RESUMO
Preventing or effectively treating metastatic uveal melanoma (UM) is critical because it occurs in about half of patients and confers a very poor prognosis. There is emerging evidence that hepatocyte growth factor (HGF) and insulin-like growth factor 1 (IGF-1) promote metastasis and contribute to the striking metastatic hepatotropism observed in UM metastasis. However, the molecular mechanisms by which HGF and IGF-1 promote UM liver metastasis have not been elucidated. ASAP1, which acts as an effector for the small GTPase ARF6, is highly expressed in the subset of uveal melanomas most likely to metastasize. Here, we found that HGF and IGF-1 hyperactivate ARF6, leading to its interaction with ASAP1, which then acts as an effector to induce nuclear localization and transcriptional activity of NFAT1. Inhibition of any component of this pathway impairs cellular invasiveness. Additionally, knocking down ASAP1 or inhibiting NFAT signaling reduces metastasis in a xenograft mouse model of UM. The discovery of this signaling pathway represents not only an advancement in our understanding of the biology of uveal melanoma metastasis but also identifies a novel pathway that could be targeted to treat or prevent metastatic uveal melanoma.
Assuntos
Melanoma , Neoplasias Uveais , Humanos , Animais , Camundongos , Fator de Crescimento de Hepatócito/genética , Fator de Crescimento de Hepatócito/metabolismo , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Melanoma/patologia , Neoplasias Uveais/metabolismo , Modelos Animais de Doenças , Proteínas Adaptadoras de Transdução de Sinal/metabolismoRESUMO
Purpose: Acute retinal necrosis (ARN) is a herpesvirus infection of the retina with blinding complications. In this study, we sought to create a reproducible mouse model of ARN that mimics human disease to better understand innate immunity within the retina during virus infection. Methods: C57Bl/6J wild type (WT) and type I interferon receptor-deficient (IFNAR-/-) mice were infected with varying amounts of herpes simplex virus type 1 (HSV-1) via subretinal injection. Viral titers, optical coherence tomography (OCT) and fundus photography, the development of encephalitis, and ocular histopathology were scored and compared between groups of WT and IFNAR-/- mice. Results: The retina of WT mice could be readily infected with HSV-1 via subretinal injection resulting in retinal whitening and full-thickness necrosis as determined by in vivo imaging and histopathology. In IFNAR-/- mice, HSV-1-induced retinal pathology was significantly worse when compared with WT mice, and viral titers were significantly elevated within two days after infection and persisted to day 5 after infection within the retina. These results were also observed in the brain where there were significantly higher viral titers and frequency of encephalitis in IFNAR-/- when compared to WT mice. Conclusions: Collectively, these findings show that our new mouse model of ARN mimics human disease and can be used to study innate immunity within the retina. We conclude that type I interferons are critical in containing HSV-1 locally within retinal tissues and prohibiting spread into the brain.
Assuntos
Encefalite , Herpes Simples , Herpesvirus Humano 1 , Interferon Tipo I , Humanos , Animais , Camundongos , Herpesvirus Humano 1/fisiologia , Camundongos Knockout , Imunidade Inata , Retina , Camundongos Endogâmicos C57BLRESUMO
Uveal melanoma is the most common primary malignancy of the eye, and a number of discoveries in the last decade have led to a more thorough molecular characterization of this cancer. However, the prognosis remains dismal for patients with metastases, and there is an urgent need to identify treatments that are effective for this stage of disease. Animal models are important tools for preclinical studies of uveal melanoma. A variety of models exist, and they have specific advantages, disadvantages, and applications. In this review article, these differences are explored in detail, and ideas for new models that might overcome current challenges are proposed.
Assuntos
Modelos Animais de Doenças , Melanoma/patologia , Neoplasias Uveais/patologia , Animais , Linhagem Celular Tumoral , Engenharia Genética , Humanos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: Mutational activation of GNAQ or GNA11 (GNAQ/11), detected in >90% of uveal melanomas, leads to constitutive activation of oncogenic pathways, including MAPK and YAP. To date, chemo- or pathway-targeted therapies, either alone or in combination, have proven ineffective in the treatment of patients with metastatic uveal melanoma. EXPERIMENTAL DESIGN: We tested the efficacy of chloroquine or hydroxychloroquine, in combination with MAPK pathway inhibition in GNAQ/11-mutated cells in vitro and in vivo and identified mechanisms of MEK1/2 inhibitor plus chloroquine-induced cytotoxicity. RESULTS: Inhibition of GNAQ/11-mediated activation of MAPK signaling resulted in the induction of autophagy. Combined inhibition of Gα and autophagy or lysosome function resulted in enhanced cell death. Moreover, the combination of MEK1/2 inhibition, using trametinib, with the lysosome inhibitor, chloroquine, also increased cytotoxicity. Treatment of mice bearing GNAQ/11-driven melanomas with trametinib plus hydroxychloroquine resulted in inhibition of tumor growth and significantly prolonged survival. Interestingly, lysosomal- and autophagy-specific inhibition with bafilomycin A1 was not sufficient to promote cytotoxicity in combination with trametinib. However, the addition of YAP inhibition with trametinib plus bafilomycin A1 resulted in cell death at comparable levels to trametinib plus chloroquine (T/CQ) treatment. Furthermore, T/CQ-treated cells displayed decreased YAP nuclear localization and decreased YAP transcriptional activity. Expression of a constitutively active YAP5SA mutant conferred resistance to T/CQ-induced cell death. CONCLUSIONS: These results suggest that YAP, MEK1/2, and lysosome function are necessary and critical targets for the therapy of GNAQ/11-driven melanoma, and identify trametinib plus hydroxychloroquine as a potential treatment strategy for metastatic uveal melanoma.
Assuntos
Cloroquina/farmacologia , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Subunidades alfa de Proteínas de Ligação ao GTP/genética , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 2/antagonistas & inibidores , Melanoma/tratamento farmacológico , Mutação , Piridonas/farmacologia , Pirimidinonas/farmacologia , Neoplasias Uveais/tratamento farmacológico , Animais , Antimaláricos/farmacologia , Apoptose , Proliferação de Células , Resistencia a Medicamentos Antineoplásicos , Quimioterapia Combinada , Humanos , Melanoma/genética , Melanoma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Inibidores de Proteínas Quinases/farmacologia , Células Tumorais Cultivadas , Neoplasias Uveais/genética , Neoplasias Uveais/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The activation of the small GTPase ARF6 has been implicated in promoting several pathological processes related to vascular instability and tumor formation, growth, and metastasis. ARF6 also plays a vital role during embryonic development. Recent studies have suggested that ARF6 carries out these disparate functions primarily by controlling protein trafficking within the cell. ARF6 helps direct proteins to intracellular or extracellular locations where they function in normal cellular responses during development and in pathological processes later in life. This transport of proteins is accomplished through a variety of mechanisms, including endocytosis and recycling, microvesicle release, and as yet uncharacterized processes. This Commentary will explore the functions of ARF6, while focusing on the role of this small GTPase in development and postnatal physiology, regulating barrier function and diseases associated with its loss, and tumor formation, growth, and metastasis.
Assuntos
Fatores de Ribosilação do ADP/fisiologia , Fator 6 de Ribosilação do ADP , Animais , Desenvolvimento Embrionário , Endotélio Vascular/fisiologia , Humanos , Neoplasias/enzimologia , Neoplasias/patologia , Transporte ProteicoRESUMO
Uveal melanoma is a rare but often lethal malignancy and is the leading cause of death due to an ophthalmic condition. Uveal melanoma is often diagnosed at a late stage and has a strong propensity to hepatic metastasis. Recently, the most common driver mutations in uveal melanoma have been identified, predominantly in the G-proteins GNAQ. This pattern differs from that of cutaneous melanoma in which Braf and Nras predominate. There are no current clinically used agents that target GNAQ mutations, unlike the use of Braf inhibitors in cutaneous melanoma. We tested the novel agent Tris DBA palladium and found that it was markedly more effective against GNAQ mutant melanomas than wild type uveal melanomas. Given that ARF6 has recently been discovered as a node in GNAQ mutations, we evaluated the efficacy of Tris DBA palladium on ARF6 signaling and found that it was effective in inhibiting ARF6 activation. Finally, Tris DBA palladium was orally effective against GNAQ mutant melanoma in vivo. Tris DBA Palladium deserves further evaluation as a systemic agent for uveal melanoma.
