RESUMO
Genotoxic stress activates checkpoint signaling pathways that activate the checkpoint kinases ATM and ATR, halt cell cycle progression, and promote DNA repair. A number of proteins act in concert with ATR to phosphorylate Chk1, including RAD17, the RAD9-RAD1-HUS1 complex, ATR/ATRIP and TopBp1. However, how these proteins involved act in concert with one another to propagate and maintain the checkpoint response is not well understood. Here, we reported that upregulation of RAD9 protein increased the quantity of ATRIP, suggesting that RAD9 activation will induce more efficient accumulation of ATRIP in vivo. Furthermore, the DNA damage-induced ATRIP foci formation was faster in the mRad9-/- ES cells. Also, ATRIP interacts specifically with RAD9, but not HUS1 and RAD1. Taken together, we suggested that RAD9 could affect both the ATRIP protein levels and DNA damage-induced ATRIP foci formation. Thus, we propose a role of RAD9 in the ATR-Chk1 pathway that is necessary for successful formation of the damage-sensing complex and DNA damage checkpoint signaling.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Ciclo Celular/genética , Reparo do DNA , Proteínas de Ligação a DNA/genética , DNA/genética , Proteínas Quinases/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Quinase 1 do Ponto de Checagem , Cromatina/química , Cromatina/efeitos dos fármacos , Cromatina/metabolismo , DNA/metabolismo , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Exonucleases/genética , Exonucleases/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Humanos , Hidroxiureia/farmacologia , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Células-Tronco Embrionárias Murinas/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilação , Proteínas Quinases/metabolismo , Transdução de Sinais , Especificidade da EspécieRESUMO
In order to elucidate the biochemical mechanism of sperm capacitation, the relationship between plasmalemma Na,K-ATPase, capacitation and acrosome reaction was investigated. Plasmalemma of guinea pig spermatozoa was isolated by sucrose gradient centrifugation for the determination of Na,K-ATPase activity. As far as the authors are aware, the Na,K-ATPase activity in plasmalemma of the guinea pig has never been detected. By treating sperm plasmalemma with 0.05% DOC (deoxycholate), enzyme activity could be quantitatively measured. After spermatozoa were incubated in capacitation medium for 8 h, Na,K-ATPase activity was found to be decreased to 35.6% as compared with that before incubation. The spermatozoa incubated for 10.5 h in capacitation medium containing 1 and 5 mumol/L ouabain showed 46.5% and 64.4% acrosome reactions respectively, while the acrosome reaction of the control group was only 27.4%. The above results suggest that the decrease in the Na,K-ATPase activity of guinea pig spermatozoa may be a prerequisite for capacitation. Experimental results demonstrated for the first time that Na,K-ATPase activity exists in the sperm plasmalemma of the guinea pig. It was further found that the decrease of Na,K-ATPase activity of plasmalemma enhances sperm capacitation. It is suggested that sperm capacitation in the guinea pig is possibly induced by the decrease in plasmalemma Na,K-ATPase and, as a consequence, the intracellular Na+ is increased, which would benefit the exchange of Na+out/Ca++in and the onset of acrosome reaction.