RESUMO
Mitogen-activated protein kinase (MAPK)-activated protein kinases (MAPKAPKs) are defined by their exclusive activation by MAPKs. They can be activated by classical and atypical MAPKs that have been stimulated by mitogens and various stresses. Genetic deletions of MAPKAPKs and availability of highly specific small-molecule inhibitors have continuously increased our functional understanding of these kinases. MAPKAPKs cooperate in the regulation of gene expression at the level of transcription; RNA processing, export, and stability; and protein synthesis. The diversity of stimuli for MAPK activation, the crosstalk between the different MAPKs and MAPKAPKs, and the specific substrate pattern of MAPKAPKs orchestrate immediate-early and inflammatory responses in space and time and ensure proper control of cell growth, differentiation, and cell behavior. Hence, MAPKAPKs are promising targets for cancer therapy and treatments for conditions of acute and chronic inflammation, such as cytokine storms and rheumatoid arthritis.
Assuntos
Quinases de Proteína Quinase Ativadas por Mitógeno , Proteínas Quinases Ativadas por Mitógeno , Humanos , Inflamação/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/química , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/química , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , FosforilaçãoRESUMO
Allosteric interactions provide precise spatiotemporal control over signaling proteins, but how allosteric activators and their targets coevolve is poorly understood. Here, we trace the evolution of two allosteric activator motifs within the yeast scaffold protein Ste5 that specifically target the mating MAP kinase Fus3. One activator (Ste5-VWA) provides pathway insulation and dates to the divergence of Fus3 from its paralog, Kss1; a second activator (Ste5-FBD) that tunes mating behavior is, in contrast, not conserved in most lineages. Surprisingly, both Ste5 activator motifs could regulate MAP kinases that diverged from Fus3 prior to the emergence of Ste5, suggesting that Ste5 activators arose by exploiting latent regulatory features already present in the MAPK ancestor. The magnitude of this latent allosteric potential drifts widely among pre-Ste5 MAP kinases, providing a pool of hidden phenotypic diversity that, when revealed by new activators, could lead to functional divergence and to the evolution of distinct signaling behaviors.
Assuntos
Ascomicetos/genética , Proteínas Quinases Ativadas por Mitógeno/química , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Saccharomyces cerevisiae/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Regulação Alostérica , Ascomicetos/enzimologia , Ascomicetos/metabolismo , Ativação Enzimática , Evolução Molecular , Proteínas Quinases Ativadas por Mitógeno/genética , Modelos Moleculares , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de SinaisRESUMO
Allosteric mechanisms provide finely-tuned control over signalling proteins. Proteins of the same family may share high sequence identity and structural similarity but show distinct traits of allosteric control and evolutionary divergent regulation. Revealing the determinants of such properties may be important to understand the molecular bases of different regulatory pathways. Herein, we investigate whether and how evolutionarily-divergent traits of allosteric regulation in homologous proteins can be decoded in terms of internal dynamics and interaction networks that support functionally oriented conformations. In this framework, we start from the comparative analysis of the dynamics and energetics of the yeast MAP Kinases (MAPKs) Fus3 and Kss1 in their native basins. Importantly, distinctive dynamic and energetic stabilization features emerge, which can be related to the two proteins' differential ability to be phosphorylated and engage with the allosteric activator Ste5. We then expanded our study to other evolutionarily-related MAPKs. We show that the dynamical and energetical traits defining the distinct regulatory profiles of Fus3 and Kss1 can be traced along their evolutionary tree. Overall, our approach is able to reconnect (latent) allostery with the principal elements of protein structural stabilization and dynamics, showing how allosteric regulation was encrypted in MAPKs structure well before Ste5 appearance.
