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1.
Mol Cells ; 42(1): 36-44, 2019 Jan 31.
Artículo en Inglés | MEDLINE | ID: mdl-30622228

RESUMEN

Alzheimer's disease (AD) is the most frequent age-related human neurological disorder. The characteristics of AD include senile plaques, neurofibrillary tangles, and loss of synapses and neurons in the brain. ß-Amyloid (Aß) peptide is the predominant proteinaceous component of senile plaques. The amyloid hypothesis states that Aß initiates the cascade of events that result in AD. Amyloid precursor protein (APP) processing plays an important role in Aß production, which initiates synaptic and neuronal damage. δ-Catenin is known to be bound to presenilin-1 (PS-1), which is the main component of the γ-secretase complex that regulates APP cleavage. Because PS-1 interacts with both APP and δ-catenin, it is worth studying their interactive mechanism and/or effects on each other. Our immunoprecipitation data showed that there was no physical association between δ-catenin and APP. However, we observed that δ-catenin could reduce the binding between PS-1 and APP, thus decreasing the PS-1 mediated APP processing activity. Furthermore, δ-catenin reduced PS-1-mediated stabilization of APP. The results suggest that δ-catenin can influence the APP processing and its level by interacting with PS-1, which may eventually play a protective role in the degeneration of an Alzheimer's disease patient.


Asunto(s)
Precursor de Proteína beta-Amiloide/metabolismo , Cateninas/metabolismo , Presenilina-1/metabolismo , Animales , Línea Celular , Línea Celular Tumoral , Humanos , Ratones , Unión Proteica , Procesamiento Proteico-Postraduccional , Estabilidad Proteica , Catenina delta
2.
Mol Cell Endocrinol ; 413: 217-27, 2015 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-26159900

RESUMEN

Yin Yang 1 (YY1) is an ubiquitously distributed transcription factor that belongs to the GLI-Kruppel class of zinc finger proteins. The mechanism by which YY1 regulates adipocyte differentiation remains unclear. In this study, we investigated the functional role of YY1 during adipocyte differentiation. During the early stage, YY1 gene and protein expression was transiently downregulated upon the induction of differentiation, however, it was consistently induced during the later stage. YY1 overexpression decreased adipocyte differentiation and blocked cell differentiation at the preadipocyte stage, while YY1 knockdown by RNA interference increased adipocyte differentiation. YY1 physically interacted with PPARγ (Peroxisome proliferator-activated receptor gamma) and C/EBPß (CCAAT/enhancer-binding protein beta) respectively in 3T3-L1 cells. Through its interaction with PPARγ, YY1 directly decreased PPARγ transcriptional activity. YY1 ectopic expression prevented C/EBPß from binding to the PPARγ promoter, resulting in the downregulation of PPARγ transcriptional activity. These results indicate that YY1 repressed adipocyte differentiation by repressing the activity of adipogenic transcriptional factors in 3T3-L1 cells.


Asunto(s)
Adipocitos/metabolismo , Diferenciación Celular/fisiología , Regulación de la Expresión Génica/fisiología , Transcripción Genética/fisiología , Factor de Transcripción YY1/metabolismo , Células 3T3-L1 , Adipocitos/citología , Animales , Ratones , PPAR gamma/genética , PPAR gamma/metabolismo , Factor de Transcripción YY1/genética
3.
Bone ; 75: 201-9, 2015 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-25744063

