Post-translation modifications play a crucial role in coordinating the cellular response to DNA damage. Double strand DNA breaks (DSBs) trigger the activation of ATM and Chk2 kinases, which represent the primary transducers in the signalling cascade. Among the high number of phosphorylated proteins, our attention was focused on Che-1, a novel ATM and Chk2 substrate whose role in DNA damage response has been recently shown. Phosphorylated Che-1 accumulates and promotes transcription of p53 and p53-responsive genes, which are critical for the maintenance of G2 arrest and for DNA repair processes . Poly(ADP-ribosyl)ation is a post-translational modification that shows an emerging role in the signal transduction to the DDR machinery. Poly(ADP-ribose) polymerase 1 (PARP-1), the main enzyme involved in this modification, is recruited on DNA lesions and catalyzes the synthesis of poly(ADP-ribose) polymers (PAR) on itself and on target proteins. In particular, a recent work demonstrated that PAR synthesis at DSBs sites is necessary to recruit ATM kinase, which can interact non-covalently with PAR. In this study we showed that poly(ADP-ribosyl)ation, beyond phosphorylation, is involved in the regulation of Che-1 stabilization following DNA damage. We demonstrated that Che-1 accumulation upon doxorubicin treatment is reduced after inhibition of PARP activity in HCT116 cells and in PARP-1 knock-out or silenced cells. In accordance, impairment in Che-1 accumulation by PARP inhibition reduced Che-1 occupancy at p21 promoter and affected the expression of the corresponding gene. Epistasis experiments showed that the effect of poly(ADP-ribosyl)ation on Che-1 stabilization is independent from ATM kinase activity. Indeed we demonstrated that Che-1 protein co-immunoprecipitates with ADP-ribose polymers and that PARP-1 directly interacts with Che-1, promoting its modification in vitro and in vivo. Altogether, these findings suggest that poly(ADP-ribosyl)ation of Che-1 represents a mechanism enabling the precise control over the level of Che-1 protein in response to DNA damage.
|Titolo:||Poly(ADP-Ribosyl)ation affects stabilization of CHE-1 protein in response to DNA damage|
|Data di pubblicazione:||2011|
|Appartiene alla tipologia:||07b Tesi di Dottorato (EX-Padis)|