Assuntos
Proteínas Quinases Ativadas por Mitógeno , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Regulação Alostérica , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/química , Proteínas Quinases Ativadas por Mitógeno/genética , Conformação Proteica , Simulação de Dinâmica Molecular , Evolução Molecular , TermodinâmicaRESUMO
The scaffold protein Ste5 is required to properly direct signaling through the yeast mating pathway to the mitogen-activated protein kinase (MAPK), Fus3. Scaffolds are thought to function by tethering kinase and substrate in proximity. We find, however, that the previously identified Fus3-binding site on Ste5 is not required for signaling, suggesting an alternative mechanism controls Fus3's activation by the MAPKK Ste7. Reconstituting MAPK signaling in vitro, we find that Fus3 is an intrinsically poor substrate for Ste7, although the related filamentation MAPK, Kss1, is an excellent substrate. We identify and structurally characterize a domain in Ste5 that catalytically unlocks Fus3 for phosphorylation by Ste7. This domain selectively increases the k(cat) of Ste7-->Fus3 phosphorylation but has no effect on Ste7-->Kss1 phosphorylation. The dual requirement for both Ste7 and this Ste5 domain in Fus3 activation explains why Fus3 is selectively activated by the mating pathway and not by other pathways that also utilize Ste7.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Regulação Alostérica , Ativação Enzimática , Sistema de Sinalização das MAP Quinases , Quinases de Proteína Quinase Ativadas por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/química , Modelos Moleculares , Fosforilação , Proteínas Quinases/metabolismo , Estrutura Terciária de Proteína , Proteínas de Saccharomyces cerevisiae/químicaRESUMO
Short linear peptide motifs that are intracellular ligands of folded proteins are a modular, incompletely understood molecular interaction language in signaling systems. Such motifs, which frequently occur in intrinsically disordered protein regions, often bind partner proteins with modest affinity and are difficult to study with conventional structural biology methods. We developed LiF-MS (ligand-footprinting mass spectrometry), a method to map peptide binding sites on folded protein domains that allows consideration of their dynamic disorder, and used it to analyze a set of D-motif peptide-mitogen-activated protein kinase (MAPK) associations to validate the approach and define unknown binding structures. LiF-MS peptide ligands carry a short-lived, indiscriminately reactive cleavable crosslinker that marks contacts close to ligand binding sites with high specificity. Each marked amino acid provides an independent constraint for a set of directed peptide-protein docking simulations, which are analyzed by agglomerative hierarchical clustering. We found that LiF-MS provides accurate ab initio identification of ligand binding surfaces and a view of potential binding ensembles of a set of D-motif peptide-MAPK associations. Our analysis provides an MKK4-JNK1 structural model, which has thus far been crystallographically unattainable, a potential alternate binding mode for part of the NFAT4-JNK interaction, and evidence of bidirectional association of MKK4 peptide with ERK2. Overall, we find that LiF-MS is an effective noncrystallographic way to understand how short linear motifs associate with specific sites on folded protein domains at the level of individual amino acids.
Assuntos
Espectrometria de Massas/métodos , Proteínas Quinases Ativadas por Mitógeno/química , Peptídeos/química , Mapeamento de Interação de Proteínas/métodos , Motivos de Aminoácidos , Sítios de Ligação , Humanos , Ligantes , Sistema de Sinalização das MAP Quinases , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Peptídeos/metabolismo , Ligação Proteica , Dobramento de ProteínaRESUMO
Preliminary bioassay-guided fractionation was performed to identify cytotoxic compounds from Hechtia glomerata, a plant that is used in Mexican ethnomedicine. Organic and aqueous extracts were prepared from H. glomerata's leaves and evaluated against two cancer cell lines. The CHCl3/MeOH (1:1) active extract was fractionated, and the resulting fractions were assayed against prostate adenocarcinoma PC3 and breast adenocarcinoma MCF7 cell lines. Active fraction 4 was further analyzed by high-performance liquid chromatography-quadrupole time-of-flight-mass spectrometry analysis to identify its active constituents. Among the compounds that were responsible for the cytotoxic effects of this fraction were flavonoids, phenolic acids, and aromatic compounds, of which p-coumaric acid (p-CA) and its derivatives were abundant. To understand the mechanisms that underlie p-CA cytotoxicity, a microarray assay was performed on PC3 cells that were treated or not with this compound. The results showed that mitogen-activated protein kinases (MAPKs) that regulate many cancer-related pathways were targeted by p-CA, which could be related to the reported effects of reactive oxygen species (ROS). A molecular docking study of p-CA showed that this phenolic acid targeted these protein active sites (MAPK8 and Serine/Threonine protein kinase 3) at the same binding site as their inhibitors. Thus, we hypothesize that p-CA produces ROS, directly affects the MAPK signaling pathway, and consequently causes apoptosis, among other effects. Additionally, p-CA could be used as a platform for the design of new MAPK inhibitors and re-sensitizing agents for resistant cancers.