RESUMO
The global obesity epidemic and associated metabolic diseases require alternative biological targets for new therapeutic strategies. In this study, we show that a phytochemical sulfuretin suppressed adipocyte differentiation of preadipocytes and administration of sulfuretin to high fat diet-fed obese mice prevented obesity and increased insulin sensitivity. These effects were associated with a suppressed expression of inflammatory markers, induced expression of adiponectin, and increased levels of phosphorylated ERK and AKT. To elucidate the molecular mechanism of sulfuretin in adipocytes, we performed microarray analysis and identified activating transcription factor 3 (Atf3) as a sulfuretin-responsive gene. Sulfuretin elevated Atf3 mRNA and protein levels in white adipose tissue and adipocytes. Consistently, deficiency of Atf3 promoted lipid accumulation and the expression of adipocyte markers. Sulfuretin's but not resveratrol's anti-adipogenic effects were diminished in Atf3 deficient cells, indicating that Atf3 is an essential factor in the effects of sulfuretin. These results highlight the usefulness of sulfuretin as a new anti-obesity intervention for the prevention of obesity and its associated metabolic diseases.
RESUMO
Melanoma has an unusual capacity to spread in early-stage disease, prompting aggressive clinical intervention in very thin primary tumors. Despite these proactive efforts, patients with low-risk, low-stage disease can still develop metastasis, indicating the presence of permissive cues for distant spread. Here, we show that constitutive activation of the small GTPase ARF6 (ARF6Q67L) is sufficient to accelerate metastasis in mice with BRAFV600E/Cdkn2aNULL melanoma at a similar incidence and severity to Pten loss, a major driver of PI3K activation and melanoma metastasis. ARF6Q67L promoted spontaneous metastasis from significantly smaller primary tumors than PTENNULL, implying an enhanced ability of ARF6-GTP to drive distant spread. ARF6 activation increased lung colonization from circulating melanoma cells, suggesting that the prometastatic function of ARF6 extends to late steps in metastasis. Unexpectedly, ARF6Q67L tumors showed upregulation of Pik3r1 expression, which encodes the p85 regulatory subunit of PI3K. Tumor cells expressing ARF6Q67L displayed increased PI3K protein levels and activity, enhanced PI3K distribution to cellular protrusions, and increased AKT activation in invadopodia. ARF6 is necessary and sufficient for activation of both PI3K and AKT, and PI3K and AKT are necessary for ARF6-mediated invasion. We provide evidence for aberrant ARF6 activation in human melanoma samples, which is associated with reduced survival. Our work reveals a previously unknown ARF6-PI3K-AKT proinvasive pathway, it demonstrates a critical role for ARF6 in multiple steps of the metastatic cascade, and it illuminates how melanoma cells can acquire an early metastatic phenotype in patients. SIGNIFICANCE: These findings reveal a prometastatic role for ARF6 independent of tumor growth, which may help explain how melanoma spreads distantly from thin, early-stage primary tumors.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/11/2892/F1.large.jpg.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Melanoma/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Neoplasias Cutâneas/patologia , Fator 6 de Ribosilação do ADP , Fatores de Ribosilação do ADP/genética , Animais , Inibidor p16 de Quinase Dependente de Ciclina/genética , Guanosina Trifosfato/metabolismo , Humanos , Neoplasias Pulmonares/secundário , Melanoma/metabolismo , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos Mutantes , Camundongos SCID , Metástase Neoplásica , PTEN Fosfo-Hidrolase/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Neoplasias Cutâneas/metabolismoRESUMO
Stimulation of white adipose tissue (WAT) browning is considered as a potential approach to treat obesity and metabolic diseases. Our previous studies have shown that phytochemical butein can stimulate WAT browning through induction of Prdm4 in adipocytes. Here, we investigated the effects of butein on diet-induced obesity and its underlying molecular mechanism. Treatment with butein prevented weight gains and improved metabolic profiles in diet-induced obese mice. Butein treatment groups also displayed higher body temperature, increased energy expenditure, and enhanced expression of thermogenic genes in adipose tissue. Butein also suppressed body weight gains and improved glucose and insulin tolerance in mice housed at thermoneutrality (30 °C). These effects were associated with adipose-selective induction of Prdm4, suggesting the role of Prdm4 in butein-mediated anti-obese effects. To directly assess the in vivo role of Prdm4, we generated aP2-Prdm4 transgenic mouse lines overexpressing Prdm4 in adipose tissues. Adipose-specific transgenic expression of Prdm4 recapitulated the butein's actions in stimulating energy expenditure, cold tolerance, and thermogenic gene expression, resulting in prevention of obesity and improvement of metabolism. Mechanistically, direct inhibition of PI3Kα activity followed by selective suppression of its downstream Akt1 mirrored butein's effect on Ucp1 expression and oxygen consumption. In addition, effects of butein were completely abolished in Akt1 KO mouse embryonic fibroblasts. Together, these studies demonstrate the role of butein in obesity and metabolic diseases, further highlighting that adipose PI3Kα-Akt1-Prdm4 axis is a regulator of energy expenditure.
