RNF8-dependent and RNF8-independent regulation of 53BP1 in response to DNA damage.
The DNA damage surveillance network orchestrates cellular responses to DNA damage through the recruitment of DNA damage-signaling molecules to DNA damage sites and the concomitant activation of protein phosphorylation cascades controlled by the ATM (ataxia-telangiectasia-mutated) and ATR (ATM-Rad3-related) kinases. Activation of ATM/ATR triggers cell cycle checkpoint activation and adaptive responses to DNA damage. Recent studies suggest that protein ubiquitylation or degradation plays an important role in the DNA damage response. In this study, we examined the potential role of the proteasome in checkpoint activation and ATM/ATR signaling in response to UV light-induced DNA damage. HeLa cells treated with the proteasome inhibitor MG-132 showed delayed phosphorylation of ATM substrates in response to UV light. UV light-induced phosphorylation of 53BP1, as well as its recruitment to DNA damage foci, was strongly suppressed by proteasome inhibition, whereas the recruitment of upstream regulators of 53BP1, including MDC1 and H2AX, was unaffected. The ubiquitin-protein isopeptide ligase RNF8 was critical for 53BP1 focus targeting and phosphorylation in ionizing radiation-damaged cells, whereas UV light-induced 53BP1 phosphorylation and targeting exhibited partial dependence on RNF8 and the ubiquitin-conjugating enzyme UBC13. Suppression of RNF8 or UBC13 also led to subtle defects in UV light-induced G2/M checkpoint activation. These findings are consistent with a model in which RNF8 ubiquitylation pathways are essential for 53BP1 regulation in response to ionizing radiation, whereas RNF8-independent pathways contribute to 53BP1 targeting and phosphorylation in response to UV light and potentially other forms of DNA replication stress.
Sakasai
Ryo
R
Department of Pharmacology, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin 53706, USA.
Tibbetts
Randal
R
D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; D004249:DNA Damage; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000502:physiology*; D005786:Gene Expression Regulation; D006657:Histones / Q000737:chemistry; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics* / Q000502:physiology*; D007976:Leupeptins / Q000494:pharmacology; D008954:Models, Biological; D009687:Nuclear Proteins / Q000737:chemistry; D010766:Phosphorylation; D061988:Proteasome Inhibitors; D015398:Signal Transduction; D015534:Trans-Activators / Q000737:chemistry; D000071857:Tumor Suppressor p53-Binding Protein 1; D025801:Ubiquitin / Q000737:chemistry; D044767:Ubiquitin-Protein Ligases; D014466:Ultraviolet Rays
D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C468783:H2AX protein, human; D006657:Histones; D047908:Intracellular Signaling Peptides and Proteins; D007976:Leupeptins; C413163:MDC1 protein, human; D009687:Nuclear Proteins; D061988:Proteasome Inhibitors; C486521:RNF8 protein, human; C498301:TP53BP1 protein, human; D015534:Trans-Activators; D000071857:Tumor Suppressor p53-Binding Protein 1; D025801:Ubiquitin; D044767:Ubiquitin-Protein Ligases; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde