RESUMEN
ATM and rad3-related protein kinase (ATR), a member of the phosphoinositide kinase-like protein kinase family, plays a critical role in cellular responses to DNA structural abnormalities in conjunction with its interacting protein, ATRIP. Here, we show that the amino-terminal portion of ATRIP is relocalized to DNA damage-induced nuclear foci in an RPA-dependent manner, despite its lack of ability to associate with ATR. In addition, ATR-free ATRIP protein can be recruited to the nuclear foci. Our results suggest that the N-terminal domain of the ATRIP protein contributes to the cell cycle checkpoint by regulating the intranuclear localization of ATR.
Asunto(s)
Proteínas de Ciclo Celular/fisiología , Núcleo Celular/metabolismo , Daño del ADN , Exodesoxirribonucleasas/fisiología , Fosfoproteínas/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Adaptadoras Transductoras de Señales , Proteínas de la Ataxia Telangiectasia Mutada , Western Blotting , Proteínas de Ciclo Celular/química , Proteínas de Unión al ADN , Exodesoxirribonucleasas/química , Técnica del Anticuerpo Fluorescente , Células HeLa , Humanos , Fosfoproteínas/química , Plásmidos , Proteínas Serina-Treonina Quinasas/química , Transporte de Proteínas , ARN Interferente PequeñoRESUMEN
PI-kinase-related protein kinase ATR forms a complex with ATRIP and plays pivotal roles in maintaining genome integrity. When DNA is damaged, the ATR-ATRIP complex is recruited to chromatin and is activated to transduce the checkpoint signal, but the precise kinase activation mechanism remains unknown. Here, we show that ATRIP is phosphorylated in an ATR-dependent manner after genotoxic stimuli. The serine 68 and 72 residues are important for the phosphorylation in vivo and are required exclusively for direct modification by ATR in vitro. Using phospho-specific antibody, we demonstrated that phosphorylated ATRIP accumulates at foci induced by DNA damage. Moreover, the loss of phosphorylation does not lead to detectable changes in the relocalization of ATRIP to nuclear foci nor in the activation of downstream effector proteins. Collectively, our results suggest that the ATR-mediated phosphorylation of ATRIP at Ser-68 and -72 is dispensable for the initial response to DNA damage.