RESUMEN
We hypothesized that fusing granulocyte-macrophage colony-stimulation factor (GMCSF) and interleukin (IL)-21 as a single bifunctional cytokine (hereafter GIFT-21) would lead to synergistic anticancer immune effects because of their respective roles in mediating inflammation. Mechanistic analysis of GIFT-21 found that it leads to IL-21Ralpha-dependent STAT3 hyperactivation while also contemporaneously behaving as a dominant-negative inhibitor of GMCSF-driven STAT5 activation. GIFT-21's aberrant interactions with its cognate receptors on macrophages resulted in production of 30-fold greater amounts of IL-6, TNF-alpha, and MCP-1 when compared to controls. Furthermore, GIFT-21 treatment of primary B and T lymphocytes leads to STAT1-dependent apoptosis of IL-21Ralpha(+) lymphocytes. B16 melanoma cells gene-enhanced to produce GIFT-21 were immune rejected by syngeneic C57Bl/6 mice comparable to the effect of IL-21 alone. However, a significant GIFT-21-driven survival advantage was seen when NOD-SCID mice were implanted with GIFT-21-secreting B16 cells, consistent with a meaningful role of macrophages in tumor rejection. Because GIFT-21 leads to apoptosis of IL-21Ralpha(+) lymphocytes, we tested its cytolytic effect on IL-21Ralpha(+) EL-4 lymphoma tumors implanted in C57Bl/6 mice and could demonstrate a significant increase in survival. These data indicate that GIFT-21 is a novel IL-21Ralpha agonist that co-opts IL-21Ralpha-dependent signaling in a manner permissive for targeted cancer immunotherapy.
Asunto(s)
Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Subunidad alfa del Receptor de Interleucina-21/metabolismo , Interleucinas/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Proteínas Recombinantes de Fusión/fisiología , Animales , Apoptosis/efectos de los fármacos , Linfocitos B/efectos de los fármacos , Linfocitos B/metabolismo , Línea Celular , Células Cultivadas , Citocinas/metabolismo , Femenino , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Interleucinas/genética , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Melanoma Experimental/metabolismo , Melanoma Experimental/terapia , Ratones , Ratones Endogámicos C57BL , Proteínas Recombinantes de Fusión/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismoRESUMEN
Phosphoinositide-3 kinase (PI3K) plays an important role in signal transduction in response to a wide range of cellular stimuli involved in cellular processes that promote cell proliferation and survival. Phosphorylation of the alpha subunit of the eukaryotic translation initiation factor eIF2 at Ser51 takes place in response to various types of environmental stress and is essential for regulation of translation initiation. Herein, we show that a conditionally active form of the eIF2alpha kinase PKR acts upstream of PI3K and turns on the Akt/PKB-FRAP/mTOR pathway leading to S6 and 4E-BP1 phosphorylation. Also, induction of PI3K signaling antagonizes the apoptotic and protein synthesis inhibitory effects of the conditionally active PKR. Furthermore, induction of the PI3K pathway is impaired in PKR(-/-) or PERK(-/-) mouse embryonic fibroblasts (MEFs) in response to various stimuli that activate each eIF2alpha kinase. Mechanistically, PI3K signaling activation is indirect and requires the inhibition of protein synthesis by eIF2alpha phosphorylation as demonstrated by the inactivation of endogenous eIF2alpha by small interfering RNA or utilization of MEFs bearing the eIF2alpha Ser51Ala mutation. Our data reveal a novel property of eIF2alpha kinases as activators of PI3K signaling and cell survival.
