Abstract
Protein kinases are central components of signal transduction pathways in the cell. They catalyze the phosphorylation of substrate proteins, resulting in changes of the activity, localization, stability, and protein interactions of the substrates, ultimately coordinating the activity of important cellular processes. CONSTITUTIVE TRIPLE RESPONSE 1 (CTR1) is a Raf-like protein kinase that functions as a negative regulator in the phytohormone ethylene signaling pathway. CTR1 physically interacts with ethylene receptors via its N-terminal domain at the endoplasmic reticulum, and is involved in suppressing ethylene signaling in the absence of ethylene. Recent studies demonstrated that CTR1 directly interacts with and differentially phosphorylates the positive regulator ETHYLENE INSENSITIVE 2 (EIN2), therefore regulating the movement of EIN2 into the nucleus. Here, we describe protocols for determining the kinase activity of CTR1 by calculating the incorporated radiolabeled phosphate [γ-32P] from ATP into its physiological substrate, EIN2 protein.
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References
Kieber JJ, Rothenberg M, Roman G, Feldmann KA, Ecker JR (1993) CTR1, a negative regulator of the ethylene response pathway in Arabidopsis, encodes a member of the raf family of protein kinases. Cell 72(3):427–441
Huang Y, Li H, Hutchison CE, Laskey J, Kieber JJ (2003) Biochemical and functional analysis of CTR1, a protein kinase that negatively regulates ethylene signaling in Arabidopsis. Plant J 33(2):221–233
Gao Z et al (2003) Localization of the Raf-like kinase CTR1 to the endoplasmic reticulum of Arabidopsis through participation in ethylene receptor signaling complexes. J Biol Chem 278(36):34725–34732
Chen R et al (2011) Proteomic responses in Arabidopsis thaliana seedlings treated with ethylene. Mol Biosyst 7(9):2637–2650
Ju C et al (2012) CTR1 phosphorylates the central regulator EIN2 to control ethylene hormone signaling from the ER membrane to the nucleus in Arabidopsis. Proc Natl Acad Sci U S A 109(47):19486–19491
Qiao H et al (2012) Processing and subcellular trafficking of ER-tethered EIN2 control response to ethylene gas. Science 338(6105):390–393
Wen X et al (2012) Activation of ethylene signaling is mediated by nuclear translocation of the cleaved EIN2 carboxyl terminus. Cell Res 22(11):1613–1616
Witt JJ, Roskoski R Jr (1975) Rapid protein kinase assay using phosphocellulose-paper absorption. Anal Biochem 66(1):253–258
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This work is supported by a startup fund from Purdue University to GMY.
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Lee, H.Y., Yoon, G.M. (2017). Kinase Assay for CONSTITUTIVE TRIPLE RESPONSE 1 (CTR1) in Arabidopsis thaliana . In: Binder, B., Eric Schaller, G. (eds) Ethylene Signaling. Methods in Molecular Biology, vol 1573. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6854-1_11
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DOI: https://doi.org/10.1007/978-1-4939-6854-1_11
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Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6852-7
Online ISBN: 978-1-4939-6854-1
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