Fig. 4

The old12 mutation impairs P2K2 kinase activity. A Schematic representation of the P2K2 protein structure highlighting the different domains and the position of the old12 mutation (C407Y, asterisk). B Multiple sequence alignment of the active site of several LecRK proteins and other receptor like kinases (RLKs). The old12 mutation affects a cysteine residue that is conserved in the kinase domain. C P2K2 autophosphorylation activity was analyzed using an expressed and purified FLAG-tagged kinase domain (KD) of the wild-type P2K2 and the old12 mutant. Autophosphorylation was measured by detecting thiophosphorylation with ATPγS and subsequent western blotting using thiophosphate ester-specific antibodies. D Transphosphorylation assays were performed by incubating purified FLAG-tagged KDs of wild-type P2K2 and old12 with the universal kinase substrate MBP. Thiophosphorylation of MBP was subsequently detected by a western blot using antibodies against the thiophosphate ester. The upper arrow indicates autophosphorylation of P2K2, while the lower arrow indicates the transphosphorylation of MBP. Mock reactions only contained the MBP substrate. The loading control involves the detection of OLD12 proteins with an anti-FLAG antibody. Images for the loading controls are cropped, full images can be found in the supplement (Fig. S6)