RESUMEN

E3 ubiquitin ligase Cbl-b and c-Cbl play important roles in bone formation and maintenance. Cbl-b and c-Cbl regulate the activity of various receptor tyrosine kinases and intracellular protein tyrosine kinases mainly by regulating the degradation of target proteins. However, the precise mechanisms of how Cbl-b and c-Cbl regulate osteoblast differentiation are not well known. In this study, we investigated potential targets of Cbl-b and c-Cbl. We found that Cbl-b and c-Cbl inhibit BMP2-induced osteoblast differentiation in mesenchymal cells. Among various osteogenic transcription factors, we identified that Cbl-b and c-Cbl suppress the protein stability and transcriptional activity of Osterix. Our results suggest that Cbl-b and c-Cbl inhibit the function of Osterix by enhancing the ubiquitin-proteasome-mediated degradation of Osterix. Taken together, we propose novel regulatory roles of Cbl-b and c-Cbl during osteoblast differentiation in which Cbl-b and c-Cbl regulate the degradation of Osterix through the ubiquitin-proteasome pathway.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Diferenciación Celular/fisiología , Osteoblastos/citología , Proteínas Proto-Oncogénicas c-cbl/metabolismo , Factores de Transcripción/metabolismo , Animales , Línea Celular , Humanos , Immunoblotting , Inmunoprecipitación , Ratones , Osteogénesis/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción Sp7 , Transfección , Ubiquitinación
4.
Biochem Biophys Res Commun ; 453(3): 557-62, 2014 Oct 24.
Artículo en Inglés | MEDLINE | ID: mdl-25285640

RESUMEN

SIRT2 is a member of the sirtuin family of NAD(+)-dependent protein deacetylases. It is involved in metabolic homeostasis and has been linked to the progression of age-related diseases. Casitas B-lineage lymphoma (Cbl) proteins regulate signal transduction through many pathways and, consequently, regulate cell function and development. Cbl proteins are ubiquitin ligases that ubiquitinate and target many signaling molecules for degradation. The function of SIRT2 is modulated by post-translational modifications. However, the precise molecular signaling mechanism of SIRT2 through interactions with Cbl proteins has not yet been established. In this study, we investigated the potential regulation of SIRT2 function by the Cbl mammalian family members Cbl-b and c-Cbl. We found that Cbl-b and c-Cbl increased the protein level and stability of SIRT2 and that Cbl-b and c-Cbl interact with SIRT2. They were also found to regulate the deacetylase activity of SIRT2. Further investigation revealed that Cbl-mediated SIRT2 regulation occurred via ubiquitination of SIRT2.


Asunto(s)
Proteínas Proto-Oncogénicas c-cbl/fisiología , Sirtuina 2/fisiología , Línea Celular , Humanos , Procesamiento Proteico-Postraduccional , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitinación
5.
Biochem Biophys Res Commun ; 450(2): 1120-5, 2014 Jul 25.
Artículo en Inglés | MEDLINE | ID: mdl-24996174

RESUMEN

SIRT2 is a mammalian member of the Sirtuin family of NAD(+)-dependent protein deacetylases. The tyrosine kinase Src is involved in a variety of cellular signaling pathways, leading to the induction of DNA synthesis, cell proliferation, and cytoskeletal reorganization. The function of SIRT2 is modulated by post-translational modifications; however, the precise molecular signaling mechanism of SIRT2 through interactions with c-Src has not yet been established. In this study, we investigated the potential regulation of SIRT2 function by c-Src. We found that the protein levels of SIRT2 were decreased by c-Src, and subsequently rescued by the addition of a Src specific inhibitor, SU6656, or by siRNA-mediated knockdown of c-Src. The c-Src interacts with and phosphorylates SIRT2 at Tyr104. c-Src also showed the ability to regulate the deacetylation activity of SIRT2. Investigation on the phosphorylation of SIRT2 suggested that this was the method of c-Src-mediated SIRT2 regulation.


Asunto(s)
Sirtuina 2/metabolismo , Familia-src Quinasas/metabolismo , Proteína Tirosina Quinasa CSK , Células HEK293 , Humanos , Células MCF-7 , Fosforilación
6.
FEBS J ; 281(16): 3656-66, 2014 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-24961731

RESUMEN

Runx2 plays essential roles in bone formation and chondrocyte maturation. Akt promotes osteoblast differentiation induced by the bone morphogenetic proteins BMP2 and enhances the function and transcriptional activity of Runx2. However, the precise molecular mechanism underlying the relationship between Runx2 and Akt is not well understood. In this study, we examined the role of Akt in regulating Runx2 function. We found that Akt increases the stability of Runx2 protein. However, the level of Runx2 mRNA was not affected by Akt, and we did not find any evidence for direct modification of Runx2 by Akt. Instead, we found evidence that Akt induces the phosphorylation of the Smad ubiquitination regulatory factor Smurf2 and decreases the level of Smurf2 protein through ubiquitin/proteasome-mediated degradation of Smurf2. Akt also alleviates Smurf2-mediated suppression of Runx2 transcriptional activity. Taken together, our results suggest that Akt regulates osteoblast differentiation, at least in part, by enhancing the protein stability and transcriptional activity of Runx2 through regulation of ubiquitin/proteasome-mediated degradation of Smurf2.


