Kinase Assay for CONSTITUTIVE TRIPLE RESPONSE 1 (CTR1) in Arabidopsis thaliana

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.