RESUMEN
Death domain-associated protein (DAXX) is involved in the activation of adipocyte apoptosis and is downregulated in response to a high-fat diet (HFD), which implies that the inhibition of adipocyte apoptosis may cause obesity. However, the anti-obesity effects of DAXX in diet-induced obesity (DIO) remain to be characterized. Here, we identified DAXX as an interacting partner of murine protein serine-threonine kinase 38 (MPK38). This interaction was mediated by the C-terminal (amino acids 270-643) domain of MPK38 and the N-terminal (amino acids 1-440) domain of DAXX and was increased by diverse signals that activate ASK1/TGF-ß/p53 signaling. MPK38 phosphorylated DAXX at Thr578. Wild-type DAXX, but not a DAXX T578A mutant, stimulated MPK38-dependent ASK1/TGF-ß/p53 signaling by increasing the stability of MPK38 and complex formation between MPK38 and its downstream targets, such as ASK1, Smad3, and p53. This mechanism was also shown in MEF cells that were null (-/-) for DAXX. Furthermore, the adenovirally-mediated reinstatement of DAXX expression activated MPK38 and ameliorated diet-induced defects in glucose and lipid metabolism in mice. These results indicate that DAXX limits obesity-induced metabolic abnormalities in DIO mice by activating MPK38.
Asunto(s)
Proteínas Co-Represoras/metabolismo , Chaperonas Moleculares/metabolismo , Obesidad/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Células Cultivadas , Dieta/efectos adversos , Humanos , Ratones , Obesidad/inducido químicamenteRESUMEN
Murine protein serine-threonine kinase 38 (MPK38) is a member of the AMP-activated protein kinase-related serine/threonine kinase family. In this study, we show that MPK38 physically associates with p53 via the carboxyl-terminal domain of MPK38 and the central DNA-binding domain of p53. This interaction is increased by 5-fluorouracil or doxorubicin treatment and is responsible for Ser(15) phosphorylation of p53. Ectopic expression of wild-type Mpk38, but not kinase-dead Mpk38, stimulates p53-mediated transcription in a dose-dependent manner and up-regulates p53 targets, including p53, p21, MDM2, and BAX. Consistently, knockdown of MPK38 shows an opposite trend, inhibiting p53-mediated transcription. MPK38 functionally enhances p53-mediated apoptosis and cell cycle arrest in a kinase-dependent manner by stimulating p53 nuclear translocation. We also demonstrate that MPK38-mediated p53 activation is induced by removing MDM2, a negative regulator of p53, from the p53-MDM2 complex as well as by stabilization of interaction between p53 and its positive regulators, including NM23-H1, serine/threonine kinase receptor-associated protein, and 14-3-3. This leads to the enhancement of p53 stability. Together, these results suggest that MPK38 may act as a novel regulator for promoting p53 activity through direct phosphorylation of p53 at Ser(15).
Asunto(s)
Proteínas Serina-Treonina Quinasas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Animales , Antibióticos Antineoplásicos/farmacología , Antimetabolitos Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Línea Celular Tumoral , Doxorrubicina/farmacología , Fluorouracilo/farmacología , Células HEK293 , Humanos , Ratones , Nucleósido Difosfato Quinasas NM23/genética , Nucleósido Difosfato Quinasas NM23/metabolismo , Fosforilación/efectos de los fármacos , Fosforilación/fisiología , Proteínas Serina-Treonina Quinasas/genética , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Transcripción Genética/efectos de los fármacos , Transcripción Genética/fisiología , Proteína p53 Supresora de Tumor/genética , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiología , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Murine protein serine-threonine kinase 38 (MPK38) is a member of the AMP-activated protein kinase-related serine/threonine kinase family, which acts as cellular energy sensors. In this study, MPK38-induced PDK1 phosphorylation was examined to elucidate the biochemical mechanisms underlying phosphorylation-dependent regulation of 3-phosphoinositide-dependent protein kinase-1 (PDK1) activity. The results showed that MPK38 interacted with and inhibited PDK1 activity via Thr(354) phosphorylation. MPK38-PDK1 complex formation was mediated by the amino-terminal catalytic kinase domain of MPK38 and the pleckstrin homology domain of PDK1. This activity was dependent on insulin, a PI3K/PDK1 stimulator, as well as various apoptotic stimuli, including TNF-α, H(2)O(2), thapsigargin, and ionomycin. MPK38 inhibited PDK1 activity in a kinase-dependent manner and alleviated PDK1-mediated suppression of TGF-ß (or ASK1) signaling, probably via the phosphorylation of PDK1 at Thr(354). In addition, MPK38-mediated inhibition of PDK1 activity was accompanied by the modulation of PDK1 binding to its positive and negative regulators, serine/threonine kinase receptor-associated protein and 14-3-3, respectively. Together, these findings suggest an important role for MPK38-mediated phosphorylation of PDK1 in the negative regulation of PDK1 activity.
