RESUMEN
The herpesvirus life cycle has two distinct phases: latency and lytic replication. The balance between these two phases is critical for viral pathogenesis. It is believed that cellular signals regulate the switch from latency to lytic replication. To systematically evaluate the cellular signals regulating this reactivation process in Kaposi sarcoma-associated herpesvirus, the effects of 26,000 full-length cDNA expression constructs on viral reactivation were individually assessed in primary effusion lymphoma-derived cells that harbor the latent virus. A group of diverse cellular signaling proteins were identified and validated in their effect of inducing viral lytic gene expression from the latent viral genome. The results suggest that multiple cellular signaling pathways can reactivate the virus in a genetically homogeneous cell population. Further analysis revealed that the Raf/MEK/ERK/Ets-1 pathway mediates Ras-induced reactivation. The same pathway also mediates spontaneous reactivation, which sets the first example to our knowledge of a specific cellular pathway being studied in the spontaneous reactivation process. Our study provides a functional genomic approach to systematically identify the cellular signals regulating the herpesvirus life cycle, thus facilitating better understanding of a fundamental issue in virology and identifying novel therapeutic targets.
Asunto(s)
Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Herpesvirus Humano 8/fisiología , Quinasas Quinasa Quinasa PAM/metabolismo , Proteína Proto-Oncogénica c-ets-1/metabolismo , Transducción de Señal/fisiología , Activación Viral/fisiología , Quinasas raf/fisiología , Línea Celular Tumoral , Quinasas MAP Reguladas por Señal Extracelular/genética , Regulación de la Expresión Génica , Genes Reporteros/fisiología , Herpesvirus Humano 8/patogenicidad , Humanos , Linfoma Relacionado con SIDA/patología , Linfoma Relacionado con SIDA/fisiopatología , Linfoma Relacionado con SIDA/virología , Quinasas Quinasa Quinasa PAM/genética , Regiones Promotoras Genéticas/fisiología , Proteína Proto-Oncogénica c-ets-1/genética , Transducción de Señal/genética , Replicación Viral/genética , Replicación Viral/fisiología , Quinasas raf/genéticaRESUMEN
Because of the central role of Ras in cancer cell signaling, there has been considerable interest in developing small molecule inhibitors of the Ras signaling pathways as potential chemotherapeutic agents. This chapter describes the use of a two-hybrid approach to identify the MCP compounds, small molecules that disrupt the interaction between Ras and its effector Raf. We first outline the reagent development and selection/counter selection methods required to successfully apply a two-hybrid approach to isolation of MCP compounds. Separately, we describe the collateral benefits of this screening approach in yielding novel antifungal compounds. We then discuss secondary physiological validation approaches to confirm the MCP compounds specifically target Ras-Raf signaling. Finally, we develop a decision tree for subsequent preclinical characterization and optimization of this class of pathway-targeted reagent.
Asunto(s)
Inhibidores de Proteínas Quinasas/farmacología , Técnicas del Sistema de Dos Híbridos , Quinasas raf/antagonistas & inhibidores , Proteínas ras/antagonistas & inhibidores , Animales , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Ratones , Mapeo de Interacción de Proteínas/métodos , Proteínas ras/genéticaRESUMEN
Rheb GTPase is a key player in the control of growth, cell cycle and nutrient uptake that is conserved from yeast to humans. To further our understanding of the Rheb pathway, we sought to identify hyperactivating mutations in the Schizosaccharomyces pombe Rheb, Rhb1. Hyperactive forms of Rhb1 were found to result from single amino acid changes at valine-17, serine-21, lysine-120 or asparagine-153. Expression of these mutants confers resistance to canavanine and thialysine, phenotypes which are similar to phenotypes exhibited by cells lacking the Tsc1/Tsc2 complex that negatively regulates Rhb1. The thialysine-resistant phenotype of the hyperactive Rhb1 mutants is suppressed by a second mutation in the effector domain. Purified mutant proteins exhibit dramatically decreased binding of GDP, while their GTP binding is not drastically affected. In addition, some of the mutant proteins show significantly decreased GTPase activities. Thus the hyperactivating mutations are expected to result in an increase in the GTP-bound/GDP-bound ratio of Rhb1. By using the hyperactive mutant, Rhb1(K120R), we have been able to demonstrate that Rhb1 interacts with Tor2, one of the two S. pombe TOR (Target of Rapamycin) proteins. These fission yeast results provide the first evidence for a GTP-dependent association of Rheb with Tor.
