Kincore: a web resource for structural classification of protein kinases and their inhibitors.

The active form of kinases is shared across different family members, as are several commonly observed inactive forms. We previously performed a clustering of the conformation of the activation loop of all protein kinase structures in the Protein Data Bank (PDB) into eight classes based on the dihedral angles that place the Phe side chain of the DFG motif at the N-terminus of the activation loop. Our clusters are strongly associated with the placement of the activation loop, the C-helix, and other structural elements of kinases. We present Kincore, a web resource providing access to our conformational assignments for kinase structures in the PDB. While other available databases provide conformational states or drug type but not both, KinCore includes the conformational state and the inhibitor type (Type 1, 1.5, 2, 3, allosteric) for each kinase chain. The user can query and browse the database using these attributes or determine the conformational labels of a kinase structure using the web server or a standalone program. The database and labeled structure files can be downloaded from the server. Kincore will help in understanding the conformational dynamics of these proteins and guide development of inhibitors targeting specific states. Kincore is available at http://dunbrack.fccc.edu/kincore.

Phosphorylation-dependent subfunctionalization of the calcium-dependent protein kinase CPK28.

Calcium (Ca2+)-dependent protein kinases (CDPKs or CPKs) are a unique family of Ca2+ sensor/kinase-effector proteins with diverse functions in plants. In Arabidopsis thaliana, CPK28 contributes to immune homeostasis by promoting degradation of the key immune signaling receptor-like cytoplasmic kinase BOTRYTIS-INDUCED KINASE 1 (BIK1) and additionally functions in vegetative-to-reproductive stage transition. How CPK28 controls these seemingly disparate pathways is unknown. Here, we identify a single phosphorylation site in the kinase domain of CPK28 (Ser318) that is differentially required for its function in immune homeostasis and stem elongation. We show that CPK28 undergoes intermolecular autophosphorylation on Ser318 and can additionally be transphosphorylated on this residue by BIK1. Analysis of several other phosphorylation sites demonstrates that Ser318 phosphorylation is uniquely required to prime CPK28 for Ca2+ activation at physiological concentrations of Ca2+, possibly through stabilization of the Ca2+-bound active state as indicated by intrinsic fluorescence experiments. Together, our data indicate that phosphorylation of Ser318 is required for the activation of CPK28 at low intracellular [Ca2+] to prevent initiation of an immune response in the absence of infection. By comparison, phosphorylation of Ser318 is not required for stem elongation, indicating pathway-specific requirements for phosphorylation-based Ca2+-sensitivity priming. We additionally provide evidence for a conserved function for Ser318 phosphorylation in related group IV CDPKs, which holds promise for biotechnological applications by generating CDPK alleles that enhance resistance to microbial pathogens without consequences to yield.

Insight into early diversification of leucine-rich repeat receptor-like kinases provided by the sequenced moss and hornwort genomes.

KEY MESSAGE: Identification of the subfamily X leucine-rich repeat receptor-like kinases in the recently sequenced moss and hornwort genomes points to their diversification into distinct groups during early evolution of land plants. Signal transduction mediated through receptor-ligand interactions plays key roles in controlling developmental and physiological processes of multicellular organisms, and plants employ diverse receptors in signaling. Leucine-rich repeat receptor-like kinases (LRR-RLKs) represent one of the largest receptor classes in plants and are structurally classified into subfamilies. LRR-RLKs of the subfamily X are unique in the variety of their signaling roles; they include receptors for steroid or peptide hormones as well as negative regulators of signaling through binding to other LRR-RLKs, raising a question as to how they diversified. However, our understanding of diversification processes of LRR-RLKs has been hindered by the paucity of genomic data in non-seed plants and limited taxa sampling in previous phylogenetic analyses. Here we analyzed the phylogeny of LRR-RLK X sequences collected from all major land plant lineages and show that this subfamily diversified into six major clades before the divergence between bryophytes and vascular plants. Notably, we have identified homologues of the brassinosteroid receptor, BRASSINOSTEROID INSENSITIVE 1 (BRI1), in the genomes of Sphagnum mosses, hornworts, and ferns, contrary to earlier reports that postulate the origin of BRI1-like LRR-RLKs in the seed plant lineage. The phylogenetic distribution of major clades illustrates that the current receptor repertoire was shaped through lineage-specific gene family expansion and independent gene losses, highlighting dynamic changes in the evolution of LRR-RLKs.

