RESUMEN
Here, we show how targeting protein phosphatase 2A (PP2A), a key regulator of cellular protein phosphorylation, can either induce or prevent apoptosis depending on what other signals the cell is receiving. The oncoprotein polyoma small T interacts with PP2A to regulate survival. In the presence of growth factors, small T induces apoptosis. Akt activity, which usually promotes survival, is required for this death response, because inhibitors of Akt or PI3 kinase protect cells from death. The activation of Akt under these conditions is partial, characterized by T308 phosphorylation but not S473 phosphorylation. In the absence of growth factors, small T protects from cell death. Here, small T uses PP2A to promote phosphorylation of Akt on both T308 and S473. This effect results in a different pattern of phosphorylation of Akt substrates and shifts Akt from a proapoptotic (presence of growth factors) to an antiapoptotic mode (absence of growth factors). An intriguing possibility is that Akt phosphorylation could be therapeutically disregulated to decrease the survival of cancer cells.
Asunto(s)
Antígenos Virales de Tumores/fisiología , Apoptosis/fisiología , Proteína Fosfatasa 2/fisiología , Proteínas Proto-Oncogénicas c-akt/fisiología , Animales , Antígenos Virales de Tumores/genética , Caspasa 3/análisis , Medio de Cultivo Libre de Suero/farmacología , Ratones , Células 3T3 NIH/efectos de los fármacos , Células 3T3 NIH/metabolismo , Fosfatidilinositol 3-Quinasas/fisiología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación/efectos de los fármacos , Fosfoserina/análisis , Fosfotirosina/análisis , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas/fisiología , Inhibidores de Proteínas Quinasas/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/fisiología , Transducción de Señal/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/fisiologíaRESUMEN
Polyomavirus T antigens share a common N-terminal sequence that comprises a DnaJ domain. DnaJ domains activate DnaK molecular chaperones. The functions of J domains have primarily been tested by mutation of their conserved HPD residues. Here, we report detailed mutagenesis of the polyomavirus J domain in both large T (63 mutants) and middle T (51 mutants) backgrounds. As expected, some J mutants were defective in binding DnaK (Hsc70); other mutants retained the ability to bind Hsc70 but were defective in stimulating its ATPase activity. Moreover, the J domain behaves differently in large T and middle T. A given mutation was twice as likely to render large T unstable as it was to affect middle T stability. This apparently arose from middle T's ability to bind stabilizing proteins such as protein phosphatase 2A (PP2A), since introduction of a second mutation preventing PP2A binding rendered some middle T J-domain mutants unstable. In large T, the HPD residues are critical for Rb-dependent effects on the host cell. Residues Q32, A33, Y34, H49, M52, and N56 within helix 2 and helix 3 of the large T J domain were also found to be required for Rb-dependent transactivation. Cyclin A promoter assays showed that J domain function also contributes to large T transactivation that is independent of Rb. Single point mutations in middle T were generally without effect. However, residue Q37 is critical for middle T's ability to form active signaling complexes. The Q37A middle T mutant was defective in association with pp60(c-src) and in transformation.
Asunto(s)
Antígenos Virales de Tumores/genética , Poliomavirus/genética , Adenosina Trifosfatasas/metabolismo , Secuencia de Aminoácidos , Animales , Antígenos Virales de Tumores/química , Antígenos Virales de Tumores/metabolismo , Análisis Mutacional de ADN , Proteínas del Choque Térmico HSC70 , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Células 3T3 NIH , Fosfoproteínas Fosfatasas/metabolismo , Poliomavirus/metabolismo , Proteína Fosfatasa 2 , Estructura Terciaria de Proteína , Proteínas/fisiología , Proteína p130 Similar a la del Retinoblastoma , Activación TranscripcionalRESUMEN
Polyomavirus large T antigen (LT) has a direct role in viral replication and a profound effect on cell phenotype. It promotes cell cycle progression, immortalizes primary cells, blocks differentiation, and causes apoptosis. While much of large T function is related to its effects on tumor suppressors of the retinoblastoma susceptibility (Rb) gene family, we have previously shown that activation of the cyclin A promoter can occur through a non-Rb-dependent mechanism. Here we show that activation occurs via an ATF/CREB site. Investigation of the mechanism indicates that large T can synergize with CREB family members to activate transcription. Experiments with Gal4-CREB constructs show that synergy is independent of CREB phosphorylation by protein kinase A. Examination of synergy with Gal4-CREB deletion constructs indicates that large T acts on the constitutive activation domain of CREB. Large T can bind to CREB in vivo. Genetic analysis shows that the DNA-binding domain (residues 264 to 420) is sufficient to activate transcription when it is localized to the nucleus. Further analysis of the DNA-binding domain shows that while site-specific DNA binding is not required, non-site-specific DNA binding is important for the activation. Thus, CREB binding and DNA binding are both important for large T activation of CREB/ATF sites. In contrast to previous models where large T transactivation occurred indirectly, these results also suggest that large T can act directly at promoters to activate transcription.