Protein Phosphorylation Assay

Protein phosphorylation reactions were performed in 50 µL of 50 mm Tris-HCl, pH 7.5, 10 mm MgCl₂, 1 mm DTT, 1 mm EDTA, 0.1 mm ATP, 1 µCi of [γ-³²P]ATP, and 25 pmol of each kinase and substrate protein. After incubation at 30°C for 30 min, the reactions were stopped by adding an equal volume of 2× SDS-PAGE sample buffer and boiled for 5 min before SDS-PAGE. Gels were stained with Coomassie Brilliant Blue and dried, and the labeled proteins were visualized by autoradiography. Alternatively, the separated proteins were transferred to nitrocellulose membrane for autoradiography and later quantified by immunoblotting with His₆ antibodies. For the preparation of activated and purified His₆-SnRK1.1 kinase domain, 2.5 nmol of each GST-GRIK1 and His₆-SnRK1.1 kinase domain was incubated in a 300-µL kinase reaction mixture with unlabeled ATP at 30°C for 1 h. GST-GRIK1 was removed by two passes through a 50-µL bed volume of glutathione-Sepharose (GE Healthcare). The supernatant was then incubated with a 50-µL bed volume of Ni-NTA agarose, and His₆-SnRK1.1 was eluted with four fractions of 50 µL of elution buffer. Pooled protein was concentrated to greater than 1 µg µL⁻¹ and stored in 0.5 mm DTT and 50% (v/v) glycerol at −20°C. Peptide kinase assays using a peptide, acetyl-KGRMRRISSVEMMK (GenScript), containing a SnRK1 phosphorylation site from spinach (Spinacia oleracea) Suc-phosphate synthase was described previously (Huang and Huber, 2001; Shen et al., 2009).