Genome-Wide Identification, Classification, Characterization, and Expression Analysis of the Wall-Associated Kinase Family during Fruit Development and under Wound Stress in Tomato (Solanum lycopersicum L.).

The wall-associated kinase (WAK) and wall-associated kinase like (WAKL) is a subfamily of receptor-like kinases associated with the cell wall, which have been suggested as sensors of the extracellular environment and triggers of intracellular signals. However, these proteins have not yet been comprehensively analyzed in tomato (Solanum lycopersicum L.). In this study, 11 SlWAK and 18 SlWAKL genes were identified in an uneven distribution in 9 of 12 chromosomes. GUB-WAK-bind (wall-associated receptor kinase galacturonan-binding) and epidermal growth factor (EGF) domains appear more often in SlWAK proteins. However, more SlWAKLs (wall-associated kinase like) have a WAK-assoc (wall-associated receptor kinase C-terminal) domain. Based on their phylogenetic relationships, 29 SlWAK-RLKs (wall associated kinase-receptor like kinases) were clustered into three distinct categories analogous to those in Arabidopsis thaliana. High similarities were found in conserved motifs of the genes within each group. Cis-elements in the promoter region of these 29 genes were found mainly in response to methyl jasmonate (MeJA), abscisic acid (ABA), salicylic acid (SA), anaerobic, light, wound, and MYB transcription factors. Public tomato genome RNA-seq data indicates that multiple SlWAK-RLKs showed different expression patterns under developmental and ripening stages of fruits, such as SlWAK4, SlWAKL11, SlWAKL9, SlWAKL15, SlWAKL14, and SlWAKL1, their RPKM (Reads Per Kilo bases per Million reads) value constantly increases during the fruit expansion period, and decreases as the fruit matures. In tomato leaves, our RNA-seq data showed that nine SlWAK-RLKs transcripts (SlWAK3, SlWAK4, SlWAK10,SlWAKL1, SlWAKL2, SlWAKL3, SlWAKL5, SlWAKL14, and SlWAKL18) were significantly induced (p < 0.001), and three transcripts (SlWAK2, SlWAK5, and SlWAKL15) were significantly inhibited (p < 0.001) under mechanical wounding. The qRT-PCR (Quantitative reverse transcription polymerase chain reaction) of SlWAKL1 and SlWAKL6 verify these results.

Genome-wide and structural analyses of pseudokinases encoded in the genome of Arabidopsis thaliana provide functional insights.

Protein Kinase-Like Non-Kinases (PKLNKs), commonly known as "pseudokinases", are homologous to eukaryotic Ser/Thr/Tyr protein kinases (PKs) but lack the crucial aspartate residue in the catalytic loop, indispensable for phosphotransferase activity. Therefore, they are predicted to be "catalytically inactive" enzyme homologs. Analysis of protein-kinase like sequences from Arabidopsis thaliana led to the identification of more than 120 pseudokinases lacking catalytic aspartate, majority of which are closely related to the plant-specific receptor-like kinase family. These pseudokinases engage in different biological processes, enabled by their diverse domain architectures and specific subcellular localizations. Structural comparison of pseudokinases with active and inactive conformations of canonical PKs, belonging to both plant and animal origin, revealed unique structural differences. The currently available crystal structures of pseudokinases show that the loop topologically equivalent to activation segment of PKs adopts a distinct-folded conformation, packing against the pseudoenzyme core, in contrast to the extended and inhibitory geometries observed for active and inactive states, respectively, of catalytic PKs. Salt-bridge between ATP-binding Lys and DFG-Asp as well as hydrophobic interactions between the conserved nonpolar residue C-terminal to the equivalent DFG motif and nonpolar residues in C-helix mediate such a conformation in pseudokinases. This results in enhanced solvent accessibility of the pseudocatalytic loop in pseudokinases that can possibly serve as an interacting surface while associating with other proteins. Specifically, our analysis identified several residues that may be involved in pseudokinase regulation and hints at the repurposing of pseudocatalytic residues to achieve mechanistic control over noncatalytic functions of pseudoenzymes.