Asunto(s)
Diferenciación Celular , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Osteoblastos/fisiología , Proteínas Proto-Oncogénicas c-akt/fisiología , Ubiquitina-Proteína Ligasas/fisiología , Animales , Células HEK293 , Humanos , Ratones , Fosforilación , Unión Proteica , Mapas de Interacción de Proteínas , Estabilidad Proteica , Proteolisis , Transcripción Genética , Ubiquitinación
7.
Biochem Biophys Res Commun ; 437(2): 245-9, 2013 Jul 26.
Artículo en Inglés | MEDLINE | ID: mdl-23806683

RESUMEN

SIRT2 is a mammalian member of the Sirtuin family of NAD-dependent protein deacetylases. The function of SIRT2 can be modulated by post-translational modification. However, the precise molecular signaling mechanisms of SIRT2 and extracellular signal-regulated kinase (ERK)1/2 have not been correlated. We investigated the potential regulation of SIRT2 function by ERK1/2. ERK activation by the over-expression of constitutively active MEK increased protein levels and enhanced the stability of SIRT2. In contrast, U0126, an inhibitor of mitogen-activated kinase kinase, suppressed SIRT2 protein level. ERK1/2 interacted with SIRT2 exogenously and endogenously. Deacetylase activity of SIRT2 was up-regulated in an ERK1/2-mediated manner. These results suggest that ERK1/2 regulates SIRT2 by increasing the protein levels, stability and activity of SIRT2.


Asunto(s)
Sistema de Señalización de MAP Quinasas , Sirtuina 2/metabolismo , Activación Enzimática , Estabilidad de Enzimas , Células HEK293 , Humanos
8.
Biochim Biophys Acta ; 1823(8): 1225-32, 2012 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-22633971

RESUMEN

Protein kinase C (PKC) signaling regulates osteoblast differentiation, but little is known about its downstream effectors. We examined the effect of modulating PKC activity on osteogenic transcription factors and found that the protein level of Msx2 is affected. Msx2 is induced by osteogenic signals such as BMPs and it plays critical roles in bone formation and osteoblast differentiation. Here, we examined the role of PKC signaling in regulating the function of Msx2. We found that the inhibition of PKC signaling enhances osteogenic differentiation in BMP2-stimulated C2C12 cells. Treatment with inhibitors of PKC activity or overexpression of kinase-defective (KD), dominant-negative mutant PKC isoforms strongly reduced the level of Msx2 protein. Several PKC isoforms (α, ß, δ, and ζ) interacted with Msx2, and PKCß phosphorylated Msx2 at Thr135 and Thr141. Msx2 repressed the transcriptional activity of the osteogenic transcription factor Runx2, and this repression was relieved by inhibition of PKC activity or overexpression of the KD mutant PKC isoforms. In addition, PKC prolonged the half-life of Msx2 protein. These results suggest that PKC signaling modulates osteoblast differentiation, at least in part, through the regulation of Msx2.


Asunto(s)
Diferenciación Celular , Proteínas de Homeodominio/metabolismo , Proteína Quinasa C/fisiología , Transducción de Señal , Fosfatasa Alcalina/metabolismo , Animales , Proteína Morfogenética Ósea 2/fisiología , Línea Celular , Proliferación Celular , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Expresión Génica , Semivida , Proteínas de Homeodominio/química , Proteínas de Homeodominio/genética , Humanos , Ratones , Osteogénesis , Fosforilación , Proteína Quinasa C/química , Proteína Quinasa C/metabolismo , Proteína Quinasa C beta , Inhibidores de Proteínas Quinasas/farmacología , Procesamiento Proteico-Postraduccional , Estabilidad Proteica , Activación Transcripcional , Ubiquitinación
9.
Arch Pharm Res ; 34(8): 1381-7, 2011 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-21910061