Asunto(s)
Complejos Multienzimáticos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Secuencia de Bases , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Células HEK293 , Humanos , Peróxido de Hidrógeno/farmacología , MAP Quinasa Quinasa Quinasa 5/genética , MAP Quinasa Quinasa Quinasa 5/metabolismo , Ratones , Datos de Secuencia Molecular , Complejos Multienzimáticos/genética , Oxidantes/farmacología , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación/efectos de los fármacos , Fosforilación/fisiología , Proteínas Serina-Treonina Quinasas/genética , Estructura Terciaria de Proteína , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora , Factor de Crecimiento Transformador beta1/genética , Factor de Crecimiento Transformador beta1/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Serine-threonine kinase receptor-associated protein (STRAP) functions as a regulator of both TGF-ß and p53 signaling. However, the regulatory mechanism of STRAP activity is not understood. In this study, we report that B-MYB is a new STRAP-interacting protein, and that an amino-terminal DNA-binding domain and an area (amino acids 373-468) between the acidic and conserved regions of B-MYB mediate the B-MYB·STRAP interaction. Functionally, B-MYB enhances STRAP-mediated inhibition of TGF-ß signaling pathways, such as apoptosis and growth inhibition, by modulating complex formation between the TGF-ß receptor and SMAD3 or SMAD7. Furthermore, coexpression of B-MYB results in a dose-dependent increase in STRAP-mediated stimulation of p53-induced apoptosis and cell cycle arrest via direct interaction. Confocal microscopy showed that B-MYB prevents the normal translocation of SMAD3 in response to TGF-ß1 and stimulates p53 nuclear translocation. These results suggest that B-MYB acts as a positive regulator of STRAP.
Asunto(s)
Apoptosis/fisiología , Proteínas de Ciclo Celular/metabolismo , Proteínas de Neoplasias/metabolismo , Transducción de Señal/fisiología , Transactivadores/metabolismo , Neoplasias de la Mama , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Núcleo Celular/metabolismo , Femenino , Células HCT116 , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Proteínas de Neoplasias/química , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , ARN Interferente Pequeño , Proteínas de Unión al ARN , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Proteína smad3/metabolismo , Proteína smad7/metabolismo , Transactivadores/química , Transactivadores/genética , Factor de Crecimiento Transformador beta/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
A zinc finger protein, ZPR9, has been identified as a physiological substrate of murine protein serine/threonine kinase 38 (MPK38), which is involved in various cellular responses, including the cell cycle, apoptosis, embryonic development, and oncogenesis. Here, ZPR9 was found to physically interact with apoptosis signal-regulating kinase 1 (ASK1) through a disulfide linkage involving Cys(1351) and Cys(1360) of ASK1 and Cys(305) and Cys(308) of ZPR9. ASK1 directly phosphorylated ZPR9 at Ser(314) and Thr(318), suggesting that ZPR9 can act as an ASK1 substrate. Ectopic expression of wild-type ZPR9, but not an S314A/T318A mutant, stimulated ASK1 kinase activity and positively regulated ASK1-mediated signaling to both JNK and p38 kinases by destabilizing complex formation between ASK1 and its negative regulators, Trx and 14-3-3, or by increasing complex formation between ASK1 and its substrate MKK3. ZPR9 functionally stimulated ASK1-induced AP-1 transcriptional activity as well as H(2)O(2)-mediated apoptosis in a phosphorylation-dependent manner. ASK1-mediated phosphorylation of ZPR9 at Ser(314) and Thr(318) was also responsible for ZPR9-induced apoptosis. Moreover, ZPR9 inhibited PDK1-mediated signaling through ASK1 activation. These results suggest that ZPR9 functions as a novel positive regulator of ASK1.
Asunto(s)
Apoptosis , Péptidos y Proteínas de Señalización Intracelular/metabolismo , MAP Quinasa Quinasa Quinasa 5/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Transducción de Señal , Animales , Línea Celular , Disulfuros , Humanos , MAP Quinasa Quinasa 3 , Ratones , Fosforilación , Unión Proteica , Transcripción Genética , Dedos de ZincRESUMEN
Density-dependent regulation of cell growth is presumed to be caused by cell-cell contact, but the underlying molecular mechanism is not yet clearly defined. Here, we report that receptor-type protein tyrosine phosphatase-kappa (R-PTP-κ) is an important regulator of cell contact-dependent growth inhibition. R-PTP-κ expression increased in proportion to cell density. siRNA-mediated R-PTP-κ downregulation led to the loss of cell contact-mediated growth inhibition, whereas its upregulation reduced anchorage-independent cell growth in soft agar as well as tumor growth in nude mice. Expression profiling and luciferase reporter system-mediated signaling pathway analysis revealed that R-PTP-κ induced under cell contact conditions distinctly suppressed E2F activity. Among the structural domains of R-PTP-κ, the cytoplasmic domain containing the tandemly repeated PTP motif acts as a potent downregulator of the E2F pathway. Specifically, R-PTP-κ suppressed CDK2 activity through the induction of p21Cip1/WAF-1 and p27Kip1, resulting in cell cycle arrest at the G1 phase. In transcriptome-based public datasets generated from four different tumor types, R-PTP-κ expression was negatively correlated with the expression pattern and prognostic value of two known E2F1 target genes (CCNE1 and CDC25A). Therefore, our results indicate that the R-PTP-κ-E2F axis plays a crucial role in cell growth-inhibitory signaling arising from cell-cell contact conditions.
RESUMEN
The present study demonstrated that murine protein serine/threonine kinase 38 (MPK38) coimmunoprecipitates with Smad proteins (Smad2, -3, -4, and -7) and that this association is mediated by the catalytic kinase domain of MPK38. The association between MPK38 and Smad2, -3, and -4 was significantly increased by TGF-ß or ASK1 signals, whereas these signals decreased association of MPK38 with Smad7. MPK38 stimulated TGF-ß-induced transcription required for TGF-ß-mediated biological functions, such as apoptosis and cell growth arrest, in a kinase-dependent manner. Knockdown of endogenous MPK38 showed an opposite effect, inhibiting TGF-ß signaling. MPK38-mediated phosphorylation of Smad proteins (Ser(245) of Smad2, Ser(204) of Smad3, Ser(343) of Smad4, and Thr(96) of Smad7) was also found to be crucial to the positive regulation of TGF-ß signaling induced by MPK38. In addition, MPK38 enhanced nuclear translocation of Smad3, as well as redistribution of Smad7 from the nucleus to the cytoplasm, in response to TGF-ß. Together, these results indicate that MPK38 functions as a stimulator of TGF-ß signaling through direct interaction with and phosphorylation of Smad proteins.
Asunto(s)
Núcleo Celular/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Proteínas Smad/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Transporte Activo de Núcleo Celular/fisiología , Animales , Núcleo Celular/genética , Células Hep G2 , Humanos , MAP Quinasa Quinasa Quinasa 5/genética , MAP Quinasa Quinasa Quinasa 5/metabolismo , Ratones , Fosforilación/fisiología , Proteínas Smad/genética , Transcripción Genética/fisiología , Factor de Crecimiento Transformador beta/genética , Proteínas Quinasas p38 Activadas por Mitógenos/genéticaRESUMEN
Cell survival and death-inducing signals are tightly associated with each other, and the decision as to whether a cell survives or dies is determined by controlling the relationship between these signals. However, the mechanism underlying the reciprocal regulation of such signals remains unclear. In this study, we reveal a functional association between PDK1 (3-phosphoinositide-dependent protein kinase 1), a critical mediator of cell survival, and ASK1 (apoptosis signal-regulating kinase 1), an apoptotic stress-activated MAPKKK. The physical association between PDK1 and ASK1 is mediated through the pleckstrin homology domain of PDK1 and the C-terminal regulatory domain of ASK1 and is decreased by ASK1-activating stimuli, such as H(2)O(2), tumor necrosis factor alpha, thapsigargin, and ionomycin, as well as insulin, a PDK1 stimulator. Wild-type PDK1, but not kinase-dead PDK1, negatively regulates ASK1 activity by phosphorylating Ser(967), a binding site for 14-3-3 protein, on ASK1. PDK1 functionally suppresses ASK1-mediated AP-1 transactivation and H(2)O(2)-mediated apoptosis in a kinase-dependent manner. On the other hand, ASK1 has been shown to inhibit PDK1 functions, including PDK1-mediated regulation of apoptosis and cell growth, by phosphorylating PDK1 at Ser(394) and Ser(398), indicating that these putative phosphorylation sites are involved in the negative regulation of PDK1 activity. These results provide evidence that PDK1 and ASK1 directly interact and phosphorylate each other and act as negative regulators of their respective kinases in resting cells.
