State Key Laboratory of Plant Genomics, Center for Agricultural Resources Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Shijiazhuang, Hebei, 050021, China.
School of Agriculture and Biology, Shanghai Jiao Tong University, 800 Dongchuan Road, Minhang District, Shanghai, 200240, China.
New Phytol. 2023 Feb;237(4):1270-1284. doi: 10.1111/nph.18596. Epub 2022 Dec 2.
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.
植物先天免疫受到严格调控。拟南芥钙依赖蛋白激酶 28（CPK28）作为一种负向免疫调节剂发挥作用。我们最近的研究表明，CPK28 发生泛素化修饰，由两个泛素连接酶 ATL31 和 ATL6 介导，导致其被蛋白酶体降解。CPK28 发生分子间自身磷酸化和 BIK1 介导的磷酸化。然而，CPK28 的磷酸化状态是否决定其泛素化和降解尚不清楚。我们采用免疫反应分析、瞬时降解系统、泛素化测定、共免疫沉淀和其他生化及遗传方法，研究 CPK28 的磷酸化状态对 ATL31/6 介导的降解的影响。我们发现，CPK28 的 Ser318 突变（分子间自身磷酸化和 BIK1 介导的磷酸化位点）或 CPK28 的 BIK1 磷酸化位点突变，导致其与 ATL31 的结合能力下降，泛素化减少。此外，我们证实了先前的发现，即两个 CPK28 可以相互作用，这可能促进分子间自身磷酸化。我们还表明，CPK28 的磷酸化状态反过来影响其分子间相互作用。我们证明了 CPK28 的磷酸化状态影响其由 ATL31 介导的降解。我们的研究结果揭示了 CPK28 的磷酸化与其泛素化和降解之间的联系。