Kinase Interaction Network Expands Functional and Disease Roles of Human Kinases.

Protein kinases are essential for signal transduction and control of most cellular processes, including metabolism, membrane transport, motility, and cell cycle. Despite the critical role of kinases in cells and their strong association with diseases, good coverage of their interactions is available for only a fraction of the 535 human kinases. Here, we present a comprehensive mass-spectrometry-based analysis of a human kinase interaction network covering more than 300 kinases. The interaction dataset is a high-quality resource with more than 5,000 previously unreported interactions. We extensively characterized the obtained network and were able to identify previously described, as well as predict new, kinase functional associations, including those of the less well-studied kinases PIM3 and protein O-mannose kinase (POMK). Importantly, the presented interaction map is a valuable resource for assisting biomedical studies. We uncover dozens of kinase-disease associations spanning from genetic disorders to complex diseases, including cancer.

Genome-wide analysis of proline-rich extension-like receptor protein kinase (PERK) in Brassica rapa and its association with the pollen development.

BACKGROUND: Proline-rich extension-like receptor protein kinases (PERKs) are an important class of receptor kinases located in the plasma membrane, most of which play a vital role in pollen development. RESULTS: Our study identified 25 putative PERK genes from the whole Brassica rapa genome (AA). Phylogenetic analysis of PERK protein sequences from 16 Brassicaceae species divided them into four subfamilies. The biophysical properties of the BrPERKs were investigated. Gene duplication and synteny analyses and the calculation of Ka/Ks values suggested that all 80 orthologous/paralogous gene pairs between B. rapa and A. thaliana, B. nigra and B. oleracea have experienced strong purifying selection. RNA-Seq data and qRT-PCR analyses showed that several BrPERK genes were expressed in different tissues, while some BrPERKs exhibited high expression levels only in buds. Furthermore, comparative transcriptome analyses from six male-sterile lines of B. rapa indicated that 7 BrPERK genes were downregulated in all six male-sterile lines. Meanwhile, the interaction networks of the BrPERK genes were constructed and 13 PERK coexpressed genes were identified, most of which were downregulated in the male sterile buds. CONCLUSION: Combined with interaction networks, coexpression and qRT-PCR analyses, these results demonstrated that two BrPERK genes, Bra001723.1 and Bra037558.1 (the orthologs of AtPERK6 (AT3G18810)), were downregulated beginning in the meiosis II period of male sterile lines and involved in anther development. Overall, this comprehensive analysis of some BrPERK genes elucidated their roles in male sterility.

Genome-wide identification and characterization of eukaryotic protein kinases.

The tropical liver fluke, Fasciola gigantica is a food-borne parasite responsible for the hepatobiliary disease fascioliasis. The recent completion of F. gigantica genome sequencing by our group has provided a platform for the systematic analysis of the parasite genome. Eukaryotic protein kinases (ePKs) are regulators of cellular phosphorylation. In the present study, we used various computational and bioinformatics tools to extensively analyse the ePKs in F. gigantica (FgePKs) genome. A total of 455 ePKs were identified that represent ~2% of the parasite genome. Out of these, 214 ePKs are typical kinases (Ser/Thr- and Tyr-specific ePKs), and 241 were other kinases. Several FgePKs were found to possess unusual domain architectures, which suggests the diverse nature of the proteins that can be exploited for designing novel inhibitors. 115 kinases showed <35% query coverage when compared to human ePKs highlighting significant divergences in their respective kinomes, further providing a platform for novel structure-based drug designing. This study provides a platform that may open new avenues into our understanding of helminth biochemistry and drug discovery.

The Sucrose Non-Fermenting 1-Related Protein Kinase 2 (SnRK2) Genes Are Multifaceted Players in Plant Growth, Development and Response to Environmental Stimuli.