Asunto(s)
Apoptosis/fisiología , MAP Quinasa Quinasa Quinasa 5/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal/fisiología , Proteínas 14-3-3/metabolismo , Caspasa 3/metabolismo , Supervivencia Celular/fisiología , Regulación Enzimológica de la Expresión Génica/fisiología , Células HeLa , Humanos , Peróxido de Hidrógeno/farmacología , MAP Quinasa Quinasa 3/metabolismo , MAP Quinasa Quinasa Quinasa 5/química , MAP Quinasa Quinasa Quinasa 5/genética , Oxidantes/farmacología , Fosforilación/fisiología , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Estructura Terciaria de Proteína , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora , Serina/metabolismo , Transducción de Señal/efectos de los fármacos , Activación Transcripcional/fisiología , Transfección , Técnicas del Sistema de Dos HíbridosRESUMEN
Serine-threonine kinase receptor-associated protein (STRAP) interacts with transforming growth factor beta (TGF-beta) receptors and inhibits TGF-beta signaling. Here, we identify STRAP as an interacting partner of ASK1 (apoptosis signal-regulating kinase 1). The association between ASK1 and STRAP is mediated through the C-terminal domain of ASK1 and the fourth and sixth WD40 repeats of STRAP. Using cysteine-to-serine amino acid substitution mutants of ASK1 (C1005S, C1351S, C1360S, and C1351S/C1360S) and STRAP (C152S, C270S, and C152S/C270S), we demonstrated that Cys(1351) and Cys(1360) of ASK1 and Cys(152) and Cys(270) of STRAP are required for ASK1-STRAP binding. ASK1 phosphorylated STRAP at Thr(175) and Ser(179), suggesting a potential role for STRAP phosphorylation in ASK1 activity regulation. Expression of wild-type STRAP, but not STRAP mutants (C152S/C270S and T175A/S179A), inhibited ASK1-mediated signaling to both JNK and p38 kinases by stabilizing complex formation between ASK1 and its negative regulators, thioredoxin and 14-3-3, or decreasing complex formation between ASK1 and its substrate MKK3. In addition, STRAP suppressed H(2)O(2)-mediated apoptosis in a dose-dependent manner by inhibiting ASK1 activity through direct interaction. These results suggest that STRAP can act as a negative regulator of ASK1.
Asunto(s)
MAP Quinasa Quinasa Quinasa 5/antagonistas & inhibidores , Proteínas de Neoplasias/metabolismo , Proteínas 14-3-3/metabolismo , Animales , Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Línea Celular , Activación Enzimática/efectos de los fármacos , Humanos , Peróxido de Hidrógeno/farmacología , Péptidos y Proteínas de Señalización Intracelular , MAP Quinasa Quinasa 3/metabolismo , MAP Quinasa Quinasa Quinasa 5/química , MAP Quinasa Quinasa Quinasa 5/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteínas de la Membrana , Ratones , Proteínas de Neoplasias/química , Fosforilación/efectos de los fármacos , Fosfoserina/metabolismo , Fosfotreonina/metabolismo , Unión Proteica/efectos de los fármacos , Mapeo de Interacción de Proteínas , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora , Proteínas de Unión al ARN , Factor de Transcripción AP-1/genética , Transcripción Genética/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Murine protein serine-threonine kinase 38 (MPK38)/maternal embryonic leucine zipper kinase (MELK) is implicated in diverse biological processes, including the cell cycle, apoptosis, and tumorigenesis; however, its physiological role is unknown. Using mice lacking MPK38 (MPK38-/-), we found that MPK38-/- male, but not female, mice (7 months of age) became obese while consuming a standard diet, displayed impairments in metabolism and inflammation, became more obese than wild-type mice while consuming a high-fat diet, and exhibited no castration/testosterone replacement-induced metabolic changes. The adenoviral restoration of MPK38 ameliorated the obesity-induced adverse metabolic profile of the obese male, but not female, mice. Seven-month-old MPK38-/- males displayed typical postcastration concentrations of serum testosterone with an accompanying decrease in serum luteinizing hormone (LH) levels, suggesting a role for MPK38 in the age-related changes in serum testosterone in aged mature adult male mice. The stability and activity of MPK38 were increased by dihydrotestosterone but reduced by estradiol (E2). These findings suggest MPK38 as a therapeutic target for obesity-related metabolic disorders in males.
Asunto(s)
Obesidad/genética , Proteínas Serina-Treonina Quinasas/genética , Animales , Glucemia/metabolismo , Dieta Alta en Grasa , Estradiol/sangre , Ácidos Grasos no Esterificados/sangre , Femenino , Prueba de Tolerancia a la Glucosa , Insulina/sangre , Lipogénesis/fisiología , Hormona Luteinizante/sangre , Masculino , Ratones , Ratones Noqueados , Obesidad/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Factores Sexuales , Testosterona/sangreRESUMEN
SHP (small heterodimer partner) is a well-known NR (nuclear receptor) co-regulator. In the present study, we have identified a new SHP-interacting protein, termed SMILE (SHP-interacting leucine zipper protein), which was previously designated as ZF (Zhangfei) via a yeast two-hybrid system. We have determined that the SMILE gene generates two isoforms [SMILE-L (long isoform of SMILE) and SMILE-S (short isoform of SMILE)]. Mutational analysis has demonstrated that the SMILE isoforms arise from the alternative usage of initiation codons. We have confirmed the in vivo interaction and co-localization of the SMILE isoforms and SHP. Domain-mapping analysis indicates that the entire N-terminus of SHP and the middle region of SMILE-L are involved in this interaction. Interestingly, the SMILE isoforms counteract the SHP repressive effect on the transactivation of ERs (estrogen receptors) in HEK-293T cells (human embryonic kidney cells expressing the large T-antigen of simian virus 40), but enhance the SHP-repressive effect in MCF-7, T47D and MDA-MB-435 cells. Knockdown of SMILE gene expression using siRNA (small interfering RNA) in MCF-7 cells increases ER-mediated transcriptional activity. Moreover, adenovirus-mediated overexpression of SMILE and SHP down-regulates estrogen-induced mRNA expression of the critical cell-cycle regulator E2F1. Collectively, these results indicate that SMILE isoforms regulate the inhibition of ER transactivation by SHP in a cell-type-specific manner and act as a novel transcriptional co-regulator in ER signalling.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Isoformas de Proteínas/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Estrógenos/metabolismo , Activación Transcripcional , Animales , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Línea Celular , Factor de Transcripción E2F1/genética , Factor de Transcripción E2F1/metabolismo , Humanos , Leucina Zippers , Ratones , Isoformas de Proteínas/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Receptores de Estrógenos/genética , Distribución Tisular , Transcripción Genética , Técnicas del Sistema de Dos HíbridosRESUMEN
Murine protein serine-threonine kinase 38 (MPK38)/maternal embryonic leucine zipper kinase (MELK), an AMP-activated protein kinase (AMPK)-related kinase, has previously been shown to interact with p53 and to stimulate downstream signaling. p21, a downstream target of p53, is also known to be involved in adipocyte and obesity metabolism. However, little is known about the mechanism by which p21 mediates obesity-associated metabolic adaptation. Here, we identify MPK38 as an interacting partner of p21. p21 and MPK38 interacted through the cyclin-dependent kinase (CDK) binding region of p21 and the C-terminal domain of MPK38. MPK38 potentiated p21-mediated apoptosis and cell cycle arrest in a kinase-dependent manner by inhibiting assembly of CDK2-cyclin E and CDK4-cyclin D complexes via induction of CDK2-p21 and CDK4-p21 complex formation and reductions in complex formation between p21 and its negative regulator mouse double minute 2 (MDM2), leading to p21 stabilization. MPK38 phosphorylated p21 at Thr55, stimulating its nuclear translocation, which resulted in greater association of p21 with peroxisome proliferator-activated receptor γ (PPARγ), preventing the PPARγ transactivation required for adipogenesis. Furthermore, restoration of p21 expression by adenoviral delivery in diet-induced obese mice ameliorated obesity-induced metabolic abnormalities in a MPK38 phosphorylation-dependent manner. These results suggest that MPK38 functions as a positive regulator of p21, regulating apoptosis, cell cycle arrest, and metabolism during obesity.
