RESUMO
Cardiomyopathy syndrome (CMS) poses a significant threat to farmed Atlantic salmon (Salmo salar), leading to high mortality rates during the seawater phase. Given that controlled experimental challenge trials with PMCV do not reproduce the mortality observed in severe field outbreaks of CMS, field trials on natural CMS outbreaks are warranted. This field study explored the impact of a clinical nutrition intervention, specifically a diet enriched with eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), on a severe CMS outbreak in a commercial sea farm. CMS was diagnosed in a single sea cage with high mortality rates. Histopathological analysis, RT-qPCR in situ hybridization for virus detection, and fatty acid composition analysis were used to monitor the impact of disease and the inclusion of EPA and DHA in heart tissue. Following the implementation of clinical nutrition, a decline in mortality rates, regression of CMS-associated changes, and a significant reduction in piscine myocarditis virus (PMCV) RNA load were observed within the salmon population. Fatty acid composition analysis of heart samples demonstrated increased levels of EPA and DHA, reinforcing the association between dietary factors, viral load dynamics, and overall fish health. Although further validation is needed in future studies, as field trials may not be sufficient to establish causation, our results indicate that optimizing the EPA + DHA levels may prove beneficial in severe CMS outbreaks.
RESUMO
The atypical MAP kinases ERK3 and ERK4 are activated by phosphorylation of a serine residue lying within the activation loop signature sequence S-E-G. However, the regulation of ERK3 and ERK4 phosphorylation and activity is poorly understood. Here we report that the inducible nuclear dual-specificity MAP kinase phosphatase (MKP) DUSP2, a known regulator of the ERK and p38 MAPKs, is unique amongst the MKP family in being able to bind to both ERK3 and ERK4. This interaction is mediated by a conserved common docking (CD) domain within the carboxyl-terminal domains of ERK3 and ERK4 and the conserved kinase interaction motif (KIM) located within the non-catalytic amino terminus of DUSP2. This interaction is direct and results in the dephosphorylation of ERK3 and ERK4 and the stabilization of DUSP2. In the case of ERK4 its ability to stabilize DUSP2 requires its kinase activity. Finally, we demonstrate that expression of DUSP2 inhibits ERK3 and ERK4-mediated activation of its downstream substrate MK5. We conclude that the activity of DUSP2 is not restricted to the classical MAPK pathways and that DUSP2 can also regulate the atypical ERK3/4-MK5 signalling pathway in mammalian cells.
Assuntos
Fosfatase 2 de Especificidade Dupla/genética , Regulação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteína Quinase 6 Ativada por Mitógeno/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Recombinantes de Fusão/genética , Animais , Baculoviridae/genética , Baculoviridae/metabolismo , Fosfatase 2 de Especificidade Dupla/metabolismo , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Células Jurkat , Proteína Quinase 6 Ativada por Mitógeno/metabolismo , Fosforilação , Plasmídeos/química , Plasmídeos/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Estabilidade Proteica , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , SpodopteraRESUMO
ERK3 is an atypical Mitogen-activated protein kinase (MAPK6). Despite the fact that the Erk3 gene was originally identified in 1991, its function is still unknown. MK5 (MAP kinase- activated protein kinase 5) also called PRAK is the only known substrate for ERK3. Recently, it was found that group I p21 protein activated kinases (PAKs) are critical effectors of ERK3. PAKs link Rho family of GTPases to actin cytoskeletal dynamics and are known to be involved in the regulation of cell adhesion and migration. In this study we demonstrate that ERK3 protein levels are elevated as MDA-MB-231 breast cancer cells adhere to collagen I which is concomitant with changes in cellular morphology where cells become less well spread following nascent adhesion formation. During this early cellular adhesion event we observe that the cells retain protrusive activity while reducing overall cellular area. Interestingly exogenous expression of ERK3 delivers a comparable reduction in cell spread area, while depletion of ERK3 expression increases cell spread area. Importantly, we have detected a novel specific endogenous ERK3 localization at the cell periphery. Furthermore we find that ERK3 overexpressing cells exhibit a rounded morphology and increased cell migration speed. Surprisingly, exogenous expression of a kinase inactive mutant of ERK3 phenocopies ERK3 overexpression, suggesting a novel kinase independent function for ERK3. Taken together our data suggest that as cells initiate adhesion to matrix increasing levels of ERK3 at the cell periphery are required to orchestrate cell morphology changes which can then drive migratory behavior.