Asunto(s)
Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de Señal , eIF-2 Quinasa/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular , Muerte Celular , Línea Celular Tumoral , Activación Enzimática , Factor 2 Eucariótico de Iniciación/metabolismo , Factores Eucarióticos de Iniciación , Humanos , Ratones , Modelos Biológicos , Fosfoproteínas/metabolismo , Fosforilación , Biosíntesis de Proteínas , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Quinasas S6 Ribosómicas/metabolismoAsunto(s)
Academias e Institutos , Farmacorresistencia Microbiana , Bibliometría , Canadá , Conducta Cooperativa , Brotes de Enfermedades/prevención & control , Farmacorresistencia Bacteriana Múltiple , Farmacorresistencia Viral , Financiación Gubernamental , Agencias Gubernamentales/organización & administración , Humanos , Control de Infecciones/economía , Control de Infecciones/organización & administración , Investigadores/economía , Investigadores/educaciónRESUMEN
Hepatitis C virus (HCV) affects at least 268,000 Canadians and causes greater disease burden than any other infectious disease in the country. The Canadian Institutes of Health Research (CIHR) and the Public Health Agency of Canada (PHAC) have identified HCV-related liver disease as a priority. In 2015, the release of well-tolerated, short course treatments (~12 weeks) able to cure the majority of treated HCV patients revolutionized HCV therapy. However, treatment is extremely costly and puts a significant burden on the Canadian healthcare system. Thus, managing treatment costs and improving treatment engagement in those most in need will be a key challenge. Diagnosis and treatment uptake are currently poor in Canada due to financial, geographical, cultural, and social barriers. The United States, Australia, and Scotland all have National Action Plans to prevent, diagnose, and treat HCV in order to efficiently reduce the burden and costs associated with HCV-related liver disease. The theme of the 4th annual symposium held on Feb 27, 2015, "Strategies to Manage HCV Infection in Canada: Moving towards a National Action Plan," was aimed at identifying strategies to maximize the impact of highly effective therapies to reduce HCV disease burden and ultimately eliminate HCV in Canada.
Asunto(s)
Política de Salud , Hepatitis C , Antivirales/uso terapéutico , Canadá , Costo de Enfermedad , HumanosRESUMEN
Vaccine biomarkers are critical to many aspects of vaccine development and licensure, including bridging findings in pre-clinical studies to clinical studies, predicting potential adverse events, and predicting vaccine efficacy. Despite advances in our understanding of various biological pathways, and advances in systems analyses of the immune response, there remains much to learn about qualitative and quantitative aspects of the human host response to vaccination. To stimulate discussion and identify opportunities for collaborative ways to advance the field of vaccine biomarkers, A Next Generation Vaccine Biomarker workshop was held in Ottawa. The two day workshop, sponsored by the National Research Council Canada, Canadian Institutes of Health Research, Public Health Agency of Canada, Pfizer, and Medicago, brought together stakeholders from Canadian and international industry, government and academia. The workshop was grouped in themes, covering vaccine biomarker challenges in the pre-clinical and clinical spaces, veterinary vaccines, regulatory challenges, and development of biomarkers for adjuvants and cancer vaccines. The use of case studies allowed participants to identify the needs and gaps requiring innovation. The workshop concluded with a discussion on opportunities for vaccine biomarker discovery, the Canadian context, and approaches for moving forward. This article provides a synopsis of these discussions and identifies steps forward for advancing vaccine biomarker research in Canada.
Asunto(s)
Biomarcadores/análisis , Vacunación , Vacunas/inmunología , Investigación Biomédica , Canadá , HumanosRESUMEN
Hepatitis C virus (HCV) currently infects approximately 250,000 individuals in Canada and causes more years of life lost than any other infectious disease in the country. In August 2011, new therapies were approved by Health Canada that have achieved higher response rates among those treated, but are poorly tolerated. By 2014/2015, short-course, well-tolerated treatments with cure rates >95% will be available. However, treatment uptake is poor due to structural, financial, geographical, cultural and social barriers. As such, 'Barriers to access to HCV care in Canada' is a crucial topic that must be addressed to decrease HCV disease burden and potentially eliminate HCV in Canada. Understanding how to better care for HCV-infected individuals requires integration across multiple disciplines including researchers, clinical services and policy makers to address the major populations affected by HCV including people who inject drugs, baby boomers, immigrants and Aboriginal and/or First Nations people. In 2012, the National CIHR Research Training Program in Hepatitis C organized the 1st Canadian Symposium on Hepatitis C Virus (CSHCV) in Montreal, Quebec. The 2nd CSHCV was held in 2013 in Victoria, British Columbia. Both symposia were highly successful, attracting leading international faculty with excellent attendance leading to dialogue and knowledge translation among attendees of diverse backgrounds. The current article summarizes the 3rd CSHCV, held February 2014, in Toronto, Ontario.