Reversible protein phosphorylation orchestrated by protein kinases and phosphatases is a major regulatory event in plants and animals. The SnRK2 subfamily consists of plant-specific protein kinases in the Ser/Thr protein kinase superfamily. Early observations indicated that SnRK2s are mainly involved in response to abiotic stress. Recent evidence shows that SnRK2s are multifarious players in a variety of biological processes. Here, we summarize the considerable knowledge of SnRK2s, including evolution, classification, biological functions and regulatory mechanisms at the epigenetic, post-transcriptional and post-translation levels.

The Landscape of Atypical and Eukaryotic Protein Kinases.

Kinases are attractive anticancer targets due to their central role in the growth, survival, and therapy resistance of tumor cells. This review explores the two primary kinase classes, the eukaryotic protein kinases (ePKs) and the atypical protein kinases (aPKs), and provides a structure-centered comparison of their sequences, structures, hydrophobic spines, mutation and SNP hotspots, and inhibitor interaction patterns. Despite the limited sequence similarity between these two classes, atypical kinases commonly share the archetypical kinase fold but lack conserved eukaryotic kinase motifs and possess altered hydrophobic spines. Furthermore, atypical kinase inhibitors explore only a limited number of binding modes both inside and outside the orthosteric binding site. The distribution of genetic variations in both classes shows multiple ways they can interfere with kinase inhibitor binding. This multilayered review provides a research framework bridging the eukaryotic and atypical kinase classes.

Thinking outside of the cell: Secreted protein kinases in bacteria, parasites, and mammals.

Previous decades have seen an explosion in our understanding of protein kinase function in human health and disease. Hundreds of unique kinase structures have been solved, allowing us to create generalized rules for catalysis, assign roles of communities within the catalytic core, and develop specific drugs for targeting various pathways. Although our understanding of intracellular kinases has developed at a fast rate, our exploration into extracellular kinases has just begun. In this review, we will cover the secreted protein kinase families found in humans, bacteria, and parasites. © 2019 IUBMB Life, 71(6):749-759, 2019.

Targeted Quantitative Kinome Analysis Identifies PRPS2 as a Promoter for Colorectal Cancer Metastasis.

Kinases are among the most important families of enzymes involved in cell signaling. In this study, we employed a recently developed parallel-reaction monitoring (PRM)-based targeted proteomic method to examine the reprogramming of the human kinome during colorectal cancer (CRC) metastasis. We were able to quantify the relative expression of 299 kinase proteins in a pair of matched primary/metastatic CRC cell lines. We also found that, among the differentially expressed kinases, phosphoribosyl pyrophosphate synthetase 2 (PRPS2) promotes the migration and invasion of cultured CRC cells through regulating the activity of matrix metalloproteinase 9 (MMP-9) and the expression of E-cadherin. Moreover, we found that the up-regulation of PRPS2 in metastatic CRC cells could be induced by the MYC proto-oncogene. Together, our unbiased kinome profiling approach led to the identification, for the first time, of PRPS2 as a promoter for CRC metastasis.

Mechanistic insights into the evolution of DUF26-containing proteins in land plants.

Large protein families are a prominent feature of plant genomes and their size variation is a key element for adaptation. However, gene and genome duplications pose difficulties for functional characterization and translational research. Here we infer the evolutionary history of the DOMAIN OF UNKNOWN FUNCTION (DUF) 26-containing proteins. The DUF26 emerged in secreted proteins. Domain duplications and rearrangements led to the appearance of CYSTEINE-RICH RECEPTOR-LIKE PROTEIN KINASES (CRKs) and PLASMODESMATA-LOCALIZED PROTEINS (PDLPs). The DUF26 is land plant-specific but structural analyses of PDLP ectodomains revealed strong similarity to fungal lectins and thus may constitute a group of plant carbohydrate-binding proteins. CRKs expanded through tandem duplications and preferential retention of duplicates following whole genome duplications, whereas PDLPs evolved according to the dosage balance hypothesis. We propose that new gene families mainly expand through small-scale duplications, while fractionation and genetic drift after whole genome multiplications drive families towards dosage balance.

Convex hull principle for classification and phylogeny of eukaryotic proteins.