Assuntos
Neoplasias da Mama/genética , Adesão Celular/genética , Movimento Celular/genética , Proteína Quinase 6 Ativada por Mitógeno/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Sistema de Sinalização das MAP Quinases , Proteína Quinase 6 Ativada por Mitógeno/metabolismo , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
ERK3 and ERK4 are atypical MAPKs in which the canonical TXY motif within the activation loop of the classical MAPKs is replaced by SEG. Both ERK3 and ERK4 bind, translocate, and activate the MAPK-activated protein kinase (MK) 5. The classical MAPKs ERK1/2 and p38 interact with downstream MKs (RSK1-3 and MK2-3, respectively) through conserved clusters of acidic amino acids, which constitute the common docking (CD) domain. In contrast to the classical MAPKs, the interaction between ERK3/4 and MK5 is strictly dependent on phosphorylation of the SEG motif of these kinases. Here we report that the conserved CD domain is dispensable for the interaction of ERK3 and ERK4 with MK5. Using peptide overlay assays, we have defined a novel MK5 interaction motif (FRIEDE) within both ERK4 and ERK3 that is essential for binding to the C-terminal region of MK5. This motif is located within the L16 extension lying C-terminal to the CD domain in ERK3 and ERK4 and a single isoleucine to lysine substitution in FRIEDE totally abrogates binding, activation, and translocation of MK5 by both ERK3 and ERK4. These findings are the first to demonstrate binding of a physiological substrate via this region of the L16 loop in a MAPK. Furthermore, the link between activation loop phosphorylation and accessibility of the FRIEDE interaction motif suggests a switch mechanism for these atypical MAPKs in which the phosphorylation status of the activation loop regulates the ability of both ERK3 and ERK4 to bind to a downstream effector.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína Quinase 6 Ativada por Mitógeno/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteínas Serina-Treonina Quinases/metabolismo , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Sítios de Ligação/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Immunoblotting , Peptídeos e Proteínas de Sinalização Intracelular/genética , Microscopia Confocal , Proteína Quinase 6 Ativada por Mitógeno/química , Proteína Quinase 6 Ativada por Mitógeno/genética , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , TransfecçãoRESUMO
ERK (extracellular-signal-regulated kinase) 4 [MAPK (mitogen-activated protein kinase) 4] and ERK3 (MAPK6) are atypical MAPKs. One major difference between these proteins and the classical MAPKs is substitution of the conserved T-X-Y motif within the activation loop by a single phospho-acceptor site within an S-E-G motif. In the present study we report that Ser(186) of the S-E-G motif in ERK4 is phosphorylated in vivo. Kinase-dead ERK4 is also phosphorylated on Ser(186), indicating that an ERK4 kinase, rather than autophosphorylation, is responsible. Co-expression of MK5 [MAPK-activated protein kinase 5; also known as PRAK (p38-regulated/activated kinase)], a physiological target of ERK4, increases phosphorylation of Ser(186). This is not dependent on MK5 activity, but does require interaction between ERK4 and MK5 suggesting that MK5 binding either prevents ERK4 dephosphorylation or facilitates ERK4 kinase activity. ERK4 mutants in which Ser(186) is replaced with either an alanine residue or a phospho-mimetic residue (glutamate) are unable to activate MK5 and Ser(186) is also required for cytoplasmic anchoring of MK5. Both defects seem to reflect an impaired ability of the ERK4 mutants to interact with MK5. We find that there are at least two endogenous pools of wild-type ERK4. One form exhibits reduced mobility when analysed using SDS/PAGE. This is due to MK5-dependent phosphorylation and only this retarded ERK4 species is both phosphorylated on Ser(186) and co-immunoprecipitates with wild-type MK5. We conclude that binding between ERK4 and MK5 facilitates phosphorylation of Ser(186) and stabilization of the ERK4-MK5 complex. This results in phosphorylation and activation of MK5, which in turn phosphorylates ERK4 on sites other than Ser(186) resulting in the observed mobility shift.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fosfosserina/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Linhagem Celular , Citoplasma/enzimologia , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Mutação/genética , Ligação Proteica , Proteínas Serina-Treonina Quinases/genéticaRESUMO
MAPK-activated protein kinase 5 (MK5) was recently identified as a physiological substrate of the atypical MAPK ERK3. Complex formation between ERK3 and MK5 results in phosphorylation and activation of MK5, concomitant stabilization of ERK3, and the nuclear exclusion of both proteins. However, ablation of ERK3 in HeLa cells using small interfering RNA or in fibroblasts derived from ERK3 null mice reduces the activity of endogenous MK5 by only 50%, suggesting additional mechanisms of MK5 regulation. Here we identify the ERK3-related kinase ERK4 as a bona fide interaction partner of MK5. Binding of ERK4 to MK5 is accompanied by phosphorylation and activation of MK5. Furthermore, complex formation also results in the relocalization of MK5 from nucleus to cytoplasm. However unlike ERK3, ERK4 is a stable protein, and its half-life is not modified by the presence or absence of MK5. Finally, although knock-down of ERK4 protein in HeLa cells reduces endogenous MK5 activity by approximately 50%, a combination of small interfering RNAs targeting both ERK4 and ERK3 causes a further reduction in the MK5 activity by more than 80%. We conclude that MK5 activation is dependent on both ERK3 and ERK4 in these cells and that these atypical MAPKs are both physiological regulators of MK5 activity.