Assuntos
Bromeliaceae/química , Ácidos Cumáricos/farmacologia , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Extratos Vegetais/química , Inibidores de Proteínas Quinases/farmacologia , Bioensaio , Morte Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Ácidos Cumáricos/química , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Humanos , Células MCF-7 , Proteínas Quinases Ativadas por Mitógeno/química , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Células PC-3 , Fenóis/farmacologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
In this study isolates from Thymelaea hirsuta, a wild plant from the Sinai Peninsula of Egypt, were identified and their selective cytotoxicity levels were evaluated. Phytochemical examination of the ethyl acetate (EtOAc) fraction of the methanolic (MeOH) extract of the plant led to the isolation of a new triflavanone compound (1), in addition to the isolation of nine previously reported compounds. These included five dicoumarinyl ethers found in Thymelaea: daphnoretin methyl ether (2), rutamontine (3), neodaphnoretin (4), acetyldaphnoretin (5), and edgeworthin (6); two flavonoids: genkwanin (7) and trans-tiliroside (8); p-hydroxy benzoic acid (9) and ß sitosterol glucoside (10). Eight of the isolated compounds were tested for in vitro cytotoxicity against Vero and HepG2 cell lines using a sulforhodamine-B (SRB) assay. Compounds 1, 2 and 5 exhibited remarkable cytotoxic activities against HepG2 cells, with IC50 values of 8.6, 12.3 and 9.4 µM, respectively, yet these compounds exhibited non-toxic activities against the Vero cells. Additionally, compound 1 further exhibited promising cytotoxic activity against both MCF-7 and HCT-116 cells, with IC50 values of 4.26 and 9.6 µM, respectively. Compound 1 significantly stimulated apoptotic breast cancer cell death, resulting in a 14.97-fold increase and arresting 40.57% of the cell population at the Pre-G1 stage of the cell cycle. Finally, its apoptosis-inducing activity was further validated through activation of BAX and caspase-9, and inhibition of BCL2 levels. In silico molecular docking experiments revealed a good binding mode profile of the isolates towards Ras activation/pathway mitogen-activated protein kinase (Ras/MAPK); a common molecular pathway in the development and progression of liver tumors.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Flavanonas/química , Flavanonas/farmacologia , Simulação de Acoplamento Molecular , Thymelaeaceae/química , Antineoplásicos/isolamento & purificação , Antineoplásicos/metabolismo , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Flavanonas/isolamento & purificação , Flavanonas/metabolismo , Humanos , Proteínas Quinases Ativadas por Mitógeno/química , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Conformação ProteicaRESUMO
An apical plasma membrane domain enriched in the regulatory phospholipid phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] is critical for polar tip growth of pollen tubes. How the biosynthesis of PtdIns(4,5)P2 by phosphatidylinositol 4-phosphate 5-kinases (PI4P 5-kinases) is controlled by upstream signaling is currently unknown. The pollen-expressed PI4P 5-kinase PIP5K6 is required for clathrin-mediated endocytosis and polar tip growth in pollen tubes. Here, we identify PIP5K6 as a target of the pollen-expressed mitogen-activated protein kinase MPK6 and characterize the regulatory effects. Based on an untargeted mass spectrometry approach, phosphorylation of purified recombinant PIP5K6 by pollen tube extracts could be attributed to MPK6. Recombinant MPK6 phosphorylated residues T590 and T597 in the variable insert of the catalytic domain of PIP5K6, and this modification inhibited PIP5K6 activity in vitro. PIP5K6 interacted with MPK6 in yeast two-hybrid tests, immuno-pull-down assays, and by bimolecular fluorescence complementation at the apical plasma membrane of pollen tubes. In vivo, MPK6 expression resulted in reduced plasma membrane association of a fluorescent PtdIns(4,5)P2 reporter and decreased endocytosis without impairing membrane association of PIP5K6. Effects of PIP5K6 expression on pollen tube growth and cell morphology were attenuated by coexpression of MPK6 in a phosphosite-dependent manner. Our data indicate that MPK6 controls PtdIns(4,5)P2 production and membrane trafficking in pollen tubes, possibly contributing to directional growth.
