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In eukaryotic organisms, protein kinases regulate diverse protein activities and signaling pathways through phosphorylation of specific protein substrates. Isolating and characterizing kinase substrates is vital for defining downstream signaling pathways. The Kinase Client (KiC) assay is an in vitro synthetic peptide LC-MS/MS phosphorylation assay that has enabled identification of protein substrates (i.e., clients) for various protein kinases. For example, previous use of a 2,100-member (2k) peptide library identified substrates for the extracellular ATP receptor-like kinase, P2K1. Many P2K1 clients were confirmed by additional in vitro and in planta studies, including Integrin-Linked Kinase 4 (ILK4), for which we provide the evidence herein. In addition, we developed a new KiC peptide library containing 8,000 (8k) peptides based on phosphorylation sites primarily from Arabidopsis thaliana datasets. The 8k peptides are enriched for sites with conservation in other angiosperm plants, with the paired goals of representing functionally conserved sites and usefulness for screening kinases from diverse plants. Screening the 8k library with the active P2K1 kinase domain identified 177 phosphopeptides, including calcineurin B-like protein (CBL9) and G protein alpha subunit 1 (GPA1), which functions in cellular calcium signaling. We confirmed that P2K1 directly phosphorylates CBL9 and GPA1 through in vitro kinase assays. This expanded 8k KiC assay will be a useful tool for identifying novel substrates across diverse plant protein kinases, ultimately facilitating the exploration of previously undiscovered signaling pathways. 

Plants are remarkable in their ability to adapt to changing environments, with receptor-like kinases (RLKs) playing a pivotal role in perceiving and transmitting environmental cues into cellular responses. Despite extensive research on RLKs from the plant kingdom, the function and activity of many kinases, i.e., their substrates or “clients”, remain uncharted. To validate a novel client prediction workflow and learn more about an important RLK, this study focuses on P2K1 (DORN1), which acts as a receptor for extracellular ATP (eATP), playing a crucial role in plant stress resistance and immunity. We designed a Kinase-Client (KiC) assay library of 225 synthetic peptides, incorporating previously identified P2K phosphorylated peptides and novel predictions from a deep-learning phosphorylation site prediction model (MUsite) and a trained hidden Markov model (HMM) based tool, HMMER. Screening the library against purified P2K1 cytosolic domain (CD), we identified 46 putative substrates, including 34 novel clients, 27 of which may be novel peptides, not previously identified experimentally. Gene Ontology (GO) analysis among phosphopeptide candidates revealed proteins associated with important biological processes in metabolism, structure development, and response to stress, as well as molecular functions of kinase activity, catalytic activity, and transferase activity. We offer selection criteria for efficient furtherin vivoexperiments to confirm these discoveries. This approach not only expands our knowledge of P2K1’s substrates and functions but also highlights effective prediction algorithms for identifying additional potential substrates. Overall, the results support use of the KiC assay as a valuable tool in unraveling the complexities of plant phosphorylation and provide a foundation for predicting the phosphorylation landscape of plant species based on peptide library results. 

Pseudokinases are thought to lack phosphotransfer activity due to altered canonical catalytic residues within their kinase domain. However, a subset of pseudokinases maintain activity through atypical phosphotransfer mechanisms. The Arabidopsis ILK1 is a pseudokinase from the Raf-like MAP3K family and is the only known plant pseudokinase with confirmed protein kinase activity. ILK1 activity promotes disease resistance and molecular pattern-induced root growth inhibition through its stabilization of the HAK5 potassium transporter with the calmodulin-like protein CML9. ILK1 also has a kinase-independent function in salt stress suggesting that it interacts with additional proteins. We determined that members of the ILK subfamily are the sole pseudokinases within the Raf-like MAP3K family and identified 179 novel putative ILK1 protein interactors. We also identified 70 novel peptide targets for ILK1, the majority of which were phosphorylated in the presence of Mn 2+ instead of Mg 2+ in line with modifications in ILK1’s DFG cofactor binding domain. Overall, the ILK1-targeted or interacting proteins included diverse protein types including transporters (HAK5, STP1), protein kinases (MEKK1, MEKK3), and a cytokinin receptor (AHK2). The expression of 31 genes encoding putative ILK1-interacting or phosphorylated proteins, including AHK2, were altered in the root and shoot in response to molecular patterns suggesting a role for these genes in immunity. We describe a potential role for ILK1 interactors in the context of cation-dependent immune signaling, highlighting the importance of K + in MAMP responses. This work further supports the notion that ILK1 is an atypical kinase with an unusual cofactor dependence that may interact with multiple proteins in the cell. 

