AtCERK1 Phosphorylation Site S493 Contributes to the Transphosphorylation of Downstream Components for Chitin-Induced Immune Signaling.

While ligand-induced autophosphorylation of receptor-like kinases (RLKs) is known to be critical for triggering the downstream responses, biochemical mechanism by which each phosphorylation site contributes to the initiation of corresponding signaling cascades is only poorly understood, except the involvement of some phosphorylation sites in the regulation of catalytic activity of these RLKs. In this article, we first confirmed that the phosphorylation of S493 of AtCERK1 is involved in the regulation of chitin-induced defense responses by the complementation of an atcerk1 mutant with AtCERK1(S493A) cDNA. In vitro kinase assay with the heterologously expressed kinase domain of AtCERK1, GST-AtCERK1cyt, showed that the S493A mutation did not affect the autophosphorylation of AtCERK1 itself but diminished the transphosphorylation of downstream signaling components, PBL27 and PUB4. On the other hand, a phosphomimetic mutant, GST-AtCERK1(S493D)cyt, transphosphorylated these substrates as similar to the wild type AtCERK1. These results suggested that the phosphorylation of S493 does not contribute to the regulation of catalytic activity but plays an important role for the transphosphorylation of the downstream signaling components, thus contributing to the initiation of chitin signaling. To our knowledge, it is a novel finding that a specific phosphorylation site contributes to the regulation of transphosphorylation activity of RLKs. Further studies on the structural basis by which S493 phosphorylation contributes to the regulation of transphosphorylation would contribute to the understanding how the ligand-induced autophosphorylation of RLKs properly regulates the downstream signaling.

Autophosphorylation site Y428 is essential for the in vivo activation of CERK1.

Autophosphorylation of PRR is a critical event for the activation of immune signaling in plant. However, the detailed function of these phosphorylation sites is still not well understood. We analyzed the function of an autophosphorylation site of Arabidopsis CERK1, Y428, in immune signaling. Biochemical characterization of CERK1 mutants transiently expressed in N. benthamiana indicated that Y428 plays a crucial role for the in vivo activation of CERK1, differently from the previous observation by the in vitro kinase assay with its cytoplasmic domain. Similar discrepancy between in vitro and in vivo kinase assay was also reported for the corresponding phosphorylation site of EFR, suggesting that these conserved tyrosine residues play important roles for the activation of both RD and non-RD RLKs.

Autophosphorylation of Specific Threonine and Tyrosine Residues in Arabidopsis CERK1 is Essential for the Activation of Chitin-Induced Immune Signaling.

Pattern recognition receptors on the plant cell surface mediate the recognition of microbe/damage-associated molecular patterns (MAMPs/DAMPs) and activate downstream immune signaling. Autophosphorylation of signaling receptor-like kinases is a critical event for the activation of downstream responses but the function of each phosphorylation site in the regulation of immune signaling is not well understood. In this study, 41 Ser/Thr/Tyr and 15 Ser/Thr residues were identified as in vitro and in vivo autophosphorylation sites of Arabidopsis CERK1, which is essential for chitin signaling. Comprehensive analysis of transgenic plants expressing mutated CERK1 genes for each phosphorylation site in the cerk1-2 background indicated that the phosphorylation of T479 in the activation segment and Y428 located upstream of the catalytic loop is important for the activation of chitin-triggered defense responses. Contribution of the phosphorylation of T573 to the chitin responses was also suggested. In vitro evaluation of kinase activities of mutated kinase domains indicated that the phosphorylation of T479 and T573 is directly involved in the regulation of kinase activity of CERK1 but the phosphorylation of Y428 regulates chitin signaling independently of the regulation of kinase activity. These results indicated that the phosphorylation of specific residues in the kinase domain contributes to the regulation of downstream signaling either through the regulation of kinase activity or the different mechanisms, e.g. regulation of protein-protein interactions.