Assuntos
Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Sequência de Aminoácidos , Sequência de Bases , Citoplasma/enzimologia , Ativação Enzimática , Regulação Enzimológica da Expressão Gênica , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Dados de Sequência Molecular , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Transporte ProteicoRESUMO
Extracellular signal-regulated kinase 3 (ERK3) is an atypical mitogen-activated protein kinase (MAPK), which is regulated by protein stability. However, its function is unknown and no physiological substrates for ERK3 have yet been identified. Here we demonstrate a specific interaction between ERK3 and MAPK-activated protein kinase-5 (MK5). Binding results in nuclear exclusion of both ERK3 and MK5 and is accompanied by ERK3-dependent phosphorylation and activation of MK5 in vitro and in vivo. Endogenous MK5 activity is significantly reduced by siRNA-mediated knockdown of ERK3 and also in fibroblasts derived from ERK3-/- mice. Furthermore, increased levels of ERK3 protein detected during nerve growth factor-induced differentiation of PC12 cells are accompanied by an increase in MK5 activity. Conversely, MK5 depletion causes a dramatic reduction in endogenous ERK3 levels. Our data identify the first physiological protein substrate for ERK3 and suggest a functional link between these kinases in which MK5 is a downstream target of ERK3, while MK5 acts as a chaperone for ERK3. Our findings provide valuable tools to further dissect the regulation and biological roles of both ERK3 and MK5.
Assuntos
Proteína Quinase 6 Ativada por Mitógeno/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/fisiologia , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Células COS , Núcleo Celular/metabolismo , Chlorocebus aethiops , Citoplasma/metabolismo , Ativação Enzimática , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Knockout , Proteína Quinase 6 Ativada por Mitógeno/genética , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Recruitment of a RNA polymerase II complex by the glutamine-rich Q2 domain of cAMP response element-binding protein (CREB) allows basal transcriptional activity, while recruitment of CBP/p300 through signal-induced phosphorylation of the kinase-inducible domain at serine-133 enhances CREB-dependent transcription. Here we demonstrate that co-administration of forskolin and phorbol ester TPA to NIH3T3 cells provoked a dose-dependent increase in phosphoserine-133. CREB- and Q2-dependent transcription, as well as transcription by other glutamine-rich transcription factors, but not by transcription factors lacking glutamine-rich regions, augmented synergistically in the presence of both stimuli. Synergistic activation was abograted by specific inhibition of protein kinase C (PKC), but not of PKA. Co-stimulation increased the basal activity of a minimal, CREB-independent promoter. Therefore, Q2, which directly interacts with the RNA polymerase II initiation complex, may transmit the increased basal promoter activity provoked by these stimuli to CREB, thereby contributing to synergistic activation of CREB-mediated transcription. This synergism may have important implications on glutamine-rich transcription factor-target genes.
Assuntos
Colforsina/farmacologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Ésteres de Forbol/farmacologia , Proteína Quinase C/metabolismo , Ativação Transcricional/genética , Animais , Glutamina/metabolismo , Camundongos , Células NIH 3T3 , Fosfosserina/metabolismo , Ativação Transcricional/efeitos dos fármacosRESUMO
The small t antigen (st-ag) of simian virus 40 can exert pleiotropic effects on biological processes such as DNA replication, cell cycle progression and gene expression. One possible mode of achieving these effects is through stimulation of NFkappaB-responsive genes encoding growth factors, cytokines, transcription factors and cell cycle regulatory proteins. Indeed, a previous study has shown that st-ag enhanced NFkappaB-mediated transcription. This study demonstrates that promoters possessing a consensus TATA box (i.e. TATAAAAG) in the context of either NFkappaB- or Sp1-binding sites are trans-activated by st-ag. Overexpressing the general transcription factor hTAF(II)130/135, but not hTAF(II)28 or hTAF(II)80, stimulated the activity of promoters in a consensus TATA box-dependent mode. Converting the consensus TATA motif into a non-consensus TATA box strongly impaired activation by st-ag and hTAF(II)130/135. Conversely, mutating a non-consensus TATA motif into the consensus TATA box rendered the mutated promoter inducible by st-ag and hTAF(II)130/135. Mutation of the TATA box had no effect on TNFalpha- or RelA/p65-mediated induction of NFkappaB-responsive promoters, indicating a specific st-ag effect on hTAF(II)130/135. St-ag stimulated the intrinsic transcriptional activity of hTAF(II)130/135. Substitutions in the conserved HPDKGG motif in the N-terminal region or a mutation that impaired the interaction with protein phosphatase 2A abrogated the ability of st-ag to activate hTAF(II)130/135-mediated transcription. These results indicate that trans-activation of promoters by st-ag may depend on a consensus TATA motif and suggest that such promoters recruit the general transcription factor hTAF(II)130/135.