Abstract Mitogen-activated protein (MAP) kinase signaling cascades play important roles in eukaryotic defense against various pathogens. Activation of the extracellular ATP (eATP) receptor P2K1 triggers MAP kinase 3 and 6 (MPK3/6) phosphorylation, which leads to an elevated plant defense response. However, the mechanism by which P2K1 activates the MAPK cascade is unclear. In this study, we show that in Arabidopsis thaliana, P2K1 phosphorylates the Raf-like MAP kinase kinase kinase (MAPKKK) INTEGRIN-LINKED KINASE 5 (ILK5) on serine 192 in the presence of eATP. The interaction between P2K1 and ILK5 was confirmed both in vitro and in planta and their interaction was enhanced by ATP treatment. Similar to P2K1 expression, ILK5 expression levels were highly induced by treatment with ATP, flg22, Pseudomonas syringae pv. tomato DC3000, and various abiotic stresses. ILK5 interacts with and phosphorylates the MAP kinase MKK5. Moreover, phosphorylation of MPK3/6 was significantly reduced upon ATP treatment in ilk5 mutant plants, relative to wild-type (WT). The ilk5 mutant plants showed higher susceptibility to P. syringae pathogen infection relative to WT plants. Plants expressing only the mutant ILK5S192A protein, with decreased kinase activity, did not activate the MAPK cascade upon ATP addition. These results suggest that eATP activation of P2K1 results in transphosphorylation of the Raf-like MAPKKK ILK5, which subsequently triggers the MAPK cascade, culminating in activation of MPK3/6 associated with an elevated innate immune response. 

Abstract S -acylation is a reversible protein post-translational modification mediated by protein S -acyltransferases (PATs). How S -acylation regulates plant innate immunity is our main concern. Here, we show that the plant immune receptor P2K1 (DORN1, LecRK-I.9; extracellular ATP receptor) directly interacts with and phosphorylates Arabidopsis PAT5 and PAT9 to stimulate their S -acyltransferase activity. This leads, in a time-dependent manner, to greater S -acylation of P2K1, which dampens the immune response. pat5 and pat9 mutants have an elevated extracellular ATP-induced immune response, limited bacterial invasion, increased phosphorylation and decreased degradation of P2K1 during immune signaling. Mutation of S -acylated cysteine residues in P2K1 results in a similar phenotype. Our study reveals that S -acylation effects the temporal dynamics of P2K1 receptor activity, through autophosphorylation and protein degradation, suggesting an important role for this modification in regulating the ability of plants in respond to external stimuli. 

Abstract In plants, light-dependent activation of de novo fatty acid synthesis (FAS) is partially mediated by acetyl-CoA carboxylase (ACCase), the first committed step for this pathway. However, it is not fully understood how plants control light-dependent FAS regulation to meet the cellular demand for acyl chains. We report here the identification of a gene family encoding for three small plastidial proteins of the envelope membrane that interact with the α-carboxyltransferase (α-CT) subunit of ACCase and participate in an original mechanism restraining FAS in the light. Light enhances the interaction between carboxyltransferase interactors (CTIs) and α-CT, which in turn attenuates carbon flux into FAS. Knockouts for CTI exhibit higher rates of FAS and marked increase in absolute triacylglycerol levels in leaves, more than 4-fold higher than in wild-type plants. Furthermore, WRINKLED1, a master transcriptional regulator of FAS, positively regulatesCTI1expression by direct binding to its promoter. This study reveals that in addition to light-dependent activation, “envelope docking” of ACCase permits fine-tuning of fatty acid supply during the plant life cycle. 

Acetyl-CoA carboxylase (ACCase) catalyzes the first committed step in the de novo synthesis of fatty acids. The multisubunit ACCase in the chloroplast is activated by a shift to pH 8 upon light adaptation and is inhibited by a shift to pH 7 upon dark adaptation. Here, titrations with the purified ACCase biotin attachment domain-containing (BADC) and biotin carboxyl carrier protein (BCCP) subunits from Arabidopsis indicated that they can competently and independently bind biotin carboxylase (BC) but differ in responses to pH changes representing those in the plastid stroma during light or dark conditions. At pH 7 in phosphate buffer, BADC1 and BADC2 gain an advantage over BCCP1 and BCCP2 in affinity for BC. At pH 8 in KCl solution, however, BCCP1 and BCCP2 had more than 10-fold higher affinity for BC than did BADC1. The pH-modulated shifts in BC preferences for BCCP and BADC partners suggest they contribute to light-dependent regulation of heteromeric ACCase. Using NMR spectroscopy, we found evidence for increased intrinsic disorder of the BADC and BCCP subunits at pH 7. We propose that this intrinsic disorder potentially promotes fast association with BC through a “fly-casting mechanism.” We hypothesize that the pH effects on the BADC and BCCP subunits attenuate ACCase activity by night and enhance it by day. Consistent with this hypothesis, Arabidopsis badc1 badc3 mutant lines grown in a light–dark cycle synthesized more fatty acids in their seeds. In summary, our findings provide evidence that the BADC and BCCP subunits function as pH sensors required for light-dependent switching of heteromeric ACCase activity.