Asunto(s)
Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Metabolismo Energético , Glucosa/metabolismo , Metabolismo de los Lípidos , Proteínas Serina-Treonina Quinasas/fisiología , Adipocitos/citología , Adipocitos/metabolismo , Animales , Diferenciación Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/química , Ratones , Ratones Obesos , Células 3T3 NIH , Obesidad/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Estabilidad Proteica , Treonina/metabolismoRESUMEN
Smad proteins have been implicated in metabolic processes, but little is known about how they regulate metabolism. Because Smad 2, 3, 4, and 7 have previously been shown to interact with murine protein serine-threonine kinase 38 (MPK38), an AMP-activated protein kinase (AMPK)-related kinase that has been implicated in obesity-associated metabolic defects, we investigated whether Smad proteins regulate metabolic processes via MPK38. Smads2/3/4 increased, but Smad7 decreased, MPK38-mediated apoptosis signal-regulating kinase-1 (ASK1)/transforming growth factor-ß (TGF-ß)/p53 signaling. However, MPK38-mediated phosphorylation-defective Smad mutants (Smad2 S245A, Smad3 S204A, Smad4 S343A, and Smad7 T96A) had no such effect. In addition, Smads2/3/4 increased, but Smad7 decreased, the stability of MPK38. Consistent with this, Smads2/3/4 attenuated complex formation between MPK38 and its negative regulator thioredoxin (Trx), whereas Smad7 increased this complex formation. However, an opposite effect was observed on complex formation between MPK38 and its positive regulator zinc-finger-like protein 9 (ZPR9). When Smads were overexpressed in high-fat diet (HFD)-fed obese mice using an adenoviral delivery system, Smads2/3/4 improved, but Smad7 worsened, obesity-associated metabolic parameters and inflammation in a MPK38 phosphorylation-dependent manner. These findings suggest that Smad proteins have class-specific impacts on obesity-associated metabolism by differentially regulating MPK38 activity in diet-induced obese mice.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Glucosa/efectos adversos , Metabolismo de los Lípidos/efectos de los fármacos , Obesidad/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Smad/metabolismo , Animales , Glucosa/farmacología , Células HEK293 , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Ratones , Obesidad/inducido químicamente , Obesidad/patologíaRESUMEN
Serine-threonine kinase receptor-associated protein (STRAP) is a transforming growth factor ß (TGF-ß) receptor-interacting protein that has been implicated in both cell proliferation and cell death in response to various stresses. However, the precise roles of STRAP in these cellular processes are still unclear. The mechanisms by which STRAP controls both cell proliferation and cell death are now beginning to be unraveled. In addition to its biological roles, this review also focuses on the dual functions of STRAP in cancers displaying redox dysregulation, where it can behave as a tumor suppressor or an oncogene (i.e., it can either inhibit or promote tumor formation), depending on the cellular context. Further studies are needed to define the functions of STRAP and the redox-sensitive intracellular signaling pathways that enhance either cell proliferation or cell death in human cancer tissues, which may help in the development of effective treatments for cancer.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Neoplasias/genética , Animales , Proliferación Celular , Humanos , Ratones , Oxidación-Reducción , Proteínas de Unión al ARN , Transducción de SeñalRESUMEN
Murine protein serine-threonine kinase 38 (MPK38), an AMP-activated protein kinase (AMPK)-related kinase, has been implicated in the induction of apoptosis signal-regulating kinase 1 (ASK1)-, transforming growth factor-ß (TGF-ß)-, and p53-mediated activity involved in metabolic homeostasis. Here, zinc finger protein ZPR9 was found to be an activator of MPK38. The association of MPK38 and ZPR9 was mediated by cysteine residues present in each of these two proteins, Cys269 and Cys286 of MPK38 and Cys305 and Cys308 of ZPR9. MPK38 phosphorylated ZPR9 at Thr252. Wild-type ZPR9, but not the ZPR9 mutant T252A, enhanced ASK1, TGF-ß, and p53 function by stabilizing MPK38. The requirement of ZPR9 Thr252 phosphorylation was validated using CRISPR/Cas9-mediated ZPR9 (T252A) knockin cell lines. The knockdown of endogenous ZPR9 showed an opposite trend, resulting in the inhibition of MPK38-dependent ASK1, TGF-ß, and p53 function. This effect was also demonstrated in mouse embryonic fibroblast (MEF) cells that were haploinsufficient (+/-) for ZPR9, NIH 3T3 cells with inducible knockdown of ZPR9, and CRISPR/Cas9-mediated ZPR9 knockout cells. Furthermore, high-fat diet (HFD)-fed mice displayed reduced MPK38 kinase activity and ZPR9 expression compared to that in mice on control chow, suggesting that ZPR9 acts as a physiological activator of MPK38 that may participate in obesity.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , MAP Quinasa Quinasa Quinasa 5/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Biomarcadores , Regulación de la Expresión Génica/efectos de los fármacos , Peróxido de Hidrógeno/farmacología , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Modelos Biológicos , Proteínas Nucleares/genética , Obesidad/metabolismo , Oxidación-Reducción , Fosfoproteínas/genética , Fosforilación , Unión Proteica , Proteínas Proto-Oncogénicas c-jun/metabolismo , Transcripción GenéticaRESUMEN
