RESUMO
Plants use leucine-rich repeat receptor kinases (LRR-RKs) to sense sequence diverse peptide hormones at the cell surface. A 3.0-Å crystal structure of the LRR-RK GSO1/SGN3 regulating Casparian strip formation in the endodermis reveals a large spiral-shaped ectodomain. The domain provides a binding platform for 21 amino acid CIF peptide ligands, which are tyrosine sulfated by the tyrosylprotein sulfotransferase TPST/SGN2. GSO1/SGN3 harbors a binding pocket for sulfotyrosine and makes extended backbone interactions with CIF2. Quantitative biochemical comparisons reveal that GSO1/SGN3-CIF2 represents one of the strongest receptor-ligand pairs known in plants. Multiple missense mutations are required to block CIF2 binding in vitro and GSO1/SGN3 function in vivo. Using structure-guided sequence analysis we uncover previously uncharacterized CIF peptides conserved among higher plants. Quantitative binding assays with known and novel CIFs suggest that the homologous LRR-RKs GSO1/SGN3 and GSO2 have evolved unique peptide binding properties to control different developmental processes. A quantitative biochemical interaction screen, a CIF peptide antagonist and genetic analyses together implicate SERK proteins as essential coreceptor kinases required for GSO1/SGN3 and GSO2 receptor activation. Our work provides a mechanistic framework for the recognition of sequence-divergent peptide hormones in plants.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Peptídeos/metabolismo , Proteínas Quinases/metabolismo , Sequência de Aminoácidos , Arabidopsis/química , Arabidopsis/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Cinética , Ligantes , Peptídeos/química , Reguladores de Crescimento de Plantas/química , Reguladores de Crescimento de Plantas/metabolismo , Ligação Proteica , Proteínas Quinases/química , Proteínas Quinases/genéticaRESUMO
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.
Assuntos
Proteínas de Ligação a DNA/genética , Embriófitas/genética , Evolução Molecular , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Proteínas de Plantas/genética , Proteínas de Ligação a DNA/classificação , Proteínas de Ligação a DNA/metabolismo , Embriófitas/classificação , Embriófitas/metabolismo , Dosagem de Genes , Duplicação Gênica , Ontologia Genética , Deriva Genética , Peptídeos e Proteínas de Sinalização Intracelular/classificação , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Anotação de Sequência Molecular , Filogenia , Proteínas de Plantas/classificação , Proteínas de Plantas/metabolismo , Proteínas Quinases/classificação , Proteínas Quinases/genética , Proteínas Quinases/metabolismoRESUMO
Arabidopsis root development is orchestrated by signaling pathways that consist of different CLAVATA3/EMBRYO SURROUNDING REGION (CLE) peptide ligands and their cognate CLAVATA (CLV) and BARELY ANY MERISTEM (BAM) receptors. How and where different CLE peptides trigger specific morphological or physiological changes in the root is poorly understood. Here, we report that the receptor-like protein CLAVATA 2 (CLV2) and the pseudokinase CORYNE (CRN) are necessary to fully sense root-active CLE peptides. We uncover BAM3 as the CLE45 receptor in the root and biochemically map its peptide binding surface. In contrast to other plant peptide receptors, we found no evidence that SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK) proteins act as co-receptor kinases in CLE45 perception. CRN stabilizes BAM3 expression and thus is required for BAM3-mediated CLE45 signaling. Moreover, protophloem-specific CRN expression complements resistance of the crn mutant to root-active CLE peptides, suggesting that protophloem is their principal site of action. Our work defines a genetic framework for dissecting CLE peptide signaling and CLV/BAM receptor activation in the root.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Proteínas de Membrana/metabolismo , Floema/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Superfície Celular/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Membrana/genética , Peptídeos/genética , Peptídeos/metabolismo , Floema/genética , Raízes de Plantas/fisiologia , Plantas Geneticamente Modificadas , Proteínas Serina-Treonina Quinases/genética , Receptores de Superfície Celular/genética , Transdução de SinaisRESUMO
A series of 2'-aminoanilides have been identified which exhibit potent and selective inhibitory activity against the cFMS tyrosine kinase. Initial SAR studies within this series are described which examine aroyl and amino group substitutions, as well as the introduction of hydrophilic substituents on the benzene core. Compound 47 inhibits the isolated enzyme (IC(50)=0.027 microM) and blocks CSF-1-induced proliferation of bone marrow-derived macrophages (IC(50)=0.11 microM) and as such, serves as a lead candidate for further optimization studies.