This study quantitatively validates the principle that the biological properties associated with a given genotype are determined by the distribution of amino acids. In order to visualize this central law of molecular biology, each protein was represented by a point in 250-dimensional space based on its amino acid distribution. Proteins from the same family are found to cluster together, leading to the principle that the convex hull surrounding protein points from the same family do not intersect with the convex hulls of other protein families. This principle was verified computationally for all available and reliable protein kinases and human proteins. In addition, we generated 2,328,761 figures to show that the convex hulls of different families were disjoint from each other. The classification performs well with high and robust accuracy (95.75% and 97.5%) together with reasonable phylogenetic trees validate our methods further.

Genome-wide identification of SERK genes in apple and analyses of their role in stress responses and growth.

BACKGROUND: Somatic embryogenesis receptor-like kinases (SERKs) are leucine-rich repeat receptor-like kinases associated with various signaling pathways. These kinases have a relationship with stress signals, and they are also believed to be important for regulating plant growth. However, information about this protein family in apple is limited. RESULTS: Twelve apple SERK genes distributed across eight chromosomes were identified. These genes clustered into three distinct groups in a phylogenetic analysis. All of the encoded proteins contained typical SERK domains. The chromosomal locations, gene/protein structures, synteny, promoter sequences, protein-protein interactions, and physicochemical characteristics of MdSERK genes were analyzed. Bioinformatics analyses demonstrated that gene duplications have likely contributed to the expansion and evolution of SERK genes in the apple genome. Six homologs of SERK genes were identified between apple and Arabidopsis. Quantitative real-time PCR analyses revealed that the MdSERK genes showed different expression patterns in various tissues. Eight MdSERK genes were responsive to stress signals, such as methyl jasmonate, salicylic acid, abscisic acid, and salt (NaCl). The application of exogenous brassinosteroid and auxin increased the growth and endogenous hormone contents of Malus hupehensis seedlings. The expression levels of seven MdSERK genes were significantly upregulated by brassinosteroid and auxin. In addition, several MdSERK genes showed higher expression levels in standard trees of 'Nagafu 2' (CF)/CF than in dwarf trees of CF/'Malling 9' (M.9), and in CF than in the spur-type bud mutation "Yanfu 6" (YF). CONCLUSION: This study represents the first comprehensive investigation of the apple SERK gene family. These data indicate that apple SERKs may function in adaptation to adverse environmental conditions and may also play roles in controlling apple tree growth.

Improved strategy for the curation and classification of kinases, with broad applicability to other eukaryotic protein groups.

Despite the substantial amount of genomic and transcriptomic data available for a wide range of eukaryotic organisms, most genomes are still in a draft state and can have inaccurate gene predictions. To gain a sound understanding of the biology of an organism, it is crucial that inferred protein sequences are accurately identified and annotated. However, this can be challenging to achieve, particularly for organisms such as parasitic worms (helminths), as most gene prediction approaches do not account for substantial phylogenetic divergence from model organisms, such as Caenorhabditis elegans and Drosophila melanogaster, whose genomes are well-curated. In this paper, we describe a bioinformatic strategy for the curation of gene families and subsequent annotation of encoded proteins. This strategy relies on pairwise gene curation between at least two closely related species using genomic and transcriptomic data sets, and is built on recent work on kinase complements of parasitic worms. Here, we discuss salient technical aspects of this strategy and its implications for the curation of protein families more generally.

Duplication and diversification of lectin receptor-like kinases (LecRLK) genes in soybean.

