Abstract
Cyclic nucleotides (CNs) are intracellular second messengers that play an important role in mediating physiological responses to environmental and developmental signals, in species ranging from bacteria to humans. In response to these signals, CNs are synthesized by nucleotidyl cyclases and then act by binding to and altering the activity of downstream target proteins known as cyclic nucleotide-binding proteins (CNBPs). A number of CNBPs have been identified across kingdoms including transcription factors, protein kinases, phosphodiesterases, and channels, all of which harbor conserved CN-binding domains. In plants however, few CNBPs have been identified as homology searches fail to return plant sequences with significant matches to known CNBPs. Recently, affinity pull-down techniques have been successfully used to identify CNBPs in animals and have provided new insights into CN signaling. The application of these techniques to plants has not yet been extensively explored and offers an alternative approach toward the unbiased discovery of novel CNBP candidates in plants. Here, an affinity pull-down technique for the identification of the plant CN interactome is presented. In summary, the method involves an extraction of plant proteins which is incubated with a CN-bait, followed by a series of increasingly stringent elutions that eliminates proteins in a sequential manner according to their affinity to the bait. The eluted and bait-bound proteins are separated by one-dimensional gel electrophoresis, excised, and digested with trypsin after which the resultant peptides are identified by mass spectrometry—techniques that are commonplace in proteomics experiments. The discovery of plant CNBPs promises to provide valuable insight into the mechanism of CN signal transduction in plants.
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References
Newton RP, Roef LUC, Witters E, Van Onckelen H (1999) Tansley review no. 106. New Phytol 143:427–455
Martinez-Atienza J, Van Ingelgem C, Roef L, Maathuis FJ (2007) Plant cyclic nucleotide signalling: facts and fiction. Plant Signal Behav 2:540–543
Newton RP, Smith CJ (2004) Cyclic nucleotides. Phytochemistry 65:2423–2437
Visser NF, Scholten A, van den Heuvel RH, Heck AJ (2007) Surface-plasmon-resonance-based chemical proteomics: efficient specific extraction and semiquantitative identification of cyclic nucleotide-binding proteins from cellular lysates by using a combination of surface plasmon resonance, sequential elution and liquid chromatography-tandem mass spectrometry. Chembiochem 8:298–305
Kim E, Park JM (2003) Identification of novel target proteins of cyclic GMP signaling pathways using chemical proteomics. J Biochem Mol Biol 36:299–304
Scholten A, Poh MK, van Veen TA, van Breukelen B, Vos MA, Heck AJ (2006) Analysis of the cGMP/cAMP interactome using a chemical proteomics approach in mammalian heart tissue validates sphingosine kinase type 1-interacting protein as a genuine and highly abundant AKAP. J Prot Res 5:1435–1447
Scholten A, van Veen TA, Vos MA, Heck AJ (2007) Diversity of cAMP-dependent protein kinase isoforms and their anchoring proteins in mouse ventricular tissue. J Proteome Res 6:1705–1717
Kolb A, Busby S, Buc H, Garges S, Adhya S (1993) Transcriptional regulation by cAMP and its receptor protein. Annu Rev Biochem 62:749–795
Francis SH, Corbin JD (1999) Cyclic nucleotide-dependent protein kinases: intracellular receptors for cAMP and cGMP action. Crit Rev Clin Lab Sci 36:275–328
Francis SH, Corbin JD (1994) Structure and function of cyclic nucleotide-dependent protein kinases. Annu Rev Physiol 56:237–272
Conti M, Beavo J (2007) Biochemistry and physiology of cyclic nucleotide phosphodiesterases: essential components in cyclic nucleotide signaling. Annu Rev Biochem 76:481–511
Cook NJ, Hanke W, Kaupp UB (1987) Identification, purification, and functional reconstitution of the cyclic GMP-dependent channel from rod photoreceptors. Proc Natl Acad Sci USA 84:585–589
Nakamura T, Gold GH (1987) A cyclic nucleotide-gated conductance in olfactory receptor cilia. Nature 325:442–444
Craven KB, Zagotta WN (2006) CNG and HCN channels: two peas, one pod. Annu Rev Physiol 68:375–401
