Phosphorylation status of CPK28 affects its ubiquitination and protein stability

Abstract

Summary Plant innate immunity is tightly regulated. The Arabidopsis thaliana CALCIUM‐DEPENDENT PROTEIN KINASE28 (CPK28) functions as a negative immune regulator. We recently demonstrate that CPK28 undergoes ubiquitination that is mediated by two ubiquitin ligases, ARABIDOPSIS TÓXICOS EN LEVADURA31 (ATL31) and ATL6, which results in its proteasomal degradation. CPK28 undergoes both intermolecular autophosphorylation and BIK1‐mediated phosphorylation. However, whether the phosphorylation status of CPK28 dictates its ubiquitination and degradation is unknown yet. We used immune response analysis, transient degradation system, ubiquitination assays, co‐immunoprecipitation, and other biochemical and genetic approaches to investigate the effect of the phosphorylation status of CPK28 on its degradation mediated by ATL31/6. We found the mutation of Ser318 (a site of both intermolecular autophosphorylation and BIK1‐mediated phosphorylation) or a BIK1 phosphorylation site on CPK28 leads to its compromised association with ATL31 and reduced ubiquitination by ATL31. Moreover, we confirm the previous findings that two CPK28s can interact with each other, which likely promotes the intermolecular autophosphorylation. We also show that the phosphorylation status of CPK28 in turn affects its intermolecular association. We demonstrate that the phosphorylation status of CPK28 affects its degradation mediated by ATL31. Our findings reveal a link between phosphorylation of CPK28 and its ubiquitination and degradation.

CALCIUM-DEPENDENT PROTEIN KINASE32 regulates cellulose biosynthesis through post-translational modification of cellulose synthase

“…Phosphorylation of CALCIUM-DEPENDENT PROTEIN KINASE 28 (CPK28) on Thr 76 and Ser 318 mediated by itself and BIK1 activates its phosphorylation of two E3 ligases, which induce BIK1 polyubiquitination and immune response. Phosphorylation of these sites is also required for CPK28 ubiquitination and degradation mediated by ARABIDOPSIS TOXICOS EN LEVADURA 31/6, which resists overactivation of immune signaling (14)(15)(16). Thus, it indicates a fine-tune immune signaling regulation via the phosphorylation statues of CPK28.…”

CALCIUM‐DEPENDENT PROTEIN KINASE32 regulates cellulose biosynthesis through post‐translational modification of cellulose synthase

“…Phosphorylation of CALCIUM‐DEPENDENT PROTEIN KINASE 28 (CPK28) on Thr 76 and Ser 318 mediated by itself and BIK1 activates its phosphorylation of two E3 ligases, which induce BIK1 polyubiquitination and immune response. Phosphorylation of these sites is also required for CPK28 ubiquitination and degradation mediated by ARABIDOPSIS TOXICOS EN LEVADURA 31/6, which resists overactivation of immune signaling (Bredow et al ., 2021; Liu et al ., 2022; Yuanyuan et al ., 2023). Thus, it indicates a fine‐tune immune signaling regulation via the phosphorylation statues of CPK28.…”

CPK1‐HSP90 phosphorylation and effector XopC2–HSP90 interaction underpin the antagonism during cassava defense‐pathogen infection

“…To investigate the upstream signals of HSP90-mediated immune responses, we screened the cassava prey library (c. 10 6 colonyforming unit ml À1 ) and identified 35 candidate interacting proteins of MeHSP90.9 through Y2H, including only one protein kinase (Wei et al, 2020(Wei et al, , 2021b. Given the importance of protein phosphorylation by protein kinases and the involvement of CPKmediated protein phosphorylation in plant immunity (Ding et al, 2023;Liu et al, 2023Liu et al, , 2024, MeCPK1 was chosen for further analysis. Yeast-two hybrid assay using the full CDS of MeCPK1 and MeHSP90.9 further verified their interaction in yeast (Fig.…”