Asunto(s)
Antivirales/uso terapéutico , Hepacivirus/efectos de los fármacos , Hepatitis C/tratamiento farmacológico , Hepatitis C/prevención & control , Canadá/epidemiología , Congresos como Asunto , Medicina Basada en la Evidencia , Hepatitis C/epidemiología , Humanos , Prevalencia , Factores de Riesgo , Abuso de Sustancias por Vía Intravenosa/epidemiologíaRESUMEN
In Canada, hepatitis C virus (HCV) infection results in considerable morbidity, mortality and health-related costs. Within the next three to 10 years, it is expected that tolerable, short-duration (12 to 24 weeks) therapies capable of curing >90% of those who undergo treatment will be approved. Given that most of those already infected are aging and at risk for progressive liver disease, building research-based interdisciplinary prevention, care and treatment capacity is an urgent priority. In an effort to increase the dissemination of knowledge in Canada in this rapidly advancing field, the National CIHR Research Training Program in Hepatitis C (NCRTP-HepC) established an annual interdisciplinary Canadian Symposium on Hepatitis C Virus. The first symposium was held in Montreal, Quebec, in 2012, and the second symposium was held in Victoria, British Columbia, in 2013. The current article presents highlights from the 2013 meeting. It summarizes recent advances in HCV research in Canada and internationally, and presents the consensus of the meeting participants that Canada would benefit from having its own national HCV strategy to identify critical gaps in policies and programs to more effectively address the challenges of expanding HCV screening and treatment.
Asunto(s)
Antivirales/uso terapéutico , Hepatitis C/tratamiento farmacológico , Tamizaje Masivo/métodos , Antivirales/administración & dosificación , Canadá/epidemiología , Política de Salud , Hepatitis C/diagnóstico , Hepatitis C/epidemiología , Humanos , Salud PúblicaRESUMEN
The anti-tumor function of Stat1 as a regulator of innate immunity and tumor immune surveillance has been long studied and is well understood; however, less clear is its tumor-site specific role. Although Stat1 phosphorylated at tyrosine (Y) 701 and serine (S) 727 is essential for interferon (IFN) signalling, its function in signalling induced in breast cancer cells is not understood. Herein, we show that Stat1 Y701 phosphorylation is increased in human breast tumor cells with elevated levels of ErbB2/HER-2 and in cells transfected with ErbB2/Neu. However, pharmacological inhibition of ErbB2/HER-2 results in the inhibition of Stat1 Y701 phosphorylation indicating an atypical role of phosphorylated Stat1 in the inhibition of ErbB2/HER-2 signalling. Consistent with this notion, we found that Stat1 suppresses tumor development by an activated form of ErbB2/Neu in mouse embryonic fibroblasts in xenograft tumor assays; however, this anti-tumor function of Stat1 does not rely on Y701 and S727 phosphorylation. Experiments with transgenic mice demonstrated that Stat1 acts to suppress Neu-mediated breast tumorigenesis through immune regulatory and tumor-site specific mechanisms. Our data reveal a previous uncharacterized anti-tumor activity of Stat1 in ErbB2/Neu-mediated cell transformation and breast oncogenesis with possible implications in the diagnosis and treatment of ErbB2-positive breast cancers.
