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1.
Cancer Cell ; 10(3): 191-202, 2006 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-16959611

RESUMEN

This article investigates the mechanistic aspects of mutant p53 "gain of function" in response to DNA damage. We show that mutant forms of p53 protein interact with NF-Y. The expression of cyclin A, cyclin B1, cdk1, and cdc25C, as well as the cdk1-associated kinase activities, is upregulated after DNA damage, provoking a mutant p53/NF-Y-dependent increase in DNA synthesis. Mutant p53 binds NF-Y target promoters and, upon DNA damage, recruits p300, leading to histone acetylation. The recruitment of mutant p53 to the CCAAT sites is severely impaired upon abrogation of NF-YA expression. Endogenous NF-Y, mutant p53, and p300 proteins form a triple complex upon DNA damage. We demonstrate that aberrant transcriptional regulation underlies the ability of mutant p53 proteins to act as oncogenic factors.


Asunto(s)
Factor de Unión a CCAAT/metabolismo , Ciclo Celular , Transcripción Genética/genética , Proteína p53 Supresora de Tumor/metabolismo , Secuencia de Bases , Factor de Unión a CCAAT/genética , Línea Celular Tumoral , Daño del ADN/genética , Humanos , Mutación/genética , Unión Proteica , Proteína p53 Supresora de Tumor/genética , Factores de Transcripción p300-CBP/metabolismo
2.
Am J Pathol ; 174(3): 1037-47, 2009 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-19179604

RESUMEN

The growth factor proepithelin has recently emerged as an important regulator of transformation in several physiological and pathological systems. In this study, we determined the biological roles of proepithelin in prostate cancer cells using purified human recombinant proepithelin as well as proepithelin-depletion strategies. Proepithelin promoted the migration of androgen-dependent and -independent human prostate cancer cells; androgen-independent DU145 cells were the more responsive. In these cells, proepithelin additionally stimulated wound closure, invasion, and promotion of cell growth in vitro. These effects required the activation of both the Akt and mitogen-activated protein kinase pathways. We have analyzed proepithelin expression levels in different available prostate cancer microarray studies using the Oncomine database and found a statistically significant increase in proepithelin mRNA expression levels in prostate cancers compared with nonneoplastic controls. Notably, depletion of endogenous proepithelin by siRNA and antisense strategies impaired the ability of DU145 cells to grow and migrate after serum withdrawal and inhibited anchorage-independent growth. Our results provide the first evidence for a role of proepithelin in stimulating the migration, invasion, proliferation, and anchorage-independent growth of prostate cancer cells. This study supports the hypothesis that proepithelin may play a critical role as an autocrine growth factor in the establishment and initial progression of prostate cancer. Furthermore, proepithelin may prove to be a useful clinical marker for the diagnosis of prostate tumors.


Asunto(s)
Péptidos y Proteínas de Señalización Intercelular/fisiología , Neoplasias de la Próstata/patología , División Celular , Línea Celular Tumoral , Movimiento Celular , Silenciador del Gen , Granulinas , Homeostasis , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Masculino , Invasividad Neoplásica , Progranulinas , Neoplasias de la Próstata/genética , Precursores de Proteínas/genética , Precursores de Proteínas/fisiología , Transducción de Señal , Cicatrización de Heridas
3.
J Cell Physiol ; 216(2): 426-37, 2008 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-18286479

RESUMEN

The adaptor protein Grb10 is an interacting partner of the IGF-I receptor (IGF-IR) and the insulin receptor (IR). Previous work from our laboratory has established the role of Grb10 as a negative regulator of IGF-IR-dependent cell proliferation. We have shown that Grb10 binds the E3 ubiquitin ligase Nedd4 and promotes IGF-I-stimulated ubiquitination, internalization, and degradation of the IGF-IR, thereby giving rise to long-term attenuation of signaling. Recent biochemical evidence suggests that tyrosine-kinase receptors (RTK) may not be polyubiquitinated but monoubiquitinated at multiple sites (multiubiquitinated). However, the type of ubiquitination of the IGF-IR is still not defined. Here we show that the Grb10/Nedd4 complex upon ligand stimulation mediates multiubiquitination of the IGF-IR, which is required for receptor internalization. Moreover, Nedd4 by promoting IGF-IR ubiquitination and internalization contributes with Grb10 to negatively regulate IGF-IR-dependent cell proliferation. We also demonstrate that the IGF-IR is internalized through clathrin-dependent and-independent pathways. Grb10 and Nedd4 remain associated with the IGF-IR in early endosomes and caveosomes, where they may participate in sorting internalized receptors. Grb10 and Nedd4, unlike the IGF-IR, which is targeted for lysosomal degradation are not degraded and likely directed into recycling endosomes. These results indicate that Grb10 and Nedd4 play a critical role in mediating IGF-IR down-regulation by promoting ligand-dependent multiubiquitination of the IGF-IR, which is required for receptor internalization and regulates mitogenesis.


Asunto(s)
Proteína Adaptadora GRB10/metabolismo , Poliubiquitina/metabolismo , Receptor IGF Tipo 1/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Caveolina 1/genética , Caveolina 1/metabolismo , Línea Celular , Proliferación Celular , Clatrina/genética , Clatrina/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte , Endosomas/metabolismo , Proteína Adaptadora GRB10/genética , Lisosomas/metabolismo , Ratones , Complejos Multiproteicos/metabolismo , Ubiquitina-Proteína Ligasas Nedd4 , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Ratas , Receptor IGF Tipo 1/genética , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo
4.
Mol Biol Cell ; 19(12): 5203-13, 2008 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-18815279

RESUMEN

NF-Y binds to CCAAT motifs in the promoter region of a variety of genes involved in cell cycle progression. The NF-Y complex comprises three subunits, NF-YA, -YB, and -YC, all required for DNA binding. Expression of NF-YA fluctuates during the cell cycle and is down-regulated in postmitotic cells, indicating its role as the regulatory subunit of the complex. Control of NF-YA accumulation is posttranscriptional, NF-YA mRNA being relatively constant. Here we show that the levels of NF-YA protein are regulated posttranslationally by ubiquitylation and acetylation. A NF-YA protein carrying four mutated lysines in the C-terminal domain is more stable than the wild-type form, indicating that these lysines are ubiquitylated Two of the lysines are acetylated in vitro by p300, suggesting a competition between ubiquitylation and acetylation of overlapping residues. Interestingly, overexpression of a degradation-resistant NF-YA protein leads to sustained expression of mitotic cyclin complexes and increased cell proliferation, indicating that a tight regulation of NF-YA levels contributes to regulate NF-Y activity.


Asunto(s)
Factor de Unión a CCAAT/metabolismo , Regulación de la Expresión Génica , Procesamiento Proteico-Postraduccional , Subunidades de Proteína/metabolismo , Transcripción Genética , Secuencia de Aminoácidos , Animales , Factor de Unión a CCAAT/genética , Línea Celular , Humanos , Lisina/genética , Lisina/metabolismo , Ratones , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma , Subunidades de Proteína/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Ubiquitinación , Factores de Transcripción p300-CBP/metabolismo
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