Assessment of BAK1 activity in different plant receptor-like kinase complexes by quantitative profiling of phosphorylation patterns.

Plant receptor-like kinases (RLKs) constitute a large family of receptors coordinating developmental programs with adaptation to environmental stresses including immune defenses. BRI1-ASSOCIATED KINASE 1 (BAK1), a member of the plant RLK family, forms receptor complexes with multiple RLK proteins including BRI1, FLS2, EFR and BIK1 to regulate responses to growth hormones or PAMPs. RLK activation and signal initiation involve protein complex formation and phosphorylation/dephosphorylation between BAK1 and its interacting partners. To gain new insight into how phosphorylation contributes to BAK1-mediated signaling specificity, we first mapped the phosphorylation patterns of BAK1 associated with different RLK partners (BRI1, FLS2, EFR and BIK1). Quantitative phospho-pattern profiling by label-free mass spectrometry revealed that differential phosphorylation patterns of RLK partners resulted from altered BAK1 phosphorylation status. More interestingly, the study of two BAK1 mutants (T450A and C408Y) both showing severe defect in immune defense yet normal growth phenotype suggested that varied phosphorylation patterns of RLK partners by BAK1 could be the molecular basis for selective regulation of multiple BAK1-dependent pathways. Taken together, this phospho-pattern profiling strategy allowed for explicit assessment of BAK1 kinase activity in different RLK complexes, which would facilitate elucidation of BAK1 diverse functions in plant development, defense, and adaptation. BIOLOGICAL SIGNIFICANCE: BAK1 is a functionally important co-receptor known to interact with different receptor-like kinases (RLKs) to coordinate plant development and immune defenses. Our study first mapped the phosphorylation patterns of BAK1 associated with four RLK partners (BRI1, FLS2, EFR and BIK1), and further revealed that differential phosphorylation patterns of multiple RLK partners resulted from altered BAK1 phosphorylation status. More interestingly, the study of two BAK1 mutants suggested that varied phosphorylation patterns of RLK partners by BAK1 could be the basis for selective regulation of signaling pathways. Taken together, this phospho-pattern profiling strategy allowed for explicit assessment of BAK1 kinase activity in different RLK complexes, which would facilitate elucidation of BAK1 diverse functions in plant development, defense, and adaptation.