Assuntos
Anilidas/síntese química , Anilidas/farmacologia , Anti-Inflamatórios/farmacologia , Piperidinas/química , Inibidores de Proteínas Quinases/farmacologia , Receptor de Fator Estimulador de Colônias de Macrófagos/antagonistas & inibidores , Anilidas/química , Anti-Inflamatórios/síntese química , Anti-Inflamatórios/química , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Humanos , Concentração Inibidora 50 , Macrófagos/efeitos dos fármacos , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Relação Estrutura-AtividadeRESUMO
The cFMS proto-oncogene encodes for the colony-stimulating factor-1 receptor, a receptor-tyrosine kinase responsible for the differentiation and maturation of certain macrophages. Upon binding its ligand colony-stimulating factor-1 cFMS autophosphorylates, dimerizes, and induces phosphorylation of downstream targets. We report the novel crystal structure of unphosphorylated cFMS in complex with two members of different classes of drug-like protein kinase inhibitors. cFMS exhibits a typical bi-lobal kinase fold, and its activation loop and DFG motif are found to be in the canonical inactive conformation. Both ATP competitive inhibitors are bound in the active site and demonstrate a binding mode similar to that of STI-571 bound to cABL. The DFG motif is prevented from switching into the catalytically competent conformation through interactions with the inhibitors. Activation of cFMS is also inhibited by the juxtamembrane domain, which interacts with residues of the active site and prevents formation of the activated kinase. Together the structures of cFMS provide further insight into the autoinhibition of receptor-tyrosine kinases via their respective juxtamembrane domains; additionally the binding mode of two novel classes of kinase inhibitors will guide the design of novel molecules targeting macrophage-related diseases.
Assuntos
Inibidores de Proteínas Quinases/química , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/química , Receptor de Fator Estimulador de Colônias de Macrófagos/antagonistas & inibidores , Receptor de Fator Estimulador de Colônias de Macrófagos/química , Amidas/química , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Humanos , Dados de Sequência Molecular , Proteínas Mutantes Quiméricas/antagonistas & inibidores , Proteínas Mutantes Quiméricas/química , Estrutura Terciária de Proteína/genética , Proto-Oncogene Mas , Quinolonas/química , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Receptor de Fator Estimulador de Colônias de Macrófagos/genética , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Receptor TIE-2/química , Receptor TIE-2/genética , Receptores de Fatores de Crescimento de Fibroblastos/química , Receptores de Fatores de Crescimento de Fibroblastos/genéticaRESUMO
2-Hydroxy-4,6-diamino-[1,3,5]triazines are described which are a novel class of potent inhibitors of the VEGF-R2 (flk-1/KDR) tyrosine kinase. 4-(Benzothiazol-6-ylamino)-6-(benzyl-isopropyl-amino)-[1,3,5]triazin-2-ol (14d) exhibited low nanomolar potency in the in vitro enzyme inhibition assay (IC(50) = 18 nM) and submicromolar inhibitory activity in a KDR-induced MAP kinase autophosphorylation assay in HUVEC cells (IC(50) = 280 nM), and also demonstrated good in vitro selectivity against a panel of growth factor receptor tyrosine kinases. Further, 14d showed antiangiogenic activity in an aortic ring explant assay by blocking endothelial outgrowths in rat aortas with an IC(50) of 1 microM.