The nuclear factor kappa B (NF-κB) pathway is pivotal in controlling survival and apoptosis of cancer cells. Macrophage migration inhibitory factor (MIF), a cytokine that regulates the immune response and tumorigenesis under inflammatory conditions, is upregulated in various tumors. However, the intracellular functions of MIF are unclear. In this study, we found that MIF directly interacted with thioredoxin-interacting protein (TXNIP), a tumor suppressor and known inhibitor of NF-κB activity, and MIF significantly induced NF-κB activation. MIF competed with TXNIP for NF-κB activation, and the intracellular MIF induced NF-κB target genes, including c-IAP2, Bcl-xL, ICAM-1, MMP2 and uPA, by inhibiting the interactions between TXNIP and HDACs or p65. Furthermore, we identified the interaction motifs between MIF and TXNIP via site-directed mutagenesis of their cysteine (Cys) residues. Cys57 and Cys81 of MIF and Cys36 and Cys120 of TXNIP were responsible for the interaction. MIF reversed the TXNIP-induced suppression of cell proliferation and migration. Overall, we suggest that MIF induces NF-κB activity by counter acting the inhibitory effect of TXNIP on the NF-κB pathway via direct interaction with TXNIP. These findings reveal a novel intracellular function of MIF in the progression of cancer.
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Proteínas Portadoras/metabolismo , Factores Inhibidores de la Migración de Macrófagos/metabolismo , FN-kappa B/metabolismo , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/genética , Proliferación Celular/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Células HEK293 , Células HeLa , Humanos , Molécula 1 de Adhesión Intercelular/metabolismo , Lipopolisacáridos/toxicidad , Factores Inhibidores de la Migración de Macrófagos/antagonistas & inhibidores , Factores Inhibidores de la Migración de Macrófagos/genética , Metaloproteinasa 2 de la Matriz/metabolismo , Mutagénesis Sitio-Dirigida , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Transducción de Señal/efectos de los fármacos , Factor de Transcripción ReIA/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Proteína bcl-X/metabolismoRESUMEN
AIMS: To explore the molecular connections between redox-dependent apoptosis signal-regulating kinase 1 (ASK1) and transforming growth factor-ß (TGF-ß) signaling pathways and to examine the physiological processes in which coordinated regulation of these two signaling pathways plays a critical role. RESULTS: We provide evidence that the ASK1 and TGF-ß signaling pathways are interconnected by a multiprotein complex harboring murine protein serine-threonine kinase 38 (MPK38), ASK1, Sma- and Mad-related proteins (SMADs), zinc-finger-like protein 9 (ZPR9), and thioredoxin (TRX) and demonstrate that the activation of either ASK1 or TGF-ß activity is sufficient to activate both the redox-dependent ASK1 and TGF-ß signaling pathways. Physiologically, the restoration of the downregulated activation levels of ASK1 and TGF-ß signaling in genetically and diet-induced obese mice by adenoviral delivery of SMAD3 or ZPR9 results in the amelioration of adiposity, hyperglycemia, hyperlipidemia, and impaired ketogenesis. INNOVATION AND CONCLUSION: Our data suggest that the multiprotein complex linking ASK1 and TGF-ß signaling pathways may be a potential target for redox-mediated metabolic complications.
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Glucosa/metabolismo , Metabolismo de los Lípidos , MAP Quinasa Quinasa Quinasa 5/metabolismo , Complejos Multiproteicos/metabolismo , Obesidad/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Línea Celular , Regulación de la Expresión Génica , Humanos , Ratones , Complejos Multiproteicos/genética , Oxidación-Reducción , Fosforilación , Unión Proteica , Transducción de SeñalRESUMEN
Insulin secretion is known to depend on an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)). However, recent studies have suggested that insulin secretion can also be evoked in a Ca(2+)-independent manner. In the present study we show that treatment of intact mouse islets and RINm5F cells with protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) or protein kinase A (PKA) activator forskolin promoted insulin secretion with no changes of [Ca(2+)](i). Moreover, insulin secretion mediated by PMA or forskolin was maintained even when extracellular or cytosolic Ca(2+) was deprived by treatment of cells with ethylene glycol bis(beta-amino ethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or 1,2-bis(2-amino phenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxy methyl ester) (BAPTA/AM) in RINm5F cells. The secretagogue actions of PMA and forskolin were blocked by GF109203X and H89, selective inhibitors for PKC and PKA, respectively. PMA treatment caused translocation of PKC-alpha and PKC- epsilon from cytosol to membrane, implying that selectively PKC-alpha and PKC- epsilon isoforms might be important for insulin secretion. Co-treatment with high K(+) and PMA showed a comparable level of insulin secretion to that of PMA alone. In addition, PMA and forskolin evoked insulin secretion in cells where Ca(2+)-dependent insulin secretion was completed. Our data suggest that PKC and PKA can elicit insulin secretion not only in a Ca(2+)-sensitive manner but also in a Ca(2+)-independent manner from separate releasable pools.