RESUMEN

Anticoagulation therapy with vitamin K antagonists such as warfarin is widely used to prevent and treat stroke in patients with chronic atrial fibrillation or mechanical heart valves. Because vitamin K is an essential factor for ggg-carboxylation of osteocalcin, vitamin K antagonists might cause bone loss. Although the association between warfarin use and bone metabolism is still controversial, several studies show that bone mineral density is decreased and fracture risk is increased with warfarin therapy. Meanwhile, attenuation of gap junctional communication (GJC) by warfarin is reported in rat liver epithelial cells. However, the effect of warfarin on osteoblasts, in which GJC is important for osteoblastic differentiation, remains unknown. Here we investigated whether warfarin has an inhibitory effect on osteoblastic differentiation using an osteoblastic cell line (C2C12). Warfarin and 18-α-glycyrrhetinic acid (AGA), which is known as a nontoxic reversible GJC inhibitor, had the same effect on osteoblastic differentiation. Warfarin and AGA inhibited the bone morphogenetic protein (BMP)2-induced mRNA levels of alkaline phosphatase (ALP), collagen I α1, osteocalcin (OC) and osterix, which are specific markers for osteoblastic differentiation, in a dose-dependent manner. Moreover, the activities of OC- and ALP-luciferase reporters, which are induced by BMP2, and the transcriptional activity of Runx2 on OC and ALP promoters were inhibited by warfarin and AGA. The amount and activity of ALP induced by BMP2 were also decreased by warfarin and AGA. These results suggest that warfarin and AGA, a GJC inhibitor, have an inhibitory effect on osteoblastic differentiation.


Asunto(s)
Anticoagulantes/farmacología , Diferenciación Celular/efectos de los fármacos , Ácido Glicirretínico/farmacología , Osteoblastos/efectos de los fármacos , Warfarina/farmacología , Fosfatasa Alcalina/metabolismo , Animales , Proteína Morfogenética Ósea 2/metabolismo , Línea Celular , Células Epiteliales , Uniones Comunicantes/metabolismo , Hígado , Luciferasas/metabolismo , Osteoblastos/citología , Osteoblastos/metabolismo , Osteocalcina/metabolismo , Ratas , Transfección
10.
Biochem Biophys Res Commun ; 411(3): 637-41, 2011 Aug 05.
Artículo en Inglés | MEDLINE | ID: mdl-21777568

RESUMEN

Osterix (Osx), a zinc-finger transcription factor is required for osteoblast differentiation and new bone formation during embryonic development. Akt is a member of the serine/threonine-specific protein kinase and plays important roles in osteoblast differentiation. The function of Osterix can be also modulated by post-translational modification. But, the precise molecular signaling mechanisms between Osterix and Akt are not known. In this study, we investigated the potential regulation of Osterix function by Akt in osteoblast differentiation. We found that Akt phosphorylates Osterix and that Akt activation increases protein stability, osteogenic activity and transcriptional activity of Osterix. We also found that BMP-2 increases the protein level of Osterix in an Akt activity-dependent manner. These results suggest that Akt activity enhances the osteogenic function of Osterix, at least in part, through protein stabilization and that BMP-2 regulates the osteogenic function of Osterix, at least in part, through Akt.


Asunto(s)
Osteoblastos/fisiología , Osteogénesis , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factores de Transcripción/metabolismo , Proteína Morfogenética Ósea 2/metabolismo , Diferenciación Celular , Células HEK293 , Humanos , Osteoblastos/citología , Fosforilación , Estabilidad Proteica , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Factor de Transcripción Sp7 , Factores de Transcripción/química , Factores de Transcripción/genética , Transcripción Genética
11.
Biochem Biophys Res Commun ; 409(4): 681-6, 2011 Jun 17.
Artículo en Inglés | MEDLINE | ID: mdl-21619873

RESUMEN

Akt, a phosphoinositide-dependent serine/threonine protein kinase, acts as a key regulator in bone formation. Akt can be activated by several osteogenic signaling molecules, but its precise function and downstream targets in bone development are unknown. Dlx5 transcription factor plays important roles during bone development and osteoblast differentiation. Its expression is regulated by several osteogenic signals. In addition, Dlx5 function is also regulated through post-translational modification by several kinases. In this report, we have investigated a potential regulation of Dlx5 function by Akt. Our results indicate that Akt interacts with and phosphorylates Dlx5. In addition, we provide evidences that Akt kinase activity is important for Akt to enhance the protein stability and transcriptional activity of Dlx5. These results suggest that Dlx5 is a novel target of Akt and that the activity of Dlx5 could be modulated by a novel mechanism involving Akt during osteoblast differentiation.


