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

Abstract

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.

Early signalling mechanisms underlying receptor kinase-mediated immunity in plants

“…A most recent study showed that flg22 and elf18 induce BAK1 phosphorylation at Ser602, Thr603, Ser604 and Ser612 in vivo, and these four phosphosites are required for flg22 but not brassinosteroid (BR) signalling [20]. In addition to serine/threonine phosphorylation, an increasing number of RLKs have been shown to undergo tyrosine phosphorylation, and this modification is important for their activation [20][21][22][23]. A Tyr residue is present in kinase subdomain VIa (Tyr-VIa) and conserved in about 80% Arabidopsis LRR-RLKs.…”

OsCERK1 plays a crucial role in the lipopolysaccharide‐induced immune response of rice

“…Arabidopsis seedlings were collected 2 h after the 100 lg ml À1 LPS (P. aeruginosa) or 20 lM (GlcNAc) 7 treatments. Gene expression was quantified by reverse transcription real-time PCR using Fast SYBR Green Master Mix (Thermo Fisher Scientific Inc., Waltham, MA, USA) (Suzuki et al, 2016). Actin was used as the internal control.…”

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

“…This band shift was previously confirmed to show the autophosphorylation of CERK1. 9 On the other hand, the band of CERK1(Y428F)-3HA did not show such an upward shift, indicating that the mutant CERK1 was not phosphorylated in N. benthamiana. These observations are different from the results of previous in vitro kinase assay where the heterologously expressed kinase domain of this mutant was normally autophosphorylated and indicated that Y428 plays a pivotal role in the in vivo activation of CERK1 kinase required for downstream defense signaling.…”

“…[5][6][7] It has been shown that the kinase activity of CERK1 is essential for the activation of immune responses and CERK1 itself is phosphorylated by chitin treatment. 8,9 Petutschnig et al identified four or five in vivo phosphorylation sites of CERK1 and showed that the phosphorylation of at least three of them was dependent on chitin treatment. 8 Recently, we also identified 41 in vitro and 15 in vivo phosphorylation sites of CERK1 and indicated that at least three of them play important roles in chitin signaling based on the complementation experiments with the CERK1 mutants of these phosphorylation sites.…”

“…8 Recently, we also identified 41 in vitro and 15 in vivo phosphorylation sites of CERK1 and indicated that at least three of them play important roles in chitin signaling based on the complementation experiments with the CERK1 mutants of these phosphorylation sites. 9 Among them, T479A and Y428F mutants completely lost the ability to complement the cerk1 mutant for chitin responses. Mutation of T573A also showed a much less complementation ability compared to the wild type CERK1.…”