Asunto(s)
Transformación Celular Neoplásica , Neoplasias Mamarias Experimentales/metabolismo , Receptor ErbB-2/metabolismo , Factor de Transcripción STAT1/metabolismo , Animales , Antineoplásicos/farmacología , Línea Celular , Chlorocebus aethiops , Progresión de la Enfermedad , Femenino , Gefitinib , Humanos , Lapatinib , Glándulas Mamarias Animales/metabolismo , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Transgénicos , Fosforilación , Quinazolinas/farmacología , Receptor ErbB-2/antagonistas & inhibidores , Transducción de Señal , Trasplante HeterólogoRESUMEN
The S-phase kinase-associated protein 2 (Skp2) is an F-box protein that serves as a subunit of the Skp1-Cullin-F-box ubiquitin protein ligase complex. Skp2 is overexpressed in many tumors and promotes tumor formation through its ability to induce the degradation of proteins with antiproliferative and tumor-suppressor functions, such as p27(Kip1). The signal transducer and activator of transcription 1 (STAT1) is a key regulator of the immune system through its capacity to act downstream of interferons. STAT1 exhibits tumor-suppressor properties by inhibiting oncogenic pathways and promoting tumor immunosurveillance. Previous work established the antitumor function of STAT1 in Ras-transformed cells through the induction of p27(Kip1) at the transcriptional level. Herein, we unveil a novel pathway used by STAT1 to upregulate p27(Kip1). Specifically, we show that STAT1 impedes Skp2 gene transcription by binding to Skp2 promoter DNA in vitro and in vivo. Decreased Skp2 expression by STAT1 is accompanied by the increased stability of p27(Kip1) in Ras-transformed cells. We further show that impaired expression of STAT1 in human colon cancer cells containing an activated form of K-Ras is associated with the upregulation of Skp2 and downregulation of p27(Kip1). Our study identifies Skp2 as a new target gene of STAT1 in Ras-transformed cells with profound implications in cell transformation and tumorigenesis.
Asunto(s)
Transformación Celular Neoplásica/genética , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Proteínas Quinasas Asociadas a Fase-S/genética , Factor de Transcripción STAT1/fisiología , Animales , Línea Celular Transformada , Línea Celular Tumoral , Transformación Celular Neoplásica/metabolismo , Regulación hacia Abajo/genética , Regulación Neoplásica de la Expresión Génica/genética , Células HCT116 , Humanos , Ratones , Ratones Noqueados , Regiones Promotoras Genéticas/genética , Estabilidad del ARN/genética , Proteínas Represoras/fisiología , Proteínas Quinasas Asociadas a Fase-S/antagonistas & inhibidores , Factor de Transcripción STAT1/genética , Regulación hacia Arriba/genética , Proteínas ras/genética , Proteínas ras/metabolismoRESUMEN
Phosphatidate phosphatase-1 (PAP-1) converts phosphatidate to diacylglycerol and plays a key role in the biosynthesis of phospholipids and triacylglycerol (TAG). PAP-1 activity is encoded by members of the lipin family, including lipin-1 (1alpha and 1beta), -2, and -3. We determined the effect of lipin-1 expression on the assembly and secretion of very low density lipoproteins (VLDL) using McA-RH7777 cells. Expression of lipin-1alpha or -1beta increased the synthesis and secretion of [(3)H]glycerol-labeled lipids under either basal- or oleate-supplemented conditions. In the presence of oleate, the increased TAG secretion was mainly associated with VLDL(1) (S(f) > 100) and VLDL(2) (S(f) 20-100). Expression of lipin-1alpha or -1beta increased secretion efficiency and decreased intracellular degradation of [(35)S]apolipoprotein B-100 (apoB100). Knockdown of lipin-1 using specific short interfering RNA decreased secretion of [(3)H]glycerolipids and [(35)S]apoB100 even though total PAP-1 activity was not decreased, owing to the presence of lipin-2 and -3 in the cells. Deletion of the nuclear localization signal sequences within lipin-1alpha not only abolished nuclear localization but also resulted in impaired association with microsomal membranes. Cells expressing the cytosolic lipin-1alpha mutant failed to promote [(35)S]apoB100 synthesis or secretion, and showed compromised stimulation in [(3)H]TAG synthesis and secretion. Thus, alteration in hepatic expression of lipin-1 and its compartmentalization control VLDL assembly/secretion.
