FIGURE 6.

Coupled kinase assays of mutated forms of AtSnAK1 and confirmation of the FP site. A, AtSnRK1.1-GST (2 μg of protein preparation) was incubated with each AtSnAK1-GST form (2 μg of protein preparation) in the reconstituted assay containing 2 μCi of[γ-³²P]ATP, 20 μm ATP, and 90 μm AMARA peptide, at 30 °C for 60 min. Radioactivity incorporated onto the AMARA peptide was measured. The results are the average of three independent experiments ± S.D. (error bars). B, AtSnAK1 WT or S260D (2 μg of protein preparation) was incubated with AtSnRK1.1 T175D (2 μg) in the same reconstituted assay. After SDS-PAGE, the proteins were submitted to autoradiography. All three lanes are from the same exposure. The results are representative of three independent experiments. WT, wild-type; Control, AtSnRK1.1 without AtSnAK; M, molecular mass in kDa.