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1.
J Virol ; 83(17): 8340-52, 2009 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-19535438

RESUMEN

Human adenovirus E4orf4 protein is toxic in human tumor cells. Its interaction with the B alpha subunit of protein phosphatase 2A (PP2A) is critical for cell killing; however, the effect of E4orf4 binding is not known. B alpha is one of several mammalian B-type regulatory subunits that form PP2A holoenzymes with A and C subunits. Here we show that E4orf4 protein interacts uniquely with B55 family subunits and that cell killing increases with the level of E4orf4 expression. Evidence suggesting that B alpha-specific PP2A activity, measured in vitro against phosphoprotein substrates, is reduced by E4orf4 binding was obtained, and two potential B55-specific PP2A substrates, 4E-BP1 and p70(S6K), were seen to be hypophosphorylated in vivo following expression of E4orf4. Furthermore, treatment of cells with low levels of the phosphatase inhibitor okadaic acid or coexpression of the PP2A inhibitor I(1)(PP2A) enhanced E4orf4-induced cell killing and G(2)/M arrest significantly. These results suggested that E4orf4 toxicity results from the inhibition of B55-specific PP2A holoenzymes, an idea that was strengthened by an observed growth arrest resulting from treatment of H1299 cells with B alpha-specific RNA interference. We believe that E4orf4 induces growth arrest resulting in cell death by reducing the global level of B55-specific PP2A activity, thus preventing the dephosphorylation of B55-specific PP2A substrates, including those involved in cell cycle progression.


Asunto(s)
Adenovirus Humanos/patogenicidad , Ciclo Celular , Muerte Celular , Proteína Fosfatasa 2/antagonistas & inhibidores , Proteínas Virales/metabolismo , Recuento de Células , Línea Celular Tumoral , Supervivencia Celular , Humanos , Unión Proteica , Proteína Fosfatasa 2/metabolismo
2.
Cancer Res ; 64(19): 6978-88, 2004 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-15466190

RESUMEN

One set of genes sufficient for transformation of primary human cells uses the combination of Ha-Ras-V12, the telomerase catalytic subunit hTERT, SV40 large tumor antigen (LT), and SV40 small tumor antigen (ST). Whereas SV40 LT inactivates the retinoblastoma protein and p53, the contribution of ST is poorly understood. The essential helper function of ST requires a functional interaction with protein phosphatase 2A (PP2A). Here we have identified changes in gene expression induced by ST and show that ST mediates these changes through both PP2A-dependent and PP2A-independent mechanisms. Knockdown of PP2A B56gamma subunit can substitute for ST expression to fully transform cells expressing LT, hTERT, and Ras-V12. We also identify those genes affected similarly in two cell lines that have been fully transformed from a common parental line by two alternative mechanisms, namely ST expression or PP2A B56gamma subunit knockdown. ST altered expression of genes involved in proliferation, apoptosis, integrin signaling, development, immune responses, and transcriptional regulation. ST reduced surface expression of MHC class I molecules, consistent with a need for SV40 to evade immune detection. ST expression enabled cell cycle progression in reduced serum and src phosphorylation in anchorage-independent media, whereas B56gamma knockdown required normal serum levels for these phenotypes. Inhibitors of integrin and src signaling prevented anchorage-independent growth of transformed cells, suggesting that integrin and src activation are key ST-mediated events in transformation. Our data support a model in which ST promotes survival through constitutive integrin signaling, src phosphorylation, and nuclear factor kappaB activation, while inhibiting cell-cell adhesion pathways.


Asunto(s)
Antígenos Transformadores de Poliomavirus/fisiología , Transformación Celular Neoplásica/genética , Regulación de la Expresión Génica/fisiología , Fosfoproteínas Fosfatasas/fisiología , Western Blotting , Adhesión Celular/genética , División Celular/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/genética , Genes cdc , Humanos , Integrinas/genética , Integrinas/fisiología , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Fosfoproteínas Fosfatasas/genética , Proteína Fosfatasa 2 , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/genética , Virus 40 de los Simios/inmunología , Transcripción Genética/genética , Familia-src Quinasas/genética , Familia-src Quinasas/fisiología
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