Lectin receptor-like kinases (LecRLKs) play important roles in plant development and stress responses. Although genome-wide studies of LecRLKs have been performed in several species, a comprehensive analysis including evolutionary, structural and functional analysis has not been carried out in soybean (Glycine max). In this study, we identified 185 putative LecRLK genes in the soybean genome, including 123 G-type, 60 L-type and 2 C-type LecRLK genes. Tandem duplication and segmental duplication appear to be the main mechanisms of gene expansion in the soybean LecRLK (GmLecRLK) gene family. According to our phylogenetic analysis, G-type and L-type GmLecRLK genes can be organized into fourteen and eight subfamilies, respectively. The subfamilies within the G-type GmLecRLKs differ from each other in gene structure and/or protein domains and motifs, which indicates that the subfamilies have diverged. The evolution of L-type GmLecRLKs has been more conservative: most genes retain the same gene structures and nearly the same protein domain and motif architectures. Furthermore, the expression profiles of G-type and L-type GmLecRLK genes show evidence of functional redundancy and divergence within each group. Our results contribute to a better understanding of the evolution and function of soybean LecRLKs and provide a framework for further functional investigation of them.

Classification and phylogenetic analyses of the Arabidopsis and tomato G-type lectin receptor kinases.

BACKGROUND: Pathogen perception by plants is mediated by plasma membrane-localized immune receptors that have varied extracellular domains. Lectin receptor kinases (LecRKs) are among these receptors and are subdivided into 3 classes, C-type LecRKs (C-LecRKs), L-type LecRKs (L-LecRKs) and G-type LecRKs (G-LecRKs). While C-LecRKs are represented by one or two members in all plant species investigated and have unknown functions, L-LecRKs have been characterized in a few plant species and have been shown to play roles in plant defense against pathogens. Whereas Arabidopsis G-LecRKs have been characterized, this family of LecRKs has not been studied in tomato. RESULTS: This investigation updates the current characterization of Arabidopsis G-LecRKs and characterizes the tomato G-LecRKs, using LecRKs from the monocot rice and the basal eudicot columbine to establish a basis for comparisons between the two core eudicots. Additionally, revisiting parameters established for Arabidopsis nomenclature for LecRKs is suggested for both Arabidopsis and tomato. Moreover, using phylogenetic analysis, we show the relationship among and between members of G-LecRKs from all three eudicot plant species. Furthermore, investigating presence of motifs in G-LecRKs we identified conserved motifs among members of G-LecRKs in tomato and Arabidopsis, with five present in at least 30 of the 38 Arabidopsis members and in at least 45 of the 73 tomato members. CONCLUSIONS: This work characterized tomato G-LecRKs and added members to the currently characterized Arabidopsis G-LecRKs. Additionally, protein sequence analysis showed an expansion of this family in tomato as compared to Arabidopsis, and the existence of conserved common motifs in the two plant species as well as conserved species-specific motifs.

STRESS INDUCED FACTOR 2, a Leucine-Rich Repeat Kinase Regulates Basal Plant Pathogen Defense.

Protein kinases play fundamental roles in plant development and environmental stress responses. Here, we identified the STRESS INDUCED FACTOR (SIF) gene family, which encodes four leucine-rich repeat receptor-like protein kinases in Arabidopsis (Arabidopsis thaliana). The four genes, SIF1 to SIF4, are clustered in the genome and highly conserved, but they have temporally and spatially distinct expression patterns. We employed Arabidopsis SIF knockout mutants and overexpression transgenics to examine SIF involvement during plant pathogen defense. SIF genes are rapidly induced by biotic or abiotic stresses, and SIF proteins localize to the plasma membrane. Simultaneous knockout of SIF1 and SIF2 led to improved plant salt tolerance, whereas SIF2 overexpression enhanced PAMP-triggered immunity and prompted basal plant defenses, significantly improving pathogen resistance. Furthermore, SIF2 overexpression plants exhibited up-regulated expression of the defense-related genes WRKY53 and flg22-INDUCED RECEPTOR-LIKE KINASE1 as well as enhanced MPK3/MPK6 phosphorylation upon pathogen and elicitor treatments. The expression of the calcium signaling-related gene PHOSPHATE-INDUCED1 also was enhanced in the SIF2-overexpressing lines upon pathogen inoculation but repressed in the sif2 mutants. Bimolecular fluorescence complementation demonstrates that the BRI1-ASSOCIATED RECEPTOR KINASE1 protein is a coreceptor of the SIF2 kinase in the signal transduction pathway during pathogen invasion. These findings characterize a stress-responsive protein kinase family and illustrate how SIF2 modulates signal transduction for effective plant pathogenic defense.