Bos JL (2003) Epac: a new cAMP target and new avenues in cAMP research. Nat Rev 4:733–738
Goldberg JM, Bosgraaf L, Van Haastert PJ, Smith JL (2002) Identification of four candidate cGMP targets in Dictyostelium. Proc Natl Acad Sci USA 99:6749–6754
Shabb JB, Corbin JD (1992) Cyclic nucleotide-binding domains in proteins having diverse functions. J Biol Chem 267:5723–5726
Kannan N, Wu J, Anand GS, Yooseph S, Neuwald AF et al (2007) Evolution of allostery in the cyclic nucleotide binding module. Genome Biol 8:R264
Zoraghi R, Corbin JD, Francis SH (2004) Properties and functions of GAF domains in cyclic nucleotide phosphodiesterases and other proteins. Mol Pharmacol 65:267–278
Leng Q, Mercier RW, Yao W, Berkowitz GA (1999) Cloning and first functional characterization of a plant cyclic nucleotide-gated cation channel. Plant Physiol 121:753–761
Hoshi T (1995) Regulation of voltage dependence of the KAT1 channel by intracellular factors. J Gen Physiol 105:309–328
Bridges D, Fraser ME, Moorhead GB (2005) Cyclic nucleotide binding proteins in the Arabidopsis thaliana and Oryza sativa genomes. BMC Bioinf 6:6
Johnson JL, Leroux MR (2010) cAMP and cGMP signaling: sensory systems with prokaryotic roots adopted by eukaryotic cilia. Trends Cell Biol 20:435–444
Talke IN, Blaudez D, Maathuis FJ, Sanders D (2003) CNGCs: prime targets of plant cyclic nucleotide signalling? Trends Plant Sci 8:286–293
Isner JC, Nuhse T, Maathuis FJ (2012) The cyclic nucleotide cGMP is involved in plant hormone signalling and alters phosphorylation of Arabidopsis thaliana root proteins. J Exp Bot 63:3199–3205
Dubovskaya LV, Volotovsky ID (2004) Affinity chromatography isolation and characterization of soluble cGMP binding proteins from Avena sativa L. seedlings. Bulg J Plant Physiol 30:14–24
Dubovskaya LV, Bakakina YS, Kolesneva EV, Sodel DL, McAinsh MR et al (2011) cGMP-dependent ABA-induced stomatal closure in the ABA-insensitive Arabidopsis mutant abi1-1. New Phytol 191:57–69
Laukens K, Roef L, Witters E, Slegers H, Van Onckelen H (2001) Cyclic AMP affinity purification and ESI-QTOF MS-MS identification of cytosolic glyceraldehyde 3-phosphate dehydrogenase and two nucleoside diphosphate kinase isoforms from tobacco BY-2 cells. FEBS Lett 508:75–79
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Rose JK, Bashir S, Giovannoni JJ, Jahn MM, Saravanan RS (2004) Tackling the plant proteome: practical approaches, hurdles and experimental tools. Plant J 39:715–733
Baerenfaller K, Grossmann J, Grobei MA, Hull R, Hirsch-Hoffmann M et al (2008) Genome-scale proteomics reveals Arabidopsis thaliana gene models and proteome dynamics. Science 320:938–941
Peckham GD, Bugos RC, Su WW (2006) Purification of GFP fusion proteins from transgenic plant cell cultures. Protein Expr Purif 49:183–189
Charmont S, Jamet E, Pont-Lezica R, Canut H (2005) Proteomic analysis of secreted proteins from Arabidopsis thaliana seedlings: improved recovery following removal of phenolic compounds. Phytochemistry 66:453–461
Luo Y, Blex C, Baessler O, Glinski M, Dreger M et al (2009) The cAMP capture compound mass spectrometry as a novel tool for targeting cAMP-binding proteins: from protein kinase A to potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channels. Mol Cell Prot 8:2843–2856
Aye TT, Mohammed S, van den Toorn HW, van Veen TA, van der Heyden MA et al (2009) Selectivity in enrichment of cAMP-dependent protein kinase regulatory subunits type I and type II and their interactors using modified cAMP affinity resins. Mol Cell Prot 8:1016–1028
Dubovskaya LV, Molchan OV, Volotovsky ID (2002) Cyclic GMP-binding activity in Avena sativa seedlings. Russ J Plant Physiol 49:216–220
Shevchenko A, Tomas H, Havlis J, Olsen JV, Mann M (2006) In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat Protoc 1:2856–2860
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Donaldson, L., Meier, S. (2013). An Affinity Pull-Down Approach to Identify the Plant Cyclic Nucleotide Interactome. In: Gehring, C. (eds) Cyclic Nucleotide Signaling in Plants. Methods in Molecular Biology, vol 1016. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-441-8_11
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DOI: https://doi.org/10.1007/978-1-62703-441-8_11
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