Extended Data Fig. 3: Plasma membrane localization and phosphorylation are required for BIK1 ubiquitination.
From: Ligand-induced monoubiquitination of BIK1 regulates plant immunity
a, The kinase inhibitor K252a blocks flg22-induced ubiquitination of BIK1. Protoplasts transfected with FLAG–UBQ and BIK1–HA were treated with 1 μM K252a for 30 min and then with 100 nM flg22. b, BIK1(G2A) no longer localizes to the plasma membrane. BIK1–YFP or BIK1(G2A)–YFP was expressed in N. benthamiana for imaging analysis. c, BIK1(G2A) show compromised flg22-induced monoubiquitination. BIK1–HA or BIK1(G2A)–HA was co-expressed with FLAG–UBQ in protoplasts. d, Single K-to-R mutations of BIK1 fail to block flg22-induced ubiquitination without altering kinase activity. HA-tagged wild-type or mutant BIK1 was co-expressed with FLAG–UBQ in protoplasts. e, BIK1(K204R) exhibits reduced autophosphorylation and phosphorylation of BAK1. An in vitro kinase assay was performed using GST–BIK1 or GST–BIK1(K204R) as a kinase and GST or GST–BAK1K (BAK1 kinase domain without detectable autophosphorylation activity) as a substrate with [γ-32P] ATP. Top, proteins were separated with SDS–PAGE and analysed by autoradiography (Autorad.); bottom, protein loading shown CBB staining. Experiments were repeated at least twice with similar results.