Asunto(s)
Sustitución de Aminoácidos , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimología , Secuencia de Aminoácidos , Antifúngicos/farmacología , Canavanina/farmacología , Proteínas de Ciclo Celular/metabolismo , Cisteína/análogos & derivados , Cisteína/farmacología , Farmacorresistencia Fúngica , GTP Fosfohidrolasas/análisis , GTP Fosfohidrolasas/química , GTP Fosfohidrolasas/aislamiento & purificación , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Datos de Secuencia Molecular , Mutación , Fosfatidilinositol 3-Quinasas/metabolismo , Unión Proteica , Inhibidores de la Síntesis de la Proteína/farmacología , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/aislamiento & purificación , Homología de Secuencia de AminoácidoRESUMEN
Farnesyltransferase inhibitors (FTI) have been developed as anticancer drugs and are currently being evaluated in clinical trials. In this study, we have examined the effects of FTIs on Tsc-null cells to gain insight into their effects on farnesylated Rheb GTPase. This protein is involved in the activation of mTOR/S6K signaling and is down-regulated by the Tsc1/Tsc2 complex. Both Tsc1(-/-) and Tsc2(-/-) mouse embryonic fibroblasts exhibit constitutive activation of S6K and grow in the absence of serum. Two different FTI compounds, the clinical compound BMS-214662 and the newly described BMS-225975, inhibit the constitutive activation of mTOR/S6K signaling and block serum-free growth of the Tsc-null mouse embryonic fibroblasts. We have also found that Tsc-null mouse embryonic fibroblasts grow under anchorage-independent conditions and that both FTI compounds inhibit this soft agar growth. These FTI effects are similar to those observed with rapamycin. Another interesting phenotype of Tsc-null mouse embryonic fibroblasts is that they are round and contain actin filaments predominantly at the cell periphery. The addition of FTIs, but not rapamycin, led to the reappearance of intracellular actin filaments and reduction of peripheral actin filaments. The ability of FTI to rearrange actin filaments seems to be largely mediated by the inhibition of Rheb protein, as induction of intracellular actin filaments by FTI was much less efficient in Tsc2-null cells expressing Rheb (M184L), a geranylgeranylated mutant Rheb that can bypass farnesylation. These results reveal that FTIs inhibit Rheb, causing two different effects in Tsc-deficient cells, one on growth and the other on actin filament distribution.
Asunto(s)
Actinas/metabolismo , Transferasas Alquil y Aril/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Transducción de Señal/efectos de los fármacos , Sirolimus/antagonistas & inhibidores , Proteínas Supresoras de Tumor/deficiencia , Transferasas Alquil y Aril/metabolismo , Animales , Línea Celular , Proliferación Celular/efectos de los fármacos , Chlorocebus aethiops , Citoesqueleto/efectos de los fármacos , Citoesqueleto/genética , Citoesqueleto/metabolismo , Farnesiltransferasa , Fibroblastos , Humanos , Ratones , Proteínas de Unión al GTP Monoméricas/genética , Proteínas de Unión al GTP Monoméricas/metabolismo , Neuropéptidos/genética , Neuropéptidos/metabolismo , Fenotipo , Fosforilación , Proteínas Quinasas/metabolismo , Proteína Homóloga de Ras Enriquecida en el Cerebro , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Proteínas Quinasas S6 Ribosómicas/metabolismo , Sirolimus/farmacología , Serina-Treonina Quinasas TOR , Proteína 1 del Complejo de la Esclerosis Tuberosa , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
The interaction of activated Ras with Raf initiates signaling cascades that contribute to a significant percentage of human tumors, suggesting that agents that specifically disrupt this interaction might have desirable chemotherapeutic properties. We used a subtractive forward two-hybrid approach to identify small molecule compounds that block the interaction of Ras with Raf. These compounds (MCP1 and its derivatives, 53 and 110) reduced serum-induced transcriptional activation of serum response element as well as Ras-induced transcription by way of the AP-1 site. They also inhibited Ras-induced Raf-1 activation in human embryonic kidney 293 cells, Raf-1 and mitogen-activated protein kinase kinase 1 activities in HT1080 fibrosarcoma cells, and epidermal growth factor-induced Raf-1 activation in A549 lung carcinoma cells. The MCP compounds caused reversion of ras-transformed phenotypes including morphology, in vitro invasiveness, and anchorage-independent growth of HT1080 cells. Decreased level of matrix metalloproteinases was also observed. Further characterization showed that MCP compounds restore actin stress fibers and cause flat reversion in NIH 3T3 cells transformed with H-Ras (V12) but not in NIH 3T3 cells transformed with constitutively active Raf-1 (RafDeltaN). Finally, we show that MCP compounds inhibit anchorage-independent growth of A549 and PANC-1 cells harboring K-ras mutation. Furthermore, MCP110 caused G(1) enrichment of A549 cells with the decrease of cyclin D level. These results highlight potent and specific effects of MCP compounds on cancer cells with intrinsic Ras activation.