinase gene families: comprehensive analysis of the CDPKs gene family in rice. Plant Cell Physiol 46:356–366

    Article  PubMed  CAS  Google Scholar 

  • Bishop AC, Ubersax JA, Petsch DT, Matheos DP, Gray NS, Blethrow J, Shimizu E, Tsien JZ, Schultz PG, Rose MD, Wood JL, Morgan DO, Shokat KM (2000) A chemical switch for inhibitor-sensitive alleles of any protein kinase. Nature 407:395–401

    Article  PubMed  CAS  Google Scholar 

  • Bishop AC, Buzko O, Shokat KM (2001) Magic bullets for protein kinases. Trends Cell Biol 11:167–172

    Article  PubMed  CAS  Google Scholar 

  • Böhmer M, Kurth J, Witte CP, Romeis T (2006) Function of plant calcium-dependent protein kinases in the activation of abiotic and pathogen-related stress responses and its potential application in the generation of stress-resistant plants floriculture, ornamental and plant biotechnology: advances and topical issues, 1st edn. Global Science Books, London, pp 367–372

  • Botella JR, Arteca JM, Somodevilla M, Arteca RN (1996) Calcium-dependent protein kinase gene expression in response to physical and chemical stimuli in mungbean (Vigna radiata). Plant Mol Biol 30:1129–1137

    Article  PubMed  CAS  Google Scholar 

  • Brodersen P, Petersen M, Bjorn Nielsen H, Zhu S, Newman MA, Shokat KM, Rietz S, Parker J, Mundy J (2006) Arabidopsis MAP kinase 4 regulates salicylic acid- and jasmonic acid/ethylene-dependent responses via EDS1 and PAD4. Plant J 47:532–546

    Article  PubMed  CAS  Google Scholar 

  • Chico JM, Raices M, Tellez-Inon MT, Ulloa RM (2002) A calcium-dependent protein kinase is systemically induced upon wounding in tomato plants. Plant Physiol 128:256–270

    Article  PubMed  CAS  Google Scholar 

  • Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743

    Article  PubMed  CAS  Google Scholar 

  • Collins MO, Yu L, Husi H, Blackstock WP, Choudhary JS, Grant SG (2005) Robust enrichment of phosphorylated species in complex mixtures by sequential protein and peptide metal-affinity chromatography and analysis by tandem mass spectrometry. Sci STKE 298:l6

    Google Scholar 

  • Davletova S, Meszaros T, Miskolczi P, Oberschall A, Torok K, Magyar Z, Dudits D, Deak M (2001) Auxin and heat shock activation of a novel member of the calmodulin like domain protein kinase gene family in cultured alfalfa cells. J Exp Bot 52:215–221

    Article  PubMed  CAS  Google Scholar 

  • Dephoure N, Howson RW, Blethrow JD, Shokat KM, O′Shea EK (2005) Combining chemical genetics and proteomics to identify protein kinase substrates. PNAS 102:17940–17945

    Article  PubMed  CAS  Google Scholar 

  • Eblen ST (2003) Identification of novel ERK2 substrates through use of an engineered kinase and ATP analogs. J Biol Chem 278:14926–14935

    Article  PubMed  CAS  Google Scholar 

  • Estruch JJ, Kadwell S, Merlin E, Crossland L (1994) Cloning and characterization of a maize pollen-specific calcium-dependent calmodulin-independent protein kinase. Proc Natl Acad Sci USA 91:8837–8841

    Article  PubMed  CAS  Google Scholar 

  • Gevaert K, Damme PV, Ghesquière B, Impens F, Martens L, Helsens K, Vandekerckhove J (2007) A la carte proteomics with an emphasis on gel-free techniques. Proteomics 7(16):2698–2718

    Article  PubMed  CAS  Google Scholar 

  • Habelhah H, Shah K, Huang L, Burlingame AL, Shokat KM, Ronai Z (2001) Identification of new JNK substrate using ATP pocket mutant JNK and a corresponding ATP analogue. J Biol Chem 276:18090–18095

    Article  PubMed  CAS  Google Scholar 

  • Hanke JH, Gardner JP, Dow RL, Changelian PS, Brissette WH, Weringer EJ, Pollok BA, Connelly PA (1996) Discovery of a novel, potent, and Src family-selective tyrosine kinase inhibitor. J Biol Chem 271:695–701

    Article  PubMed  CAS  Google Scholar 

  • Harmon AC, Gribskov M, Gubrium E, Harper JF (2001) The CDPK superfamily of protein kinases. New Phytol 151:175–183

    Article  CAS  Google Scholar 

  • Harmon AC, Yoo BC, McCaffery C (1994) Pseudosubstrate inhibition of CDPK, a protein kinase with a calmodulin-like domain. Biochemistry 33:7278–7287

    Article  PubMed  CAS  Google Scholar 

  • Harper JF, Huang JF, Lloyd SJ (1994) Genetic identification of an autoinhibitor in CDPK, a protein kinase with a calmodulin-like domain. Biochemistry 33:7267–7277

