SUMOylation of ATRIP potentiates DNA damage signaling by boosting multiple protein interactions in the ATR pathway

Abstract

The ATR (ATM [ataxia telangiectasia-mutated]-and Rad3-related) checkpoint is a crucial DNA damage signaling pathway. While the ATR pathway is known to transmit DNA damage signals through the ATR-Chk1 kinase cascade, whether post-translational modifications other than phosphorylation are important for this pathway remains largely unknown. Here, we show that protein SUMOylation plays a key role in the ATR pathway. ATRIP, the regulatory partner of ATR, is modified by SUMO2/3 at K234 and K289. An ATRIP mutant lacking the SUMOylation sites fails to localize to DNA damage and support ATR activation efficiently. Surprisingly, the ATRIP SUMOylation mutant is compromised in the interaction with a protein group, rather than a single protein, in the ATR pathway. Multiple ATRIP-interacting proteins, including ATR, RPA70, TopBP1, and