Asunto(s)
Lipoproteínas VLDL/metabolismo , Hígado/metabolismo , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/genética , Animales , Apolipoproteína B-100/metabolismo , Transporte Biológico , Eliminación de Gen , Regulación de la Expresión Génica , Glicerol/metabolismo , Humanos , Lípidos/química , Ratones , Ratones Noqueados , Microscopía Fluorescente/métodos , Modelos Biológicos , Proteínas Asociadas a Pancreatitis , Fosfatidato FosfatasaRESUMEN
Regulating gene expression is an effective way for cells to deal with various stresses. The outcome of this regulation differs with the type of stress, and can promote either cell survival or cell death depending on the severity of the injury incurred. Gene expression can be controlled at several steps, including transcription, translation and degradation. An extensively studied protein involved in translational control is the eukaryotic translation initiation factor 2 (eIF2). When eIF2 becomes phosphorylated on a specific serine residue located within the alpha (alpha) subunit, global protein synthesis is halted. This phosphorylation occurs following periods of environmental stress, and plays a significant role in the cellular response to these events. The eIF2alpha kinase family consists of four members, which are each activated in response to different stimuli. Our group has recently discovered that two members of this family, the protein kinase activated by double-stranded RNA (PKR) and the PKR-like endoplasmic reticulum (ER) kinase (PERK) can also regulate the expression of specific proteins by promoting their degradation by the 26S proteasome. Specifically, we demonstrated that degradation of the cell cycle regulator cyclin D1, and the tumour suppressor p53 was promoted by PERK and PKR during periods of ER stress. This novel function may allow the eIF2alpha kinases to affect a larger number of cellular processes than previously believed.
Asunto(s)
Factor 2 Eucariótico de Iniciación/metabolismo , Biosíntesis de Proteínas/genética , eIF-2 Quinasa/metabolismo , Animales , Ciclina D1/genética , Ciclina D1/metabolismo , Factor 2 Eucariótico de Iniciación/genética , Humanos , Fosforilación , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , eIF-2 Quinasa/genéticaRESUMEN
The eIF2alpha kinases have been involved in the inhibition of vesicular stomatatis virus replication but the contribution of each kinase to this process has not been fully investigated. Using mouse embryonic fibroblasts (MEFs) from knock-out mice we show that PKR and HRI have no effects on VSV replication as opposed to PERK and GCN2, which exhibit strong inhibitory effects. When MEFs containing the serine 51 to alanine mutation of eIF2alpha were used, we found that VSV replication is independent of eIF2alpha phosphorylation. Nevertheless, the kinase domain of the eIF2alpha kinases is both necessary and sufficient to inhibit VSV replication in cultured cells. Induction of PI3K-Akt/PKB pathway by eIF2alpha kinase activation plays no role in the inhibition of VSV replication. Our data provide strong evidence that VSV replication is not affected by eIF2alpha phosphorylation or downstream effector pathways such as the PI3K-Akt/PKB pathway. Thus, the anti-viral properties of eIF2alpha kinases are not always related to their inhibitory effects on host protein synthesis as previously thought and are possibly mediated by phosphorylation of proteins other than eIF2alpha.