    Article  PubMed  CAS  Google Scholar 

  • Hegeman AD, Rodriguez M, Han BW, Uno Y, Phillips GN Jr, Hrabak EM, Cushman JC, Harper JF, Harmon AC, Sussman MR (2006) A phyloproteomic characterization of in vitro autophosphorylation in calcium-dependent protein kinases. Proteomics 6:3649–3664

    Article  PubMed  CAS  Google Scholar 

  • Ivashuta S, Liu J, Liu J, Lohar DP, Haridas S, Bucciarelli B, Vandenbosch KA, Vance CP, Harrison MJ, Gantt JS (2005) RNA interference identifies a calcium-dependent protein kinase involved in Medicago truncatula root development. Plant Cell 17(11):2911–2921

    Article  PubMed  CAS  Google Scholar 

  • Kange R, Selditz U, Granberg M, Lindberg U, Ekstrand G, Ek B, Gustafsson M (2005) Comparison of different IMAC techniques used for enrichment of phosphorylated peptides. J Biomol Technol 16:91–103

    Google Scholar 

  • Komatsu S, Li W, Konishi H, Yoshikawa M, Konishi T, Yang G (2001) Characterization of a Ca2+-dependent protein kinase from rice root: differential response to cold and regulation by abscisic acid. Biol Pharm Bull 24:1316–1319

    Article  PubMed  CAS  Google Scholar 

  • Kumar NV, Eblen ST, Weber MJ (2004) Identifying specific kinase substrates through engineered kinases and ATP analogs. Methods 32:389–397

    Article  PubMed  CAS  Google Scholar 

  • Liu Y, Shah K, Yang F, Witucki L, Shokat KM (1998) Engineering Src family protein kinases with unnatural nucleotide specificity. Chem Biol 5:91–101

    Article  PubMed  CAS  Google Scholar 

  • Lu SX, Hrabak EM (2002) An Arabidopsis calcium-dependent protein kinase is associated with the endoplasmic reticulum. Plant Physiol 128:1008–1021

    Article  PubMed  CAS  Google Scholar 

  • Ludwig AA, Saitoh H, Felix G, Freymark G, Miersch O, Wasternack C, Boller T, Jones JD, Romeis T (2005) Ethylene-mediated cross-talk between calcium-dependent protein kinase and MAPK signaling controls stress responses in plants. Proc Natl Acad Sci USA 102:10736–10741

    Article  PubMed  CAS  Google Scholar 

  • Martin ML, Busconi L (2001) A rice membrane-bound calcium-dependent protein kinase is activated in response to low temperature. Plant Physiol 125:1442–1449

    Article  PubMed  CAS  Google Scholar 

  • Molloy MP, Herbert BR, Walsh BJ, Tyler MI, Traini M, Sanchez JC, Hochstrasser DF, Williams KL, Gooley AA (1998) Extraction of membrane proteins by differential solubilization for separation using two-dimensional gel electrophoresis. Electrophoresis 19:837–844

    Article  PubMed  CAS  Google Scholar 

  • Monroy AF, Dhindsa RS (1995) Low-temperature signal transduction: induction of cold acclimation-specific genes of alfalfa by calcium at 25 degrees C. Plant Cell 7:321–331

    Article  PubMed  CAS  Google Scholar 

  • Mori IC, Murata Y, Yang Y, Munemasa S, Wang Y-F, Andreoli S, Tiriac H, Alonso JM, Harper JF, Ecker JR, Kwak JM, Schroeder JI (2006) CDPKs CPK6 and CPK3 function in ABA regulation of guard cell S-type anion- and Ca2+ permeable channels and stomatal closure. PLoS Biol 4:e327

    Article  PubMed  CAS  Google Scholar 

  • Nühse TS, Bottrill AR, Jones AME, Peck SC (2007) Quantitative phosphoproteomic analysis of plasma membrane proteins reveals regulatory mechanisms of plant innate immune responses. Plant J 51:931–940

    Article  PubMed  CAS  Google Scholar 

  • Papa F, Zhang C, Shokat K, Walter P (2003) Bypassing a kinase activity with an ATP-competitive drug. Science 302:1533–1537

    Article  PubMed  CAS  Google Scholar 

  • Patharkar OR, Cushman JC (2000) A stress-induced calcium-dependent protein kinase from Mesembryanthemum crystallinum phosphorylates a two-component pseudo-response regulator. Plant J 24:679–691

    Article  PubMed  CAS  Google Scholar 

  • Romeis T, Piedras P, Jones JD (2000) Resistance gene-dependent activation of a calcium-dependent protein kinase in the plant defense response. Plant Cell 12:803–816

    Article  PubMed  CAS  Google Scholar 

  • Romeis T, Ludwig AA, Martin R, Jones JD (2001) Calcium-dependent protein kinases play an essential role in a plant defence response. Embo J 20:5556–5567