Assuntos
Tecido Adiposo/metabolismo , Proteínas de Ligação a DNA/metabolismo , Metabolismo Energético/fisiologia , Resistência à Insulina/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fatores de Transcrição/metabolismo , Aumento de Peso/fisiologia , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Adipócitos/fisiologia , Tecido Adiposo/efeitos dos fármacos , Animais , Linhagem Celular , Chalconas/farmacologia , Dieta Hiperlipídica/efeitos adversos , Metabolismo Energético/efeitos dos fármacos , Camundongos , Camundongos Knockout , Camundongos Obesos , Obesidade/metabolismo , Termogênese/efeitos dos fármacos , Termogênese/fisiologia , Proteína Desacopladora 1/metabolismo , Aumento de Peso/efeitos dos fármacosRESUMO
The devastating sequelae of diabetes mellitus include microvascular permeability, which results in retinopathy. Despite clinical and scientific advances, there remains a need for new approaches to treat retinopathy. Here, we have presented a possible treatment strategy, whereby targeting the small GTPase ARF6 alters VEGFR2 trafficking and reverses signs of pathology in 4 animal models that represent features of diabetic retinopathy and in a fifth model of ocular pathological angiogenesis. Specifically, we determined that the same signaling pathway utilizes distinct GEFs to sequentially activate ARF6, and these GEFs exert distinct but complementary effects on VEGFR2 trafficking and signal transduction. ARF6 activation was independently regulated by 2 different ARF GEFs - ARNO and GEP100. Interaction between VEGFR2 and ARNO activated ARF6 and stimulated VEGFR2 internalization, whereas a VEGFR2 interaction with GEP100 activated ARF6 to promote VEGFR2 recycling via coreceptor binding. Intervening in either pathway inhibited VEGFR2 signal output. Finally, using a combination of in vitro, cellular, genetic, and pharmacologic techniques, we demonstrated that ARF6 is pivotal in VEGFR2 trafficking and that targeting ARF6-mediated VEGFR2 trafficking has potential as a therapeutic approach for retinal vascular diseases such as diabetic retinopathy.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Retinopatia Diabética/metabolismo , Transdução de Sinais , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Fator 6 de Ribosilação do ADP , Fatores de Ribosilação do ADP/genética , Linhagem Celular , Retinopatia Diabética/genética , Retinopatia Diabética/patologia , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Humanos , Transporte Proteico , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
Activating mutations in Gαq proteins, which form the α subunit of certain heterotrimeric G proteins, drive uveal melanoma oncogenesis by triggering multiple downstream signaling pathways, including PLC/PKC, Rho/Rac, and YAP. Here we show that the small GTPase ARF6 acts as a proximal node of oncogenic Gαq signaling to induce all of these downstream pathways as well as ß-catenin signaling. ARF6 activates these diverse pathways through a common mechanism: the trafficking of GNAQ and ß-catenin from the plasma membrane to cytoplasmic vesicles and the nucleus, respectively. Blocking ARF6 with a small-molecule inhibitor reduces uveal melanoma cell proliferation and tumorigenesis in a mouse model, confirming the functional relevance of this pathway and suggesting a therapeutic strategy for Gα-mediated diseases.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Melanoma/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/administração & dosagem , Neoplasias Uveais/tratamento farmacológico , beta Catenina/metabolismo , Fator 6 de Ribosilação do ADP , Fatores de Ribosilação do ADP/antagonistas & inibidores , Fatores de Ribosilação do ADP/genética , Animais , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Citoplasma/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Humanos , Melanoma/genética , Melanoma/metabolismo , Camundongos , Transplante de Neoplasias , Transporte Proteico/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Neoplasias Uveais/genética , Neoplasias Uveais/metabolismoRESUMO