Assuntos
Arabidopsis/enzimologia , Membrana Celular/enzimologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Nicotiana/enzimologia , Fosfatidilinositol 4,5-Difosfato/metabolismo , Tubo Polínico/enzimologia , Tubo Polínico/crescimento & desenvolvimento , Motivos de Aminoácidos , Sequência de Aminoácidos , Arabidopsis/citologia , Biocatálise , Endocitose , Corantes Fluorescentes/metabolismo , Proteínas Quinases Ativadas por Mitógeno/química , Modelos Biológicos , Fosforilação , Fosfotreonina/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Tubo Polínico/citologia , Ligação Proteica/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Recombinantes , Nicotiana/citologiaRESUMO
Advances in the use of targeted and immune therapies have revolutionized the clinical management of melanoma patients, prolonging significantly their overall and progression-free survival. However, both targeted and immune therapies suffer limitations due to genetic mutations and epigenetic modifications, which determine a great heterogeneity and phenotypic plasticity of melanoma cells. Acquired resistance of melanoma patients to inhibitors of BRAF (BRAFi) and MEK (MEKi), which block the mitogen-activated protein kinase (MAPK) pathway, limits their prolonged use. On the other hand, immune checkpoint inhibitors improve the outcomes of patients in only a subset of them and the molecular mechanisms underlying lack of responses are under investigation. There is growing evidence that altered expression levels of microRNAs (miRNA)s induce drug-resistance in tumor cells and that restoring normal expression of dysregulated miRNAs may re-establish drug sensitivity. However, the relationship between specific miRNA signatures and acquired resistance of melanoma to MAPK and immune checkpoint inhibitors is still limited and not fully elucidated. In this review, we provide an updated overview of how miRNAs induce resistance or restore melanoma cell sensitivity to mitogen-activated protein kinase inhibitors (MAPKi) as well as on the relationship existing between miRNAs and immune evasion by melanoma cell resistant to MAPKi.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Inibidores de Checkpoint Imunológico/uso terapêutico , Melanoma/genética , MicroRNAs/genética , Proteínas Quinases Ativadas por Mitógeno/química , Inibidores de Proteínas Quinases/uso terapêutico , Animais , Humanos , Melanoma/tratamento farmacológico , Melanoma/patologiaRESUMO
The fungal cell wall is very important for cell growth and survival during stress, and the target of rapamycin (TOR) pathway plays a major role in regulating cell growth in response to environmental cues. Ganoderma lucidum is an important edible and medicinal fungus, and the function of TOR in this organism remains unclear. As shown in the present study, the TOR pathway regulates cell wall integrity (CWI) in G. lucidum. Inhibition of TOR signaling by RNA interference (RNAi) or rapamycin treatment reduced the growth of G. lucidum mycelia, increased contents of the cell wall components chitin and ß-1,3-glucan, and increased cell wall thickness. Furthermore, inhibition of TOR signaling enhanced the relative level of phosphorylated Slt2, a member of the MAPK cascade involved in CWI signaling. Moreover, when treated with rapamycin, significantly lower chitin and ß-1,3-glucan contents were observed in Slt2-silenced strains than in WT strains, indicating that TOR regulates the synthesis of these cell wall components through the Slt2-MAPK pathway. These results indicate a potential relationship between TOR signaling and CWI signaling. Additionally, participation of Slt2-MAPK in TOR-mediated regulation of cell wall component production has not previously been reported in a microorganism.
Assuntos
Parede Celular/metabolismo , Reishi/genética , Sirolimo/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Parede Celular/genética , Quitina/química , Quitina/genética , Sistema de Sinalização das MAP Quinases/genética , Proteínas Quinases Ativadas por Mitógeno/química , Proteínas Quinases Ativadas por Mitógeno/genética , Fosforilação , Interferência de RNA , Reishi/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Serina-Treonina Quinases TOR/genética , beta-Glucanas/químicaRESUMO
Emerging evidences exhibit that mitogen-activated protein kinase (MAPK/MPK) signaling pathways are connected with many aspects of plant development. The complexity of MAPK cascades raises challenges not only to identify the MAPK module in planta but also to define the specific role of an individual module. So far, our knowledge of MAPK signaling has been largely restricted to a small subset of MAPK cascades. Our previous study has characterized an Arabidopsis bushy and dwarf1 (bud1) mutant, in which the MAP Kinase Kinase 7 (MKK7) was constitutively activated, resulting in multiple phenotypic alterations. In this study, we found that MPK3 and MPK6 are the substrates for phosphorylation by MKK7 in planta. Genetic analysis showed that MKK7-MPK6 cascade is specifically responsible for the regulation of shoot branching, hypocotyl gravitropism, filament elongation, and lateral root formation, while MKK7-MPK3 cascade is mainly involved in leaf morphology. We further demonstrated that the MKK7-MPK6 cascade controls shoot branching by phosphorylating Ser 337 on PIN1, which affects the basal localization of PIN1 in xylem parenchyma cells and polar auxin transport in the primary stem. Our results not only specify the functions of the MKK7-MPK6 cascade but also reveal a novel mechanism for PIN1 phosphorylation, establishing a molecular link between the MAPK cascade and auxin-regulated plant development.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , MAP Quinase Quinase 7/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Brotos de Planta/enzimologia , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/química , Ácidos Indolacéticos/metabolismo , MAP Quinase Quinase 7/química , Sistema de Sinalização das MAP Quinases , Proteínas de Membrana Transportadoras/química , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/química , Fosforilação , Desenvolvimento Vegetal , Brotos de Planta/crescimento & desenvolvimento , Processamento de Proteína Pós-Traducional , Transporte ProteicoRESUMO