    Article  PubMed  CAS  Google Scholar 

  • Saijo Y, Hata S, Kyozuka J, Shimamoto K, Izui K (2000) Over-expression of a single Ca2+-dependent protein kinase confers both cold and salt/drought tolerance on rice plants. Plant J 23:319–327

    Article  PubMed  CAS  Google Scholar 

  • Shah K, Liu Y, Deirmengian C, Shokat KM (1997) Engineering unnatural nucleotide specificity for Rous sarcoma virus tyrosine kinase to uniquely label its direct substrates. Proc Natl Acad Sci USA 94:3565–3570

    Article  PubMed  CAS  Google Scholar 

  • Sheen J (1996) Ca2+-dependent protein kinases and stress signal transduction in plants. Science 274:1900–1902

    Article  PubMed  CAS  Google Scholar 

  • Ubersax JA (2003) Targets of the cyclin-dependent kinase Cdk1. Nature 425:859–864

    Article  PubMed  CAS  Google Scholar 

  • Ulrich SM, Buzko O, Shah K, Shokat KM (2000) Towards the engineering of an orthogonal protein kinase/nucleotide triphosphate pair. Tetrahedron 56:9495–9502

    Article  CAS  Google Scholar 

  • Urao T, Katagiri T, Mizoguchi T, Yamaguchi-Shinozaki K, Hayashida N, Shinozaki K (1994) Two genes that encode Ca2+-dependent protein kinases are induced by drought and high-salt stresses in Arabidopsis thaliana. Mol Gen Genet 244:331–340

    Article  PubMed  CAS  Google Scholar 

  • Vitart V, Christodoulou J, Huang JF, Chazin WJ, Harper JF (2000) Intramolecular activation of a Ca2+-dependent protein kinase is disrupted by insertions in the tether that connects the calmodulin-like domain to the kinase. Biochemistry 39:4004–4011

    Article  PubMed  CAS  Google Scholar 

  • Vogel JT, Zarka DG, Van Buskirk HA, Fowler SG, Thomashow MF (2005) Roles of the CBF2 and ZAT12 transcription factors in configuring the low temperature transcriptome of Arabidopsis. Plant J 41:195–211

    Article  PubMed  CAS  Google Scholar 

  • Wang W, Scali M, Vignani R, Spadafora A, Sensi E, Mazzuca S, Cresti M (2003) Protein extraction for two-dimensional electrophoresis from olive leaf, a plant tissue containing high levels of interfering compounds. Electrophoresis 24:2369–2375

    Article  PubMed  CAS  Google Scholar 

  • Weiner MP, Costa GL, Schoettlin W, Cline J, Mathur E, Bauer JC (1994) Site-directed mutagenesis of double-stranded DNA by the polymerase chain reaction. Gene 151:119–123

    Article  PubMed  CAS  Google Scholar 

  • Weiss EL, Bishop A, Shokat K, Drubin DG (2000) Chemical genetic analysis of the budding yeast p21-activated kinase Cla4p. Mol Biol Cell 11:1813

    Google Scholar 

  • Wiener M, Sachs J, Deyanova E, Yates N (2004) Differential mass spectrometry: a label-free LC-MS method for finding significant differences in complex peptide and protein mixtures. Anal Chem 76:6085–6096

    Article  PubMed  CAS  Google Scholar 

  • Witte CP, Noel LD, Gielbert J, Parker JE, Romeis T (2004) Rapid one-step protein purification from plant material using the eight-amino acid StrepII epitope. Plant Mol Biol 55:135–147

    Article  PubMed  CAS  Google Scholar 

  • Yoon GM, Cho HS, Ha HJ, Liu JR, Lee HSP (1999) Characterization of NtCDPK1, a calcium-dependent protein kinase gene in Nicotiana tabacum, and the activity of its encoded protein. Plant Mol Biol 39:991–1001

    Article  PubMed  CAS  Google Scholar 

  • Zimmermann P, Hirsch-Hoffmann M, Hennig L, Gruissem W (2004) GENEVESTIGATOR. Arabidopsis microarray database and analysis toolbox. Plant Physiol 136:2621–2632

    Article  PubMed  CAS  Google Scholar 

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Acknowledgements

The authors wish to thank C.-P. Witte for helpful discussions and A.-C. Cazalé for providing CDPK entry vectors. We are also grateful to A. Bräutigam for assistance in the proteomics analysis. This project was supported by the Deutsche Forschungsgemeinschaft (SFB 635), the ZIP program from the German ministry of education and science, the Alexander von Humboldt-Foundation, and the Max-Planck Society.

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Correspondence to Tina Romeis.

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Böhmer, M., Romeis, T. A chemical-genetic approach to elucidate protein kinase function in planta . Plant Mol Biol 65, 817–827 (2007). https://doi.org/10.1007/s11103-007-9245-9

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