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1. MPK12 in stomatal CO₂ signaling: function beyond its kinase activity

   https://doi.org/10.1111/nph.18913  

   Yeh, Chung‐Yueh; Wang, Yuh‐Shuh; Takahashi, Yohei; Kuusk, Katarina; Paul, Karnelia; Arjus, Triinu; Yadlos, Oleksii; Schroeder, Julian I.; Ilves, Ivar; Garcia‐Sosa, Alfonso T.; et al (April 2023, New Phytologist)

   Summary Protein phosphorylation is a major molecular switch involved in the regulation of stomatal opening and closure. Previous research defined interaction between MAP kinase 12 and Raf‐like kinase HT1 as a required step for stomatal movements caused by changes in CO₂concentration. However, whether MPK12 kinase activity is required for regulation of CO₂‐induced stomatal responses warrants in‐depth investigation.We apply genetic, biochemical, and structural modeling approaches to examining the noncatalytic role of MPK12 in guard cell CO₂signaling that relies on allosteric inhibition of HT1.We show that CO₂/HCO₃⁻‐enhanced MPK12 interaction with HT1 is independent of its kinase activity. By analyzing gas exchange of plant lines expressing various kinase‐dead and constitutively active versions of MPK12 in a plant line whereMPK12is deleted, we confirmed that CO₂‐dependent stomatal responses rely on MPK12's ability to bind to HT1, but not its kinase activity. We also demonstrate that purified MPK12 and HT1 proteins form a heterodimer in the presence of CO₂/HCO₃⁻and present structural modeling that explains the MPK12:HT1 interaction interface.These data add to the model that MPK12 kinase‐activity‐independent interaction with HT1 functions as a molecular switch by which guard cells sense changes in atmospheric CO₂concentration. 

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2. Divergent kinase WNG1 is regulated by phosphorylation of an atypical activation sub-domain

   https://doi.org/10.1042/BCJ20220076  

   Dewangan, Pravin S.; Beraki, Tsebaot G.; Paiz, E. Ariana; Appiah Mensah, Delia; Chen, Zhe; Reese, Michael L. (September 2022, Biochemical Journal)

   Apicomplexan parasites like Toxoplasma gondii grow and replicate within a specialized organelle called the parasitophorous vacuole. The vacuole is decorated with parasite proteins that integrate into the membrane after trafficking through the parasite secretory system as soluble, chaperoned complexes. A regulator of this process is an atypical protein kinase called WNG1. Phosphorylation by WNG1 appears to serve as a switch for membrane integration. However, like its substrates, WNG1 is secreted from the parasite dense granules, and its activity must, therefore, be tightly regulated until the correct membrane is encountered. Here, we demonstrate that, while another member of the WNG family can adopt multiple multimeric states, WNG1 is monomeric and therefore not regulated by multimerization. Instead, we identify two phosphosites on WNG1 that are required for its kinase activity. Using a combination of in vitro biochemistry and structural modeling, we identify basic residues that are also required for WNG1 activity and appear to recognize the activating phosphosites. Among these coordinating residues are the ‘HRD’ Arg, which recognizes activation loop phosphorylation in canonical kinases. WNG1, however, is not phosphorylated on its activation loop, but rather on atypical phosphosites on its C-lobe. We propose a simple model in which WNG1 is activated by increasing ATP concentration above a critical threshold once the kinase traffics to the parasitophorous vacuole. 

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3. Kinase‐catalyzed Biotinylation with Inactivated Lysates for Discovery of Substrates (K‐BILDS)

   https://doi.org/10.1002/cpz1.851  

   Gary, Chelsea R.; Pflum, Mary Kay (August 2023, Current Protocols)

   Protein phosphorylation is catalyzed by kinases to regulate a large variety of cellular activities, including growth and signal transduction. Methods to identify kinase substrates are crucial to fully understand phosphorylation‐mediated cellular events and disease states. Here, we report a set of protocols to identify substrates of a target kinase using Kinase‐catalyzed Biotinylation with Inactivated Lysates for Discovery of Substrates (K‐BILDS). As described in these protocols, K‐BILDS involves inactivation of endogenous kinases in lysates, followed by addition of an active exogenous kinase and the γ‐phosphate‐modified ATP analog ATP‐biotin for kinase‐catalyzed biotinylation of cellular substrates. Avidin enrichment isolates biotinylated substrates of the active kinase, which can be monitored by western blot. Substrates of the target kinase can also be discovered using mass spectrometry analysis. Key advantages of K‐BILDS include compatibility with any lysate, tissue homogenate, or complex mixture of biological relevance and any active kinase of interest. K‐BILDS is a versatile method for studying or discovering substrates of a kinase of interest to characterize biological pathways thoroughly. 

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4. DNA-encoded probe-based assay for profiling plant kinase activities

   https://doi.org/10.1093/pnasnexus/pgae281  

   Chien, Yuan-Chi; Valencia, C_Alexander; Lee, Han_Yong; Yoon, Gyeong_Mee; Kim, Dongwook; Bayer, ed., Edward (July 2024, PNAS Nexus)

   Abstract Elucidating kinase–substrate relationships is pivotal for deciphering cellular signaling mechanisms, yet it remains challenging due to the complexity of kinase networks. Herein, we report the development of a versatile DNA-based kinase assay platform for high-throughput profiling of plant protein kinase activities and substrate preferences. Our approach employs DNA-linked peptide substrates, facilitating quantitative and specific kinase activity detection through next-generation DNA sequencing. Leveraging DNA barcodes as quantitative readouts, our approach establishes a high-throughput, sensitive, and specific platform for dissecting kinase–substrate networks in plants, representing a powerful tool for elucidating signaling mechanisms in plants. 

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5. Protein kinase sensors: an overview of new designs for visualizing kinase dynamics in single plant cells

   https://doi.org/10.1093/plphys/kiab277  

   Zhang, Li; Takahashi, Yohei; Schroeder, Julian I. (June 2021, Plant Physiology)

   Abstract Protein kinase dynamics play key roles in regulation of cell differentiation, growth, development and in diverse cell signaling networks. Protein kinase sensors enable visualization of protein kinase activity in living cells and tissues in time and space. These sensors have therefore become important and powerful molecular tools for investigation of diverse kinase activities and can resolve long-standing and challenging biological questions. In the present Update, we review new advanced approaches for genetically encoded protein kinase biosensor designs developed in animal systems together with the basis of each biosensor’s working principle and components. In addition, we review recent first examples of real time plant protein kinase activity biosensor development and application. We discuss how these sensors have helped to resolve how stomatal signal transduction in response to elevated CO2 merges with abscisic acid signaling downstream of a resolved basal SnRK2 kinase activity in guard cells. Furthermore, recent advances, combined with the new strategies described in this Update, can help deepen the understanding of how signaling networks regulate unique functions and responses in distinct plant cell types and tissues and how different stimuli and signaling pathways can interact. 

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