RESUMEN
Many transcription factors undergo transcription-coupled proteolysis. Although ligand binding activates ubiquitin proteolysis of estrogen receptor α (ERα), mechanisms governing this and its relationship to transcriptional activation were unclear. Data presented link cross talk between the Src kinase and liganded ERα with ERα activation and its ubiquitylation. Liganded ERα rapidly activates and recruits Src, which phosphorylates ERα at tyrosine 537 (Y537). This enhances ERα binding to the ubiquitin ligase/ERα coactivator, E6-associated protein (E6-AP), stimulating ERα ubiquitylation, target gene activation, and ultimately ERα loss. ERα phosphorylation by Src promotes ERα ubiquitylation by E6-AP and proteasomal degradation in vitro. Src inhibition impairs estrogen (E2)-activated ERα:E6-AP binding, reducing ERα degradation. ERα-Y537F shows little E2-stimulated degradation and activates native ERα target genes poorly. Src activation enhances ERα and E6-AP binding and their occupancy at ERα target gene promoters to enhance transcription. Thus, ERαY537 phosphorylation drives ERα:E6-AP binding to at least a subset of target promoters, linking transcriptional activation to ERα degradation and providing a novel mechanism to fine tune ERα action. The observation that ERα transcriptional activity can be briskly maintained in a context of reduced ERα levels raises the possibility that hormonally sensitive tissues may not always show robust ERα protein levels.
Asunto(s)
Receptor alfa de Estrógeno/genética , Receptor alfa de Estrógeno/metabolismo , Regiones Promotoras Genéticas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Familia-src Quinasas/metabolismo , Línea Celular Tumoral , Humanos , Fosforilación , Unión Proteica , UbiquitinaciónRESUMEN
Geminiviruses primarily encode only few factors, such as replication initiator protein (Rep), and need various host cellular machineries for rolling-circle replication (RCR) and/or recombination-dependent replication (RDR). We have identified a host factor, RAD54, in a screen for Rep-interacting partners and observed its role in DNA replication of the geminivirus mungbean yellow mosaic India virus (MYMIV). We identified the interacting domains ScRAD54 and MYMIV-Rep and observed that ScRAD54 enhanced MYMIV-Rep nicking, ATPase, and helicase activities. An in vitro replication assay demonstrated that the geminiviral DNA replication reaction depends on the viral Rep protein, viral origin of replication sequences, and host cell-cycle proteins. Rad54-deficient yeast nuclear extract did not support in vitro viral DNA replication, while exogenous addition of the purified ScRAD54 protein enhanced replication. The role of RAD54 in in planta replication was confirmed by the transient replication assay; i.e., agroinoculation studies. RAD54 is a well-known recombination/repair protein that uses its DNA-dependent ATPase activity in conjunction with several other host factors. However, this study demonstrates for the first time that the eukaryotic rolling-circle replicon depends on the RAD54 protein.
Asunto(s)
Begomovirus/metabolismo , ADN Helicasas/metabolismo , Replicación del ADN , Proteínas no Estructurales Virales/metabolismo , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/virología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Secuencia de Bases , Begomovirus/genética , Begomovirus/fisiología , ADN Helicasas/genética , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , ADN Viral/genética , ADN Viral/metabolismo , ADN Viral/ultraestructura , Interacciones Huésped-Patógeno , Microscopía Electrónica , Datos de Secuencia Molecular , Biblioteca de Péptidos , Unión Proteica , Dominios y Motivos de Interacción de Proteínas/genética , Mapeo de Interacción de Proteínas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/virología , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de Aminoácido , Proteínas no Estructurales Virales/genética , Proteínas Virales/genética , Replicación ViralRESUMEN
Geminiviruses replicate by rolling circle mode of replication (RCR) and the viral Rep protein initiates RCR by the site-specific nicking at a conserved nonamer (TAATATT downward arrow AC) sequence. The mechanism of subsequent steps of the replication process, e.g. helicase activity to drive fork-elongation, etc. has largely remained obscure. Here we show that Rep of a geminivirus, namely, Mungbean yellow mosaic India virus (MYMIV), acts as a replicative helicase. The Rep-helicase, requiring > or =6 nt space for its efficient activity, translocates in the 3'-->5' direction, and the presence of forked junction in the substrate does not influence the activity to any great extent. Rep forms a large oligomeric complex and the helicase activity is dependent on the oligomeric conformation ( approximately 24mer). The role of Rep as a replicative helicase has been demonstrated through ex vivo studies in Saccharomyces cerevisiae and in planta analyses in Nicotiana tabacum. We also establish that such helicase activity is not confined to the MYMIV system alone, but is also true with at least two other begomoviruses, viz., Mungbean yellow mosaic virus (MYMV) and Indian cassava mosaic virus (ICMV).