Asunto(s)
Proteínas de Homeodominio/metabolismo , Osteoblastos/citología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Diferenciación Celular/genética , Línea Celular , Humanos , Ratones , Osteoblastos/metabolismo , Osteogénesis/genética , Fosforilación , Procesamiento Proteico-Postraduccional , Estabilidad Proteica , Transcripción Genética
12.
Biochem Biophys Res Commun ; 409(1): 82-9, 2011 May 27.
Artículo en Inglés | MEDLINE | ID: mdl-21565172

RESUMEN

Xanthohumol (XN), the principal prenylated flavonoid from the hop plant, an additive that contributes bitterness and flavor to beer, is known to be a potent phytoestrogen. Although XN has been identified as a chemopreventive agent and as an anti-infective agent, its effects on bone are unknown. In the present study, the effects of XN on osteoblast differentiation and function were determined by analyzing the activity of alkaline phosphatase (ALP), an osteoblast marker, and the regulation of RUNX2, a master gene of osteoblast differentiation, in a mesenchymal stem cell line. XN upregulated ALP activity and the expression of osteogenic marker genes. Additionally, XN increased the expression and transcriptional activity of RUNX2. To determine which signaling pathways are involved in the osteogenic effects of XN, we tested the effect of inhibitors of kinases known to regulate RUNX2. Enhancement of the transcriptional activity and expression of RUNX2 were inhibited by treatment with a p38 and an ERK inhibitor. These findings suggest that XN stimulates osteoblast differentiation by activation of RUNX2 via mechanisms related to the p38 MAPK and ERK signaling pathway. Regulation of RUNX2 activation by XN may be an important therapeutic target for osteoporosis.


Asunto(s)
Diferenciación Celular/efectos de los fármacos , Subunidad alfa 1 del Factor de Unión al Sitio Principal/agonistas , Flavonoides/farmacología , Humulus/química , Osteoblastos/citología , Osteogénesis/efectos de los fármacos , Propiofenonas/farmacología , Diferenciación Celular/genética , Línea Celular , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Flavonoides/aislamiento & purificación , Humanos , Osteoblastos/metabolismo , Osteogénesis/genética , Propiofenonas/aislamiento & purificación , Transcripción Genética
13.
Biochem Biophys Res Commun ; 407(3): 461-5, 2011 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-21406180

RESUMEN

Dlx5 transcription factor plays important roles in osteoblast differentiation and its transcription is regulated by many osteogenic signals including BMP-2. Recent studies suggest that the function of Dlx5 is also regulated post-translationally by protein kinases such as p38 and CaMKII. Protein kinase A (PKA) is involved in several steps of osteoblast differentiation and its activity has been shown necessary, yet not sufficient, for BMP-induced osteoblast differentiation. PKA is a ubiquitous cellular kinase that phosphorylates serine and threonine residues(s) of target proteins. In this study, we investigated the potential regulation of Dlx5 function by PKA in osteoblast differentiation. We found that PKA phosphorylates Dlx5 and that PKA activation increases the protein stability, osteogenic activity and transcriptional activity of Dlx5. We also found that BMP-2 increases the protein level of Dlx5 in a PKA activity-dependent manner. These results suggest that PKA activity enhances the osteogenic function of Dlx5, at least in part, through protein stabilization and that BMP-2 regulates the osteogenic function of Dlx5, at least in part, through PKA.


