Department of Biology, Queen's University, Kingston, ON, K7L 3N6, Canada.
The Sainsbury Laboratory, University of East Anglia, Norwich Research Park, Norwich, NR4 7UH, UK.
New Phytol. 2024 Dec;244(6):2278-2294. doi: 10.1111/nph.20198. Epub 2024 Oct 24.
The calcium-dependent protein kinase CPK28 regulates several stress pathways in multiple plant species. Here, we aimed to discover CPK28-associated proteins in Arabidopsis thaliana. We used affinity-based proteomics and identified several potential CPK28 binding partners, including the C7 Raf-like kinases MRK1, RAF26, and RAF39. We used biochemistry, genetics, and physiological assays to gain insight into their function. We define redundant roles for these kinases in stomatal opening, immune-triggered reactive oxygen species (ROS) production, and resistance to a bacterial pathogen. We report that CPK28 associates with and trans-phosphorylates RAF26 and RAF39, and that MRK1, RAF26, and RAF39 are active kinases that localize to endomembranes. Although Raf-like kinases share some features with mitogen-activated protein kinase kinase kinases (MKKKs), we found that MRK1, RAF26, and RAF39 are unable to trans-phosphorylate any of the 10 Arabidopsis mitogen-activated protein kinase kinases (MKKs). Overall, our study suggests that C7 Raf-like kinases associate with and are phosphorylated by CPK28, function redundantly in stomatal opening and immunity, and possess substrate specificities distinct from canonical MKKKs.
钙依赖蛋白激酶 CPK28 调节多种植物物种中的几种应激途径。在这里，我们旨在发现拟南芥中与 CPK28 相关的蛋白质。我们使用基于亲和性的蛋白质组学方法鉴定了几个潜在的 CPK28 结合伴侣，包括 C7 Raf 样激酶 MRK1、RAF26 和 RAF39。我们使用生物化学、遗传学和生理学测定来深入了解它们的功能。我们定义了这些激酶在气孔开放、免疫触发的活性氧（ROS）产生和抵抗细菌病原体方面的冗余作用。我们报告 CPK28 与 RAF26 和 RAF39 结合并发生转磷酸化，MRK1、RAF26 和 RAF39 是定位于内质网的活性激酶。尽管 Raf 样激酶与丝裂原激活蛋白激酶激酶激酶（MKKK）具有一些共同特征，但我们发现 MRK1、RAF26 和 RAF39 不能磷酸化任何 10 种拟南芥丝裂原激活蛋白激酶激酶（MKK）。总体而言，我们的研究表明 C7 Raf 样激酶与 CPK28 结合并被其磷酸化，在气孔开放和免疫中冗余发挥作用，并且具有与典型 MKKK 不同的底物特异性。