Calmodulin (CaM), a key Ca(2+) sensor in eukaryotes, regulates diverse cellular processes by interacting with many proteins. To identify Ca(2+)/CaM-mediated signaling components, we screened an Arabidopsis expression library with horseradish peroxidase-conjugated Arabidopsis calmodulin2 (AtCaM2) and isolated a homolog of the UBP6 deubiquitinating enzyme family (AtUBP6) containing a Ca(2+)-dependent CaM-binding domain (CaMBD). The CaM-binding activity of the AtUBP6 CaMBD was confirmed by CaM mobility shift assay, phosphodiesterase competition assay and site-directed mutagenesis. Furthermore, expression of AtUBP6 restored canavanine resistance to the Deltaubp6 yeast mutant. This is the first demonstration that Ca(2+) signaling via CaM is involved in ubiquitin-mediated protein degradation and/or stabilization in plants.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/enzimologia , Calmodulina/metabolismo , Endopeptidases/fisiologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Ligação Competitiva , Cálcio/metabolismo , Canavanina/química , Canavanina/farmacologia , Relação Dose-Resposta a Droga , Endopeptidases/metabolismo , Biblioteca Gênica , Teste de Complementação Genética , Glutationa Transferase/metabolismo , Peroxidase do Rábano Silvestre/metabolismo , Humanos , Modelos Genéticos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Peptídeos/química , Diester Fosfórico Hidrolases/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Ubiquitina/química , Ubiquitina/metabolismo , Proteases Específicas de UbiquitinaRESUMO
Calmodulin (CaM) is a ubiquitous Ca(2+)-binding protein known to regulate diverse cellular functions by modulating the activity of various target proteins. We isolated a cDNA encoding AtWRKY7, a novel CaM-binding transcription factor, from an Arabidopsis expression library with horseradish peroxidase-conjugated CaM. CaM binds specifically to the Ca(2+)-dependent CaM-binding domain (CaMBD) of AtWRKY7, as shown by site-directed mutagenesis, a gel mobility shift assay, a split-ubiquitin assay, and a competition assay using a Ca2+/CaM-dependent enzyme. Furthermore, we show that the CaMBD of AtWRKY7 is a conserved structural motif (C-motif) found in group IId of the WRKY protein family.
Assuntos
Proteínas de Arabidopsis/metabolismo , Calmodulina/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Arabidopsis/química , Arabidopsis/classificação , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/isolamento & purificação , Proteínas de Ligação a Calmodulina/química , Proteínas de Ligação a Calmodulina/classificação , Proteínas de Ligação a Calmodulina/genética , Proteínas de Ligação a Calmodulina/metabolismo , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Filogenia , Ligação Proteica , Alinhamento de Sequência , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/isolamento & purificação , Ubiquitina/metabolismoRESUMO
Plants produce numerous calmodulin isoforms that exhibit differential gene expression patterns and sense different Ca2+ signals. This diversity results in different physiological responses to particular stimuli. Gm-CaM-4 and -5 are two divergent calmodulin isoforms from the soybean (Glycine max) that have been reported to be involved in plant disease resistance. However, little is known about the pathway by which these specific isoforms transduce the defense signal and up-regulate pathogenesis-related (PR) genes. Here we report that overexpression of GmCaM-4/-5 induces constitutive PR gene expression and enhances disease resistance in wild-type Arabidopsis, but not in the nim1 mutant of Arabidopsis. GmCaM-4/-5 also appear to activate trans-acting elements that bind to cis-acting elements in the Arabidopsis PR-1 promoter. Thus up-regulation of PR genes by these GmCaM isoforms is dependent on NIM1 (Non immunity 1) and unknown transcription factors.
