RESUMEN
DNA viruses have evolved a number of mechanisms to inhibit the major cellular tumor-suppressor pathways. Viral oncogenes can override growth suppressive signals and extend the virus proliferative capacity. The Kaposi sarcoma-associated human herpesvirus 8 (KSHV) encodes a protein, cyclin K, that is similar to cellular cyclin D1 but behaves atypically. Cyclin K resists the actions of the p16 INK4a and p27Kip1 inhibitors and extends the range of cdk6 substrates, thereby inducing cell-cycle progression toward S phase. In this study, we show that cyclin K overrides growth suppressive signals through signal transducer and activator of transcription 3 (STAT3) inactivation. Cyclin K was found to associate with the activation domain of STAT3 to inhibit its DNA-binding and transcriptional activities. Overexpression of cyclin K and inhibition of STAT3 prevents the growth suppressive effect imposed by the interleukin 6-type cytokine, oncostatin M. Altogether, these results suggest that KSHV is able to override growth suppressive effects through multiple mechanisms, and they further indicate that cyclin K plays an important role in the oncogenic activity of these viruses.
Asunto(s)
Ciclinas/farmacología , Proteínas de Unión al ADN/antagonistas & inhibidores , Inhibidores de Crecimiento/farmacología , Herpesvirus Humano 8/fisiología , Péptidos/farmacología , Sarcoma de Kaposi/virología , Transactivadores/antagonistas & inhibidores , Proteínas Virales/farmacología , División Celular/efectos de los fármacos , Humanos , Melanoma , Oncostatina M , Péptidos/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/farmacología , Factor de Transcripción STAT3 , Sarcoma de Kaposi/patología , Células Tumorales CultivadasRESUMEN
Uracil-DNA glycosylase catalyzes the excision of uracils from DNA via a mechanism where the uracil is extrahelically flipped out of the DNA helix into the enzyme active site. A conserved leucine is inserted into the DNA duplex space vacated by the uracil leading to the paradigmatic "push-pull" mechanism of nucleotide flipping. However, the order of these two steps during catalysis has not been conclusively established. We report a complete kinetic analysis of a single catalytic turnover using a hydrolyzable duplex oligodeoxyribonucleotide substrate containing a uracil:2-aminopurine base pair. Rapid chemical-quenched-flow methods defined the kinetics of excision at the active site during catalysis. Stopped-flow fluorometry monitoring the 2-aminopurine fluorescence defined the kinetics of uracil flipping. Parallel experiments detecting the protein fluorescence showed a slower Leu(191) insertion step occurring after nucleotide flipping but before excision. The inserted Leu(191) acts as a doorstop to prevent the return of the flipped-out uracil residue, thereby facilitating the capture of the uracil in the active site and does not play a direct role in "pushing" the uracil out of the DNA helix. The results define for the first time the proper sequence of events during a catalytic cycle and establish a "pull-push", as opposed to a "push-pull", mechanism for nucleotide flipping.