Asunto(s)
Factor 2 Eucariótico de Iniciación/metabolismo , Vesiculovirus/fisiología , Replicación Viral/fisiología , eIF-2 Quinasa/fisiología , Animales , Antivirales/metabolismo , Células Cultivadas , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Isoenzimas/fisiología , Ratones , Ratones Noqueados , Fosforilación , Estomatitis Vesicular/enzimología , Estomatitis Vesicular/metabolismo , eIF-2 Quinasa/química , eIF-2 Quinasa/genética , eIF-2 Quinasa/metabolismoRESUMEN
Inactivation of p27 Kip1 is implicated in tumorigenesis and has both prognostic and treatment-predictive values for many types of human cancer. The transcription factor Stat1 is essential for innate immunity and tumor immunosurveillance through its ability to act downstream of interferons. Herein, we demonstrate that Stat1 functions as a suppressor of Ras transformation independently of an interferon response. Inhibition of Ras transformation and tumorigenesis requires the phosphorylation of Stat1 at tyrosine 701 but is independent of Stat1 phosphorylation at serine 727. Stat1 induces p27 Kip1 expression in Ras transformed cells at the transcriptional level through mechanisms that depend on Stat1 phosphorylation at tyrosine 701 and activation of Stat3. The tumor suppressor properties of Stat1 in Ras transformation are reversed by the inactivation of p27 Kip1. Our work reveals a novel functional link between Stat1 and p27 Kip1, which act in coordination to suppress the oncogenic properties of activated Ras. It also supports the notion that evaluation of Stat1 phosphorylation in human tumors may prove a reliable prognostic factor for patient outcome and a predictor of treatment response to anticancer therapies aimed at activating Stat1 and its downstream effectors.
Asunto(s)
Transformación Celular Neoplásica , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Neoplasias/etiología , Factor de Transcripción STAT1/fisiología , Proteínas ras/fisiología , Animales , Células Madre Embrionarias , Humanos , Ratones , Ratones Noqueados , Fosforilación , Valor Predictivo de las Pruebas , Pronóstico , Factor de Transcripción STAT1/metabolismo , Transcripción Genética , TirosinaRESUMEN
Cyclin D1 plays a critical role in controlling the G(1)/S transition via the regulation of cyclin-dependent kinase activity. Several studies have indicated that cyclin D1 translation is decreased upon activation of the eukaryotic initiation factor 2alpha (eIF2alpha) kinases. We examined the effect of activation of the eIF2alpha kinases PKR and PKR-like endoplasmic reticulum kinase (PERK) on cyclin D1 protein levels and translation and determined that cyclin D1 protein levels decrease upon the induction of PKR and PERK catalytic activity but that this decrease is not due to translation. Inhibition of the 26 S proteasome with MG132 rescued cyclin D1 protein levels, indicating that rather than inhibiting translation, PKR and PERK act to increase cyclin D1 degradation. Interestingly, this effect still requires eIF2alpha phosphorylation at serine 51, as cyclin D1 remains unaffected in cells containing a non-phosphorylatable form of the protein. This proteasome-dependent degradation of cyclin D1 requires an intact ubiquitination pathway, although the ubiquitination of cyclin D1 is not itself affected. Furthermore, this degradation is independent of phosphorylation of cyclin D1 at threonine 286, which is mediated by the glycogen synthase kinase 3beta and mitogen-activated protein kinase pathways as described in previous studies. Our study reveals a novel functional cross-talk between eIF2alpha phosphorylation and the proteasomal degradation of cyclin D1 and that this degradation is dependent upon eIF2alpha phosphorylation during short, but not prolonged, periods of stress.