Asunto(s)
Diferenciación Celular , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas de Homeodominio/metabolismo , Osteoblastos/citología , Osteogénesis , Animales , Proteína Morfogenética Ósea 2/metabolismo , Diferenciación Celular/genética , Línea Celular , Proteínas de Homeodominio/genética , Humanos , Ratones , Osteoblastos/metabolismo , Fosforilación , Procesamiento Proteico-Postraduccional , Estabilidad Proteica , Transcripción Genética
14.
Biochem Biophys Res Commun ; 383(1): 88-92, 2009 May 22.
Artículo en Inglés | MEDLINE | ID: mdl-19344692

RESUMEN

Protein acetyltransferases and deacetylases affect the activities of each other. This is well documented by the acetylation and inhibition of HDAC1 by p300, a transcriptional co-activator with protein acetyltransferase activity. However, the relationship between HDAC6 and p300 is poorly understood. HDAC6 is a class II histone deacetylase and differs from other members of HDAC family in that it contains two HDAC domains and an ubiquitin-binding motif. HDAC6 is a microtubule-associated deacetylase. It predominantly deacetylates non-histone proteins, including alpha-tubulin, and regulates cell motility. Here, we report that p300 interacts with and acetylates HDAC6 resulting down-regulation of HDAC6 deacetylase activity. Furthermore, we provide evidences that acetylation of HDAC6 by p300 inhibits tubulin deacetylation and suppression of Sp1 transcriptional activity by HDAC6. Our results demonstrate that p300 can inactivate HDAC6 by acetylation, and that p300 may regulate the activity of Sp1 indirectly through HDAC6 in addition to its direct modification of Sp1.


Asunto(s)
Histona Desacetilasas/metabolismo , Factores de Transcripción p300-CBP/metabolismo , Acetilación , Línea Celular , Regulación de la Expresión Génica , Genes Reporteros , Histona Desacetilasa 6 , Histona Desacetilasas/genética , Humanos , Luciferasas/química , Mapeo de Interacción de Proteínas , Factor de Transcripción Sp1/metabolismo , Proteínas de Unión al GTP rho/genética
15.
Biochem Biophys Res Commun ; 384(1): 100-4, 2009 Jun 19.
Artículo en Inglés | MEDLINE | ID: mdl-19393622

RESUMEN

Calmodulin-dependent kinase II (CaMKII) acts as a key regulator of osteoblast differentiation. CaMKII is a Ca(2+)-activated serine/threonine kinase and it regulates the activity of target proteins by phosphorylation. Dlx5 transcription factor plays crucial roles in osteoblast differentiation. The expression of Dlx5 is regulated by several osteogenic signaling pathways from early stages of osteoblastogenesis. In addition, Dlx5 can be phosphorylated and activated by p38, suggesting that the function of Dlx5 can be also modulated by post-translational modification. Although CaMKII and Dlx5 both play crucial roles during osteoblast differentiation, the interaction between CaMKII and Dlx5 has not been investigated. In the current study, we examined the effects CamKII on the function of Dlx5. We found that CaMKII phosphorylates Dlx5, and that CaMKII increases the protein stability and the osteoblastogenic transactivation activity of Dlx5. Conversely, a CaMKII inhibitor KN-93 decreased the osteogenic and transactivation activities of Dlx5. These results indicate that CaMKII regulates osteoblast differentiation, at least in part, by increasing the protein stability and the transcriptional activity of Dlx5.


Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Diferenciación Celular , Proteínas de Homeodominio/metabolismo , Osteoblastos/citología , Activación Transcripcional , Animales , Proteína Morfogenética Ósea 2/farmacología , Diferenciación Celular/genética , Línea Celular , Humanos , Ratones , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo
16.
Mol Cells ; 27(1): 15-9, 2009 Jan 31.
Artículo en Inglés | MEDLINE | ID: mdl-19214430

RESUMEN

Notch signaling is controlled at multiple levels. In particular, stabilized Notch receptor activation directly affects the transcriptional activations of Notch target genes. Although some progress has been made in terms of defining the regulatory mechanism that alters Notch stability, it has not been determined whether Notch1/NICD stability is regulated by GSK-3alpha. Here, we show that Notch1/NICD levels are significantly regulated by GSK-3beta and by GSK-3alpha. Treatment with LiCl (a specific GSK-3 inhibitor) or the overexpression of the kinase-inactive forms of GSK-3alpha/beta significantly increased Notch1/NICD levels. Endogenous NICD levels were also increased by either GSK-3alpha/beta- or GSK-3alpha-specific siRNA. Furthermore, it was found that GSK-3alpha binds to Notch1. Deletion analysis showed that at least three Thr residues in Notch1 (Thr-1851, 2123, and 2125) are critical for its response to LiCl, which increased not only the transcriptional activity of endogenous NICD but also Hes1 mRNA levels. Taken together, our results indicate that GSK-3alpha is a negative regulator of Notch1/NICD.


Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Proteínas de Homeodominio/metabolismo , Receptor Notch1/química , Receptor Notch1/metabolismo , Animales , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3 beta , Humanos , Ratones , Unión Proteica , Estructura Terciaria de Proteína , Receptor Notch1/genética , Treonina/metabolismo , Factor de Transcripción HES-1 , Transcripción Genética
17.
Biochim Biophys Acta ; 1793(2): 290-9, 2009 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-19000719

RESUMEN

Wnt and Notch1 signaling pathways play an important role in a variety of biological processes including embryonic induction, the polarity of cell division, cell fate, and cell growth. Although there is evidence that the two main signaling pathways can modulate each other, the precise mechanism is not completely understood. This report shows that beta-catenin can regulate the level and transcriptional activity of the Notch1 and Notch1 intracellular domain (NICD). The in vivo and in vitro results demonstrate that beta-catenin binds with Notch1 and NICD, for which its Armadillo repeat domain is essential. It was further demonstrated that beta-catenin could upregulate the level of Notch1 and NICD, possibly by competing the common ubiquitin-dependent degradation machinery. In addition, beta-catenin enhanced the transcriptional activity of NICD on the hairy and enhancer of split 1 (HES1) and CSL through its C-terminal transactivation domain. This effect of cooperative regulation by beta-catenin could also be observed in bone morphogenetic protein 2 (BMP2) induced osteogenic differentiation of C2C12 cells. beta-catenin coexpression with NICD enhanced the alkaline phosphatase (ALP) activity in C2C12 cells compared with either beta-catenin or NICD expression alone. Culturing C2C12 cells on Delta-1 coated dishes together with Wnt3-conditioned media induced noticeable increases in ALP staining, verifying that employed physiological levels of NICD and beta-catenin are sufficient to induce ALP activation. Furthermore, effects of beta-catenin on Notch1 were dramatically diminished by overexpressed LEF1. Overall, our data suggest that beta-catenin can act as a switching molecule between the classical TCF/LEF1 mediated pathway and NICD mediated pathway.


Asunto(s)
Receptores Notch/genética , Receptores Notch/metabolismo , Transcripción Genética , beta Catenina/metabolismo , Fosfatasa Alcalina/metabolismo , Animales , Proteína Morfogenética Ósea 2/farmacología , Línea Celular , Activación Enzimática/efectos de los fármacos , Genes Reporteros , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Factor de Unión 1 al Potenciador Linfoide/metabolismo , Ratones , Regiones Promotoras Genéticas , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Estabilidad Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Receptores Notch/química , Transcripción Genética/efectos de los fármacos , Activación Transcripcional/efectos de los fármacos , Ubiquitinación/efectos de los fármacos , beta Catenina/química
18.
Biochem Biophys Res Commun ; 375(4): 576-80, 2008 Oct 31.
Artículo en Inglés | MEDLINE | ID: mdl-18722353

RESUMEN

Histone deacetylases (HDACs) are subdivided into three classes--HDAC I, HDAC II, and Sir2. Sirt proteins are mammalian members of the Sir2 family of NAD+ (nicotinamide adenine dinucleotide)-dependent protein deacetylases. The balance between acetylation and deacetylation of histone and non-histone proteins, regulated by protein acetyltransferases and deacetylases, affects the expression of genes involved in a variety of cellular processes. In addition, HDAC1 is acetylated and regulated by p300, a transcriptional co-activator with protein acetyltransferase activity, suggesting that protein acetyltransferases and deacetylases they control the activities of each other. Although the regulation of HDAC1 by p300 is well characterized, the relationship between Sir2 homologs and p300 is not understood. Here, we report that p300 interacts with Sirt2, a member of the Sir2 family, and triggers the acetylation and subsequent down-regulation of the deacetylation activity of Sirt2, and that the acetylation of Sirt2 by p300 relieves the inhibitory effect of Sirt2 on the transcriptional activity of p53. These observations demonstrate that p300 can inactivate Sirt2 by acetylation and that p300 may regulate the activity of p53 indirectly through Sirt2 in addition to its direct modification of p53.


