Extended Data Fig. 9: Analysis of phosphosite-specific kinase activities of BAK1.
From: Phosphocode-dependent functional dichotomy of a common co-receptor in plant signalling
a, Specific detection of BAK1 Y403 phosphorylation on affinity-purified recombinant BAK1CD wild-type but not Y403F or kinase-dead (D416N) proteins, using anti-pY403 antibodies and following an in vitro kinase assay with cold ATP. Blots were probed with anti-BAK1 antibodies for loading control. b, Detection of BAK1 Y403, S612, threonine and tyrosine phosphorylation using phosphorylation-specific antibodies on affinity-purified recombinant BAK1CD following an in vitro kinase assay with cold ATP. Membranes were immunoblotted with BAK1 antibodies for loading control. c, [32P]γ-ATP kinase assays showing autophosphorylation of the indicated MBP-BAK1CD phosphosite mutants. d, trans-autophosphorylation of the kinase-dead BAK1(D416N) (6×His-BAK1*CD) by the indicated BAK1 phosphosite mutants (MBP–BAK1CD). e, Trans-phosphorylation activity of the indicated affinity purified recombinant BAK1CD mutants against a kinase-dead (K105E) BIK1 substrate (GST–BIK1*). f, Trans-autophosphorylation of kinase-dead (D416N) BAK1 substrate (6×His-BAK1*CD) at S612 and Y403 by wild-type BAK1 (MBP–BAK1CD). BAK1 Y403 or S612 phosphorylation was detected using phosphorylation-specific antibodies following an in vitro incubation of the purified proteins in the presence or in the absence of cold ATP. Membranes were immunoblotted with BAK1 antibodies for loading control. In c–e, numbers indicate autoradiograph band intensity relative to the wild type. Protein loading control was determined with CBB staining. For a–f, all experiments were repeated independently at least three times. For blot source data, see Supplementary Fig. 1.