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Calcio/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Proteína Quinasa C/fisiología , Animales , Línea Celular , Células Cultivadas , Colforsina/farmacología , Exocitosis , Secreción de Insulina , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/enzimología , Isoenzimas/fisiología , Cinética , Ratones , Ratones Endogámicos BALB C , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Small heterodimer partner (SHP; NR0B2) is an atypical orphan nuclear receptor that lacks a conventional DNA binding domain (DBD) and represses the transcriptional activity of various nuclear receptors. In this study, we examined the novel cross talk between SHP and BETA2/NeuroD, a basic helix-loop-helix transcription factor. In vitro and in vivo protein interaction studies showed that SHP physically interacts with BETA2/NeuroD, but not its heterodimer partner E47. Moreover, confocal microscopic study and immunostaining results demonstrated that SHP colocalized with BETA2 in islets of mouse pancreas. SHP inhibited BETA2/NeuroD-dependent transactivation of an E-box reporter, whereas SHP was unable to repress the E47-mediated transactivation and the E-box mutant reporter activity. In addition, SHP repressed the BETA2-dependent activity of glucokinase and cyclin-dependent kinase inhibitor p21 gene promoters. Gel shift and in vitro protein competition assays indicated that SHP inhibits neither dimerization nor DNA binding of BETA2 and E47. Rather, SHP directly repressed BETA2 transcriptional activity and p300-enhanced BETA2/NeuroD transcriptional activity by inhibiting interaction between BETA2 and coactivator p300. We also showed that C-terminal repression domain within SHP is also required for BETA2 repression. However, inhibition of BETA2 activity was not observed by naturally occurring human SHP mutants that cannot interact with BETA2/NeuroD. Taken together, these results suggest that SHP acts as a novel corepressor for basic helix-loop-helix transcription factor BETA2/NeuroD by competing with coactivator p300 for binding to BETA2/NeuroD and by its direct transcriptional repression function.
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Proteínas de Unión al ADN/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Transactivadores/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Células COS , Dimerización , Proteína p300 Asociada a E1A , Regulación de la Expresión Génica/fisiología , Humanos , Ratones , Mutación , Proteínas Nucleares/metabolismo , Estructura Terciaria de Proteína , Receptores Citoplasmáticos y Nucleares/genética , Factores de Transcripción TCF , Proteína 1 Similar al Factor de Transcripción 7 , Factores de Transcripción/metabolismo , Activación TranscripcionalRESUMEN
Small heterodimer partner (SHP; NR0B2) is an atypical orphan nuclear receptor and acts as a coregulator of various nuclear receptors. Herein, we examined a novel cross talk between SHP and a forkhead transcription factor HNF3 (hepatocyte nuclear factor 3/Foxa. Transient transfection assay demonstrated that SHP inhibited the transcriptional activity of all three isoforms of HNF3, HNF3alpha, beta, and gamma. In vivo and in vitro protein interaction studies showed that SHP physically interacted with HNF3. Adenovirus-mediated overexpression of SHP significantly decreased the mRNA levels of glucose-6-phosphase (G6Pase), cholesterol 7-alpha-hydroxylase (CYP7A1), and phosphoenolpyruvate carboxykinase (PEPCK) in HepG2 cells and rat primary hepatocytes. Moreover, the mRNA level of G6Pase was notably increased by down-regulation of SHP with small interfering RNA. Interestingly, HNF3 transactivity was still repressed by SHPDelta128-139 that fails to repress nuclear receptors. Mapping of interaction domain revealed that SHP interacted with forkhead DNA binding domain of HNF3alpha. Gel mobility shift and chromatin immunoprecipitation assays demonstrated that SHP inhibits DNA binding of HNF3. These results suggest that SHP is involved in the regulation of G6Pase, CYP7A1, and PEPCK gene expression via novel mechanism of inhibition of HNF3 activity and expand the role of SHP as a coregulator of other family of transcription factors in addition to nuclear receptors.