Assuntos
Calmodulina/farmacologia , Regulação da Expressão Gênica de Plantas/imunologia , Imunidade Inata/genética , Arabidopsis/imunologia , Proteínas de Arabidopsis/fisiologia , Calmodulina/genética , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Plantas Geneticamente Modificadas , Isoformas de Proteínas , Glycine max/genética , Ativação Transcricional , TransfecçãoRESUMO
ß-Catenin has a dual function in cells: fortifying cadherin-based adhesion at the plasma membrane and activating transcription in the nucleus. We found that in melanoma cells, WNT5A stimulated the disruption of N-cadherin and ß-catenin complexes by activating the guanosine triphosphatase adenosine diphosphate ribosylation factor 6 (ARF6). Binding of WNT5A to the Frizzled 4-LRP6 (low-density lipoprotein receptor-related protein 6) receptor complex activated ARF6, which liberated ß-catenin from N-cadherin, thus increasing the pool of free ß-catenin, enhancing ß-catenin-mediated transcription, and stimulating invasion. In contrast to WNT5A, the guidance cue SLIT2 and its receptor ROBO1 inhibited ARF6 activation and, accordingly, stabilized the interaction of N-cadherin with ß-catenin and reduced transcription and invasion. Thus, ARF6 integrated competing signals in melanoma cells, thereby enabling plasticity in the response to external cues. Moreover, small-molecule inhibition of ARF6 stabilized adherens junctions, blocked ß-catenin signaling and invasiveness of melanoma cells in culture, and reduced spontaneous pulmonary metastasis in mice, suggesting that targeting ARF6 may provide a means of inhibiting WNT/ß-catenin signaling in cancer.
Assuntos
Fatores de Ribosilação do ADP/fisiologia , Melanoma/patologia , Invasividade Neoplásica , Metástase Neoplásica , Proteínas Proto-Oncogênicas/fisiologia , Ativação Transcricional/fisiologia , Proteínas Wnt/fisiologia , beta Catenina/fisiologia , Fator 6 de Ribosilação do ADP , Inativação Gênica , Humanos , Transdução de Sinais , Proteína Wnt-5a , beta Catenina/metabolismoRESUMO
Calmodulin-regulated protein phosphorylation plays a pivotal role in amplifying and diversifying the action of calcium ion. In this study, we identified a calmodulin-binding receptor-like protein kinase (CBRLK1) that was classified into an S-locus RLK family. The plasma membrane localization was determined by the localization of CBRLK1 tagged with a green fluorescence protein. Calmodulin bound specifically to a Ca(2+)-dependent calmodulin binding domain in the C-terminus of CBRLK1. The bacterially expressed CBRLK1 kinase domain could autophosphorylate and phosphorylates general kinase substrates, such as myelin basic proteins. The autophosphorylation sites of CBRLK1 were identified by mass spectrometric analysis of phosphopeptides.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Calmodulina/metabolismo , Membrana Celular/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Proteínas de Arabidopsis/genética , Dados de Sequência Molecular , Proteínas Serina-Treonina Quinases/genética , Estrutura Terciária de ProteínaRESUMO
The mitogen-activated protein kinases (MAPKs) are key signal transduction molecules, which respond to various external stimuli. The MAPK phosphatases (MKPs) are known to be negative regulators of MAPKs in eukaryotes. We screened an Arabidopsis cDNA library using horseradish peroxidase-conjugated calmodulin (CaM), and isolated AtMKP1 as a CaM-binding protein. Recently, tobacco NtMKP1 and rice OsMKP1, two orthologs of Arabidopsis AtMKP1, were reported to bind CaM via a single putative CaM binding domain (CaMBD). However, little is known about the regulation of phosphatase activity of plant MKP1s by CaM binding. In this study, we identified two Ca(2+)-dependent CaMBDs within AtMKP1. Specific binding of CaM to two different CaMBDs was verified using a gel mobility shift assay, a competition assay with a Ca(2+)/CaM-dependent enzyme, and a split-ubiquitin assay. The peptides for two CaMBDs, CaMBDI and CaMBDII, bound CaM in a Ca(2+)-dependent manner, and the binding affinity of CaMBDII was found to be higher than that of CaMBDI. CaM overlay assays using mutated CaMBDs showed that four amino acids, Trp(453) and Leu(456) in CaMBDI and Trp(678) and Ile(684) in CaMBDII, play a pivotal role in CaM binding. Moreover, the phosphatase activity of AtMKP1 was increased by CaM in a Ca(2+)-dependent manner. Our results suggest that two important signaling pathways, Ca(2+) signaling and the MAPK signaling cascade, are connected in plants via the regulation of AtMKP1 activity. To our knowledge, this is the first report to show that the biochemical activity of MKP1 in plants is regulated by CaM.