Asunto(s)
Ciclina D1/metabolismo , Factor 2 Eucariótico de Iniciación/metabolismo , Regulación de la Expresión Génica , eIF-2 Quinasa/metabolismo , Línea Celular Tumoral , Regulación hacia Abajo , Retículo Endoplásmico/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Humanos , Fosforilación , Complejo de la Endopetidasa Proteasomal/química , Complejo de la Endopetidasa Proteasomal/metabolismo , Serina/química , Ubiquitina/químicaRESUMEN
Global control of mRNA translation plays key roles in cell regulation, including growth, differentiation and apoptosis. Human monocyte-like U937 cells differentiate into macrophage-like cells upon 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment, a process which is known to be accompanied with a large decrease in general protein synthesis. Here, we found that protein levels of AMAP1 (also called ASAP1 or DDEF1), a GTPase-activating protein for Arf GTPases, increase several fold during U937 cell differentiation. This increase was not accompanied with a notable increase in the AMAP1 gene transcript, nor seemed to be due to 5'-Cap-dependent mRNA translational activities in differentiated U937 cells. We identified the 5'-untranslated region (5'-UTR) of AMAP1 mRNA, and found that this 5'-UTR exhibits significant internal ribosome entry site (IRES)-dependent translational activity in differentiated U937 cells, but not in undifferentiated cells. Our results indicate that monocyte differentiation involves enhancement of IRES activity, by which protein levels of AMAP1 are primarily upregulated.
Asunto(s)
Proteínas Portadoras/metabolismo , Diferenciación Celular/fisiología , Monocitos/fisiología , Biosíntesis de Proteínas , Regiones no Traducidas 5' , Animales , Secuencia de Bases , Proteínas Portadoras/genética , Regulación de la Expresión Génica , Humanos , Datos de Secuencia Molecular , Monocitos/citología , Células U937RESUMEN
The interferon (IFN)-inducible, double-stranded RNA activated protein kinase (PKR) is a dual-specificity kinase, which has an essential role in the regulation of protein synthesis by phosphorylating the translation eukaryotic initiation factor 2 (eIF2). Here, we show the tyrosine (Tyr) phosphorylation of PKR in response to type I or type II IFNs. We show that PKR physically interacts with either Jak1 or Tyk2 in unstimulated cells and that these interactions are increased in IFN-treated cells. We also show that PKR acts as a substrate of activated Jaks, and is phosphorylated at Tyr 101 and Tyr 293 both in vitro and in vivo. Moreover, we provide strong evidence that both the induction of eIF2alpha phosphorylation and inhibition of protein synthesis by IFN are impaired in cells lacking Jak1 or Tyk2, which corresponds to a lack of induction of PKR tyrosine phosphorylation. We conclude that PKR tyrosine phosphorylation provides an important link between IFN signalling and translational control through the regulation of eIF2alpha phosphorylation.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Interferones/metabolismo , Janus Quinasa 1/metabolismo , Transducción de Señal/fisiología , TYK2 Quinasa/metabolismo , Tirosina/metabolismo , eIF-2 Quinasa/metabolismo , Animales , Línea Celular , Electroforesis en Gel de Poliacrilamida , Immunoblotting , Ratones , FosforilaciónRESUMEN
Tyrosine phosphorylation of the transcription factors Stat1 and Stat3 is required for them to dimerize, translocate to the nucleus, and induce gene transcription. Nuclear Stat1 and Stat3 are dephosphorylated and deactivated by the T-cell protein-tyrosine phosphatase (TC-PTP), which facilitates the return of both proteins to the cytoplasm. The protein kinase PKR plays an important role in translational control through the modulation of eukaryotic initiation factor-2alpha phosphorylation. Previous data have implicated PKR in cell signaling via regulation of Stat1 and Stat3, but the molecular mechanisms underlying these events have remained elusive. Using PKR(-/-) mouse embryonic fibroblasts and a conditionally active form of human PKR, we demonstrate herein that tyrosine (but not serine) phosphorylation of either Stat1 or Stat3 is impaired in cells with activated kinase. This reduction in Stat1 and Stat3 tyrosine phosphorylation by active PKR proceeds through TC-PTP, which is a substrate of the eukaryotic initiation factor-2alpha kinase both in vitro and in vivo. TC-PTP phosphorylation alone is insufficient to increase its in vivo phosphatase activity unless accompanied by the inhibition of protein synthesis as a result of PKR activation. These data reveal a novel function of PKR as a negative regulator of Stat1 and Stat3 with important implications in cell signaling.