Asunto(s)
Inhibidores de Histona Desacetilasas , Histona Desacetilasas/metabolismo , Sirtuinas/antagonistas & inhibidores , Sirtuinas/metabolismo , Factores de Transcripción p300-CBP/metabolismo , Acetilación , Línea Celular , Regulación hacia Abajo , Humanos , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína , Sirtuina 2 , Sirtuinas/genética , Transcripción Genética , Proteína p53 Supresora de Tumor/metabolismo , Factores de Transcripción p300-CBP/genética
19.
Biochem Biophys Res Commun ; 368(3): 690-5, 2008 Apr 11.
Artículo en Inglés | MEDLINE | ID: mdl-18249187

RESUMEN

Sirt2 is a mammalian member of the Sirtuin family of NAD(+) (nicotinamide adenine dinucleotide)-dependent protein deacetylases. Although Sir-2.1 (a Caenorhabditis elegans Sirt2 ortholog) has been reported to interact with PAR-5/FTT-2 (a C. elegans 14-3-3 homolog), the molecular significance of the interaction between Sirt2 and 14-3-3 proteins in mammalian cell is not understood. Here, we report that Sirt2 interacts with 14-3-3 beta and gamma among various 14-3-3 isoforms, and that this interaction is strengthened by AKT. Furthermore, Sirt2 deacetylates and down-regulates the transcriptional activity of p53, and 14-3-3 beta/gamma augment deacetylation and down-regulation of the p53 transcriptional activity by Sirt2 in an AKT-dependent manner. Treatment of cells with nicotinamide, an inhibitor of Sirtuins, relieves the inhibition of p53 by Sirt2 and 14-3-3 beta/gamma. Therefore, our results suggest that the interaction between Sirt2 and 14-3-3 beta/gamma is a novel mechanism for the negative regulation of p53 beside the well-characterized Mdm2-mediated repression.


Asunto(s)
Proteínas 14-3-3/metabolismo , Riñón/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/fisiología , Sirtuinas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Línea Celular , Regulación hacia Abajo , Humanos , Sirtuina 2
20.
Biochem Biophys Res Commun ; 351(4): 903-8, 2006 Dec 29.
Artículo en Inglés | MEDLINE | ID: mdl-17097608

RESUMEN

Although delta-catenin/neural plakophilin-related armadillo protein (NPRAP) was reported to interact with presenilin-1 (PS-1), the effects of PS-1 on delta-catenin have not been established. In this study, we report that overexpression of PS-1 inhibits the delta-catenin-induced dendrite-like morphological changes in NIH 3T3 cells and promotes delta-catenin processing and turnover. The effects of PS-1 on endogenous delta-catenin processing were confirmed in hippocampal neurons overexpressing PS-1, as well as in the transgenic mice expressing the disease-causing mutant PS-1 (M146V). In addition, disease-causing mutant PS-1 (M146V and L286V) enhanced delta-catenin processing, whereas PS-1/gamma-secretase inhibitors could block the formation of processed forms of delta-catenin. Together, our findings suggest that PS-1 can affect delta-catenin-induced morphogenesis possibly through the regulation of its processing and stability.


Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Dendritas/ultraestructura , Fosfoproteínas/metabolismo , Presenilina-1/metabolismo , Enfermedad de Alzheimer/enzimología , Enfermedad de Alzheimer/genética , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Animales , Cateninas , Moléculas de Adhesión Celular/genética , Dendritas/enzimología , Inhibidores Enzimáticos/farmacología , Fibroblastos/enzimología , Hipocampo/citología , Hipocampo/enzimología , Ratones , Ratones Transgénicos , Células 3T3 NIH , Neuronas/metabolismo , Fosfoproteínas/genética , Presenilina-1/antagonistas & inhibidores , Presenilina-1/genética , Activación Transcripcional , Catenina delta
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