Figure 3. Identification of RHA3A-mediated BIK1 ubiquitination sites.

a. BIK1 is ubiquitinated by RHA3A at multiple lysine residues. Ubiquitinated lysine residues with a diglycine remnant by LC-MS/MS analysis are marked as red with amino acid positions. b. MS/MS spectrum of the peptide containing K³⁵⁸ is shown. c. Structure of BIK1 labeled with six lysine identified as ubiquitination sites. Structural information was obtained from protein data bank (PDB ID: 5TOS) and analyzed by PyMOL. d. BIK1^9KR is compromised in flg22-induced ubiquitination. FLAG-UBQ and HA-tagged BIK1 mutants were expressed in protoplasts followed by 100 nM flg22 for 30 min. Quantification of BIK1 ubiquitination fold change is shown as overlay of dot plot and means ± SEM (middle). Different letters indicate significant difference with others (P<0.05, One-way ANOVA, n=3). Mutated lysine for BIK1 mutants are in red (bottom). e. RHA3A is unable to ubiquitinate BIK1^9KR. The assay was performed as in Fig. 2d. f. BIK1^9KR exhibits normal in vitro kinase activity. The kinase assay was performed using GST-BIK1 or GST-BIK1^9KR as the kinase and GST or GST-BAK1^K (kinase domain) as the substrate. The experiments except MS analyses were repeated three times with similar results.