FIG 2.

AL2/C2 proteins do not inhibit GRIK or SnRK1 kinase activity. (A) GRIK phosphorylation of the SnRK1 T loop in the presence of AL2/C2 proteins. His₆-tagged kinase domain mutants of SnRK1.1 (K48A) (lanes 1 to 5) and SnRK1.2 (K49A) (lanes 6 to 10) were phosphorylated by GST-GRIK1 (top) or GST-GRIK2 (bottom) either alone or in reaction mixtures supplemented with GST (lanes 2 and 7) or GST-tagged AL2/C2 from CaLCuV (lanes 3 and 8), TGMV (lanes 4 and 9), or BCTV (lanes 5 and 10). After SDS-PAGE, SnRK1 phosphorylation was detected by immunoblotting with antibodies against the human AMPK phospho-T172 peptide. (B) Phosphorylation of the SPS peptide by the unactivated SnRK1.1 kinase domain alone (top), GRIK1-activated SnRK1.1 with the presence of GRIK1 (middle, SnRK1.1 plus GRIK1), or GRIK1-activated SnRK1.1 with GRIK removed (bottom). [γ-³²P]ATP was used as the phosphate donor. The kinase reactions were performed in the presence of GST or the GST-tagged AL2/C2 protein from CaLCuV, ToMoV, TGMV, TYLCV, or BCTV, as indicated on the left. The molar ratios of the effector proteins to SnRK1.1 were 2:1 for all proteins except GST-tagged BCTV C2, for which the molar ratio was 1:1. Kinase activities are expressed as the counts per minute (cpm) of the radioactivity incorporated into the peptide relative to that in the reactions with GST as the effector protein. The average radioactivity and SD from three experiments are shown. The relative activities of the different reaction conditions with GST are indicated on the right.