Assuntos
Antígenos Transformantes de Poliomavirus/fisiologia , Regiões Promotoras Genéticas/genética , Vírus 40 dos Símios/imunologia , TATA Box/fisiologia , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Fator de Transcrição TFIID/metabolismo , Ativação Transcricional , Células 3T3 , Animais , Sequência de Bases , Regulação da Expressão Gênica , Humanos , Camundongos , Dados de Sequência Molecular , NF-kappa B/genética , NF-kappa B/metabolismo , Vírus 40 dos Símios/patogenicidade , Fatores Associados à Proteína de Ligação a TATA/genética , Fator de Transcrição TFIID/genéticaRESUMO
Previous studies have demonstrated that the serine/threonine protein phosphatase 2A (PP2A) can modulate the transcriptional activity of several sequence-specific DNA-binding proteins. However, less is known about the effect of PP2A on the activities of general transcription factors and transcriptional coregulators. Here we describe that the activity of a general coactivator, the four-and-a-half-LIM-only protein 2 (FHL2), is regulated in a PP2A-dependent manner. Specific inhibition of PP2A by simian virus 40 (SV40) small t-antigen (st-ag) stimulated the intrinsic transcriptional activity of FHL2 more than 10-fold, while a st-ag mutant unable to bind PP2A had no effect. Overexpression of the B56 subunits alpha, beta, and gamma1 of PP2A impaired the induction of FHL2 by st-ag. FHL2 functioned as a coactivator for CREB-mediated transcription, and inactivation of PP2A further increased FHL2-induced CREB-directed transcription. Overexpression of FHL2 readily enhanced the transcription of the luciferase reporter gene driven by the c-fos promoter, and inhibition of PP2A further stimulated FHL2-induced transactivation of this promoter. These results suggest that dephosphorylation of the general coactivator FHL2 may represent a novel mechanism by which PP2A modulates the transcription of FHL2-responsive genes.
Assuntos
Colforsina/farmacologia , Inibidores Enzimáticos/farmacologia , Genes fos/genética , Proteínas de Homeodomínio/metabolismo , Proteínas Musculares , Fosfoproteínas Fosfatases/antagonistas & inibidores , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo , Células 3T3 , Animais , Sequência de Bases , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Genes fos/efeitos dos fármacos , Proteínas de Homeodomínio/efeitos dos fármacos , Proteínas com Homeodomínio LIM , Luciferases/genética , Camundongos , Mutagênese Sítio-Dirigida , Plasmídeos , Proteína Fosfatase 2 , Subunidades Proteicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-fos/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/genética , Fatores de Transcrição/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , TransfecçãoRESUMO
The p38 mitogen-activated protein kinase (MAPK) pathway is an important mediator of cellular responses to environmental stress. Targets of p38 include transcription factors, components of the translational machinery, and downstream serine/threonine kinases, including MAPK-activated protein kinase 5 (MK5). Here we have used enhanced green fluorescent protein fusion proteins to analyze the subcellular localization of MK5. Although this protein is predominantly nuclear in unstimulated cells, MK5 shuttles between the nucleus and the cytoplasm. Furthermore, we have shown that the C-terminal domain of MK5 contains both a functional nuclear localization signal (NLS) and a leucine-rich nuclear export signal (NES), indicating that the subcellular distribution of this kinase reflects the relative activities of these two signals. In support of this, we have shown that stress-induced activation of the p38 MAPK stimulates the chromosomal region maintenance 1 protein-dependent nuclear export of MK5. This is regulated by both binding of p38 MAPK to MK5, which masks the functional NLS, and stress-induced phosphorylation of MK5 by p38 MAPK, which either activates or unmasks the NES. These properties may define the ability of MK5 to differentially phosphorylate both nuclear and cytoplasmic targets or alternatively reflect a mechanism whereby signals initiated by activation of MK5 in the nucleus may be transmitted to the cytoplasm.