Paracoccidioides brasiliensis is a temperature-dependent dimorphic fungus that cause paracoccidioidomycosis (PCM), the major systemic mycosis in Latin America. The capacity to evade the innate immune response of the host is due to P. brasiliensis ability to respond and to survive the nitrosative stress caused by phagocytic cells. However, the regulation of signal transduction pathways associated to nitrosative stress response are poorly understood. Ras GTPase play an important role in the various cellular events in many fungi. Ras, in its activated form (Ras-GTP), interacts with effector proteins and can initiate a kinase cascade. In this report, we investigated the role of Ras GTPase in P. brasiliensis after in vitro stimulus with nitric oxide (NO). We observed that low concentrations of NO induced cell proliferation in P. brasiliensis, while high concentrations promoted decrease in fungal viability, and both events were reversed in the presence of a NO scavenger. We observed that high levels of NO induced Ras activation and its S-nitrosylation. Additionally, we showed that Ras modulated the expression of antioxidant genes in response to nitrosative stress. We find that the Hog1 MAP kinase contributed to nitrosative stress response in P. brasiliensis in a Ras-dependent manner. Taken together, our data demonstrate the relationship between Ras-GTPase and Hog1 MAPK pathway allowing for the P. brasiliensis adaptation to nitrosative stress.
Assuntos
Proteínas Fúngicas/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Estresse Nitrosativo/fisiologia , Paracoccidioides/fisiologia , Proteínas ras/fisiologia , Sequência de Aminoácidos , Animais , Morte Celular/fisiologia , Proliferação de Células/fisiologia , Expressão Gênica/fisiologia , Masculino , Camundongos Endogâmicos BALB C , Proteínas Quinases Ativadas por Mitógeno/química , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Óxido Nítrico/química , Óxido Nítrico/farmacologia , Processamento de Proteína Pós-TraducionalRESUMO
Phosphothreonine lyases are bacterial effector proteins secreted into host cells to facilitate the infection process. This enzyme family catalyzes an irreversible elimination reaction that converts phosphothreonine or phosphoserine to dehydrobutyrine or dehydroalanine, respectively. Herein, we report a study of substrate selectivity for each of the four known phosphothreonine lyases. This was accomplished using a combination of mass spectrometry and enzyme kinetics assays for a series of phosphorylated peptides derived from the mitogen-activated protein kinase (MAPK) activation loop. These studies provide the first experimental evidence that VirA, a putative phosphothreonine lyase identified through homology, is indeed capable of catalyzing phosphate elimination. These studies further demonstrate that OspF is the most promiscuous phosphothreonine lyase, whereas SpvC is the most specific for the MAPK activation loop. Our studies reveal that phospholyases are dramatically more efficient at catalyzing elimination from phosphothreonine than from phosphoserine. Together, our data suggest that each enzyme likely has preferred substrates, either within the MAPK family or beyond. Fully understanding the extent of selectivity is key to understanding the impact of phosphothreonine lyases during bacterial infection and to exploiting their unique chemistry for a range of applications.
Assuntos
Bactérias/enzimologia , Liases/metabolismo , Fosfosserina/metabolismo , Fosfotreonina/metabolismo , Sequência de Aminoácidos , Bactérias/metabolismo , Cinética , Proteínas Quinases Ativadas por Mitógeno/química , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Modelos Moleculares , Peptídeos/química , Peptídeos/metabolismo , Fosfatos/metabolismo , Fosforilação , Especificidade por SubstratoRESUMO
Smk1 is a mitogen-activated protein kinase (MAPK) family member in the yeast Saccharomyces cerevisiae that controls the postmeiotic program of spore formation. Ssp2 is a meiosis-specific protein that activates Smk1 and triggers the autophosphorylation of its activation loop. A fragment of Ssp2 that is sufficient to activate Smk1 contains two segments that resemble RNA recognition motifs (RRMs). Mutations in either of these motifs eliminated Ssp2's ability to activate Smk1. In contrast, deletions and insertions within the segment linking the RRM-like motifs only partially reduced the activity of Ssp2. Moreover, when the two RRM-like motifs were expressed as separate proteins in bacteria, they activated Smk1. We also find that both motifs can be cross-linked to Smk1 and that at least one of the motifs binds near the ATP-binding pocket of the MAPK. These findings demonstrate that motifs related to RRMs can directly activate protein kinases.
Assuntos
Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Motivo de Reconhecimento de RNA/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Sítios de Ligação/genética , Ativação Enzimática/genética , Meiose/genética , Proteínas Quinases Ativadas por Mitógeno/química , Modelos Moleculares , Mutação , Conformação Proteica , RNA Fúngico/genética , RNA Fúngico/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Esporos Fúngicos/genética , Esporos Fúngicos/metabolismoRESUMO
Adaptation to stress triggers the most dramatic shift in gene expression in fission yeast (Schizosaccharomyces pombe), and this response is driven by signaling via the MAPK Sty1. Upon activation, Sty1 accumulates in the nucleus and stimulates expression of hundreds of genes via the nuclear transcription factor Atf1, including expression of atf1 itself. However, the role of stress-induced, Sty1-mediated Atf1 phosphorylation in transcriptional activation is unclear. To this end, we expressed Atf1 phosphorylation mutants from a constitutive promoter to uncouple Atf1 activity from endogenous, stress-activated Atf1 expression. We found that cells expressing a nonphosphorylatable Atf1 variant are sensitive to oxidative stress because of impaired transcription of a subset of stress genes whose expression is also controlled by another transcription factor, Pap1. Furthermore, cells expressing a phospho-mimicking Atf1 mutant display enhanced stress resistance, and although expression of the Pap1-dependent genes still relied on stress induction, another subset of stress-responsive genes was constitutively expressed in these cells. We also observed that, in cells expressing the phospho-mimicking Atf1 mutant, the presence of Sty1 was completely dispensable, with all stress defects of Sty1-deficient cells being suppressed by expression of the Atf1 mutant. We further demonstrated that Sty1-mediated Atf1 phosphorylation does not stimulate binding of Atf1 to DNA but, rather, establishes a platform of interactions with the basal transcriptional machinery to facilitate transcription initiation. In summary, our results provide evidence that Atf1 phosphorylation by the MAPK Sty1 is required for oxidative stress responses in fission yeast cells by promoting transcription initiation.
Assuntos
Fator 1 Ativador da Transcrição/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Modelos Moleculares , Fosfoproteínas/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Ativação Transcricional , Fator 1 Ativador da Transcrição/química , Fator 1 Ativador da Transcrição/genética , Substituição de Aminoácidos , Deleção de Genes , Regulação Fúngica da Expressão Gênica , Sistema de Sinalização das MAP Quinases , Viabilidade Microbiana , Proteínas Quinases Ativadas por Mitógeno/química , Proteínas Quinases Ativadas por Mitógeno/genética , Mutação , Estresse Oxidativo , Proteínas Associadas a Pancreatite , Fosfoproteínas/química , Fosfoproteínas/genética , Fosforilação , Regiões Promotoras Genéticas , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Schizosaccharomyces/enzimologia , Schizosaccharomyces/crescimento & desenvolvimento , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Serina/química , Serina/metabolismo , Treonina/química , Treonina/metabolismoRESUMO
Environmental conditions modulate cell cycle progression in many cell types. A key component of the eukaryotic cell cycle is the protein kinase Wee1, which inhibits the cyclin-dependent kinase Cdk1 in yeast through human cells. In the fission yeast Schizosaccharomyces pombe, the protein kinase Cdr1 is a mitotic inducer that promotes mitotic entry by phosphorylating and inhibiting Wee1. Cdr1 and Wee1 both localize to punctate structures, termed nodes, on the medial cortex, but it has been unknown whether node localization can be altered by physiological signals. Here we investigated how environmental conditions regulate Cdr1 signaling for cell division. Osmotic stress induced hyperphosphorylation of the mitotic inducer Cdr1 for several hours, and cells delayed division for the same time period. This stress-induced hyperphosphorylation required both Cdr1 autophosphorylation and the stress-activated protein kinase Sty1. During osmotic stress, Cdr1 exited cortical nodes and localized in the cytoplasm. Using a series of truncation mutants, we mapped a C-terminal domain that is necessary and sufficient for Cdr1 node localization and found that Sty1 directly phosphorylates this domain in vitro Sty1 was not required for Cdr1 exit from nodes, indicating the existence of additional regulatory signals. Both Cdr1 phosphorylation and node localization returned to basal levels when cells adapted to osmotic conditions and resumed cell cycle progression. In summary, we identified a mechanism that prevents Cdr1 colocalization with its inhibitory target Wee1 during osmotic stress. Dynamic regulation of protein localization to cortical nodes might represent a strategy to modulate entry into mitosis under differing environmental conditions.
Assuntos
Citoplasma/enzimologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimologia , Estresse Fisiológico , Regulação para Cima , Substituição de Aminoácidos , Citoplasma/metabolismo , Deleção de Genes , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas Quinases Ativadas por Mitógeno/química , Proteínas Quinases Ativadas por Mitógeno/genética , Mitose , Mutagênese Sítio-Dirigida , Mutação , Pressão Osmótica , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fosforilação , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Transporte Proteico , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Schizosaccharomyces/citologia , Schizosaccharomyces/crescimento & desenvolvimento , Schizosaccharomyces/fisiologia , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genéticaRESUMO
In the yeast Saccharomyces cerevisiae, the exposure to mating pheromone activates a prototypic mitogen-activated protein kinase (MAPK) cascade and triggers a dose-dependent differentiation response. Whereas a high pheromone dose induces growth arrest and formation of a shmoo-like morphology in yeast cells, lower pheromone doses elicit elongated cell growth. Previous population-level analysis has revealed that the MAPK Fus3 plays an important role in mediating this differentiation switch. To further investigate how Fus3 controls the fate decision process at the single-cell level, we developed a specific translocation-based reporter for monitoring Fus3 activity in individual live cells. Using this reporter, we observed strikingly different dynamic patterns of Fus3 activation in single cells differentiated into distinct fates. Cells committed to growth arrest and shmoo formation exhibited sustained Fus3 activation. In contrast, most cells undergoing elongated growth showed either a delayed gradual increase or pulsatile dynamics of Fus3 activity. Furthermore, we found that chemically perturbing Fus3 dynamics with a specific inhibitor could effectively redirect the mating differentiation, confirming the causative role of Fus3 dynamics in driving cell fate decisions. MAPKs mediate proliferation and differentiation signals in mammals and are therapeutic targets in many cancers. Our results highlight the importance of MAPK dynamics in regulating single-cell responses and open up the possibility that MAPK signaling dynamics could be a pharmacological target in therapeutic interventions.
Assuntos
Sistema de Sinalização das MAP Quinases , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Substituição de Aminoácidos , Proteínas do Citoesqueleto/agonistas , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Ativação Enzimática/efeitos dos fármacos , Deleção de Genes , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Genes Reporter/efeitos dos fármacos , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Cinética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Fator de Acasalamento/agonistas , Fator de Acasalamento/metabolismo , Proteínas de Membrana/agonistas , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Quinases Ativadas por Mitógeno/química , Proteínas Quinases Ativadas por Mitógeno/genética , Mutação , Fragmentos de Peptídeos/agonistas , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Feromônios/farmacologia , Transporte Proteico/efeitos dos fármacos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/agonistas , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Análise de Célula ÚnicaRESUMO
Esophageal squamous cell carcinoma (ESCC) is highly prevalent in Asia, especially in China. Research findings indicate that nitrosamines, malnutrition, unhealthy living habits, and genetics contribute to esophageal carcinogenesis. Currently, the 5-year survival rate for ESCC patients remains low, owing in part to a lack of a clear understanding of mechanisms involved. Chemoprevention using natural or synthesized compounds might be a promising strategy to reduce esophageal cancer incidence. The epidermal growth factor receptor (EGFR) can activate downstream pathways including the phosphatidylinositol 3-kinase (PI3K) pathway and the Ras/mitogen-activated protein kinase (MAPK) pathways. Among the important players, AKT and ERKs have an important relationship with cancer initiation and progression. Here, we found that phosphorylated (p)-AKT and p-ERKs were highly expressed in esophageal cancer cell lines and in esophageal cancer patients. Human phospho-kinase array and pull-down assay results showed that quercetin-3-methyl ether (Q3ME) is a natural flavonoid compound that interacted with AKT and ERKs and inhibited their kinase activities. At the cellular level, Q3ME attenuated esophageal cancer cell proliferation and anchorage-independent growth. Western blot analysis showed that this compound suppressed the activation of AKT and ERKs downstream signaling pathways, subsequently inhibiting activating protein-1 (AP-1) activity. Importantly, Q3ME inhibited the formation of esophageal preneoplastic lesions induced by N-nitrosomethylbenzylamine (NMBA). The inhibition by Q3ME was associated with decreased inflammation and esophageal cancer cell proliferation in vivo. Collectively, our data suggest that Q3ME is a promising chemopreventive agent against esophageal carcinogenesis by targeting AKT and ERKs.
Assuntos
Transformação Celular Neoplásica/efeitos dos fármacos , Neoplasias Esofágicas/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Quercetina/análogos & derivados , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Animais , Biomarcadores , Linhagem Celular Tumoral , Mucosa Esofágica/metabolismo , Neoplasias Esofágicas/patologia , Humanos , Inflamação/metabolismo , Proteínas Quinases Ativadas por Mitógeno/química , Estrutura Molecular , Proteínas Proto-Oncogênicas c-akt/química , Quercetina/química , Quercetina/farmacologia , Ratos , Proteínas Quinases S6 Ribossômicas 70-kDa/química , Relação Estrutura-Atividade , Serina-Treonina Quinases TOR/químicaRESUMO
Proper control of immune-related gene expression is crucial for the host to launch an effective defense response. Perception of microbe-associated molecular patterns (MAMPs) induces rapid and profound transcriptional reprogramming via unclear mechanisms. Here, we show that ASR3 (ARABIDOPSIS SH4-RELATED3) functions as a transcriptional repressor and plays a negative role in regulating pattern-triggered immunity (PTI) in Arabidopsis thaliana. ASR3 belongs to a plant-specific trihelix transcription factor family for which functional studies are lacking. MAMP treatments induce rapid phosphorylation of ASR3 at threonine 189 via MPK4, a mitogen-activated protein kinase that negatively regulates PTI responses downstream of multiple MAMP receptors. ASR3 possesses transcriptional repressor activity via its ERF-associated amphiphilic repression motifs and negatively regulates a large subset of flg22-induced genes. Phosphorylation of ASR3 by MPK4 enhances its DNA binding activity to suppress gene expression. Importantly, the asr3 mutant shows enhanced disease resistance to virulent bacterial pathogen infection, whereas transgenic plants overexpressing the wild-type or phospho-mimetic form of ASR3 exhibit compromised PTI responses. Our studies reveal a function of the trihelix transcription factors in plant innate immunity and provide evidence that ASR3 functions as a transcriptional repressor regulated by MAMP-activated MPK4 to fine-tune plant immune gene expression.
Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/imunologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Imunidade Vegetal/efeitos dos fármacos , Proteínas Repressoras/metabolismo , Fatores Genéricos de Transcrição/química , Fatores Genéricos de Transcrição/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/enzimologia , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , DNA de Plantas/metabolismo , Resistência à Doença/efeitos dos fármacos , Resistência à Doença/genética , Flagelina/farmacologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes de Plantas , Proteínas Quinases Ativadas por Mitógeno/química , Mutação/genética , Fosforilação/efeitos dos fármacos , Fosfotreonina/metabolismo , Imunidade Vegetal/genética , Ligação Proteica/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína , Especificidade por Substrato/efeitos dos fármacos , Fatores Genéricos de Transcrição/genética , Transcrição Gênica/efeitos dos fármacos , Virulência/efeitos dos fármacosRESUMO
Upon nutrient limitation, budding yeasts like Saccharomyces cerevisiae can be induced to adopt alternate filament-like growth patterns called diploid pseudohyphal or invasive haploid growth. Here, we report a novel constitutive pseudohyphal growth state, sharing some characteristics with classic forms of filamentous growth, but differing in crucial aspects of morphology, growth conditions and genetic regulation. The constitutive pseudohyphal state is observed in fus3 mutants containing various septin assembly defects, which we refer to as sadF growth (septin assembly defect induced filamentation) to distinguish it from classic filamentation pathways. Similar to other filamentous states, sadF cultures comprise aggregated chains of highly elongated cells. Unlike the classic pathways, sadF growth occurs in liquid rich media, requiring neither starvation nor the key pseudohyphal proteins, Flo8p and Flo11p. Moreover sadF growth occurs in haploid strains of S288C genetic background, which normally cannot undergo pseudohyphal growth. The sadF cells undergo highly polarized bud growth during prolonged G2 delays dependent on Swe1p. They contain septin structures distinct from classical pseudo-hyphae and FM4-64 labeling at actively growing tips similar to the Spitzenkörper observed in true hyphal growth. The sadF growth state is induced by synergism between Kss1p-dependent signaling and septin assembly defects; mild disruption of mitotic septins activates Kss1p-dependent gene expression, which exacerbates the septin defects, leading to hyper-activation of Kss1p. Unlike classical pseudo-hyphal growth, sadF signaling requires Ste5, Ste4 and Ste18, the scaffold protein and G-protein ß and γ subunits from the pheromone response pathway, respectively. A swe1 mutation largely abolished signaling, breaking the positive feedback that leads to amplification of sadF signaling. Taken together, our findings show that budding yeast can access a stable constitutive pseudohyphal growth state with very few genetic and regulatory changes.