Assuntos
Arabidopsis/enzimologia , Sinalização do Cálcio/fisiologia , Calmodulina/metabolismo , Fosfatase 1 de Especificidade Dupla/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Arabidopsis/genética , Cálcio/química , Cálcio/metabolismo , Calmodulina/química , Calmodulina/genética , Clonagem Molecular , Fosfatase 1 de Especificidade Dupla/química , Fosfatase 1 de Especificidade Dupla/genética , MAP Quinases Reguladas por Sinal Extracelular/química , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Biblioteca Gênica , Mutação , Oryza/enzimologia , Oryza/genética , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína/fisiologia , Nicotiana/enzimologia , Nicotiana/genéticaRESUMO
Calmodulin (CaM), a ubiquitous calcium-binding protein, regulates diverse cellular functions by modulating the activity of a variety of proteins. However, little is known about how CaM directly regulates transcription. Screening of an Arabidopsis cDNA expression library using horseradish peroxidase-conjugated calmodulin as a probe identified a calmodulin-binding NAC protein (CBNAC). Using gel overlay assays, a Ca2+-dependent CaM-binding domain was identified in the C terminus of this protein. Specific binding of CaM to CaM-binding domain was corroborated by site-directed mutagenesis and a split-ubiquitin assay. Using a PCR-mediated random binding site selection method, we identified a DNA-binding sequence (CBNACBS) for CBNAC, which consisted of a GCTT core sequence flanked on both sides by other frequently repeating sequences (TTGCTTANNNNNNAAG). CBNAC was able to bind to CBNACBS, which resulted in the repression of transcription in Arabidopsis protoplasts. Interestingly, the transcriptional repression mediated by CBNAC was enhanced by CaM. These results suggest that CBNAC may be a CaM-regulated transcriptional repressor in Arabidopsis.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas de Ligação a Calmodulina/genética , Proteínas de Ligação a DNA/genética , Proteínas Repressoras/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Calmodulina/genética , Calmodulina/metabolismo , Proteínas de Ligação a Calmodulina/metabolismo , Clonagem Molecular , Proteínas de Ligação a DNA/metabolismo , Biblioteca Gênica , Ligação Proteica/genética , Estrutura Terciária de Proteína/fisiologia , Proteínas Repressoras/metabolismo , Elementos de Resposta/fisiologia , Transcrição Gênica/fisiologiaRESUMO
Calmodulin (CaM), a ubiquitous calcium-binding protein, regulates diverse cellular functions by modulating the activity of a variety of enzymes and proteins. Plants express numerous CaM isoforms that exhibit differential activation and/or inhibition of CaM-dependent enzymes in vitro. However, the specific biological functions of plant CaM are not well known. In this study, we isolated a cDNA encoding a CaM binding transcription factor, MYB2, that regulates the expression of salt- and dehydration-responsive genes in Arabidopsis. This was achieved using a salt-inducible CaM isoform (GmCaM4) as a probe from a salt-treated Arabidopsis expression library. Using domain mapping, we identified a Ca2+-dependent CaM binding domain in MYB2. The specific binding of CaM to CaM binding domain was confirmed by site-directed mutagenesis, a gel mobility shift assay, split ubiquitin assay, and a competition assay using a Ca2+/CaM-dependent enzyme. Interestingly, the specific CaM isoform GmCaM4 enhances the DNA binding activity of AtMYB2, whereas this was inhibited by a closely related CaM isoform (GmCaM1). Overexpression of Gm-CaM4 in Arabidopsis up-regulates the transcription rate of AtMYB2-regulated genes, including the proline-synthesizing enzyme P5CS1 (Delta1-pyrroline-5-carboxylate synthetase-1), which confers salt tolerance by facilitating proline accumulation. Therefore, we suggest that a specific CaM isoform mediates salt-induced Ca2+ signaling through the activation of an MYB transcriptional activator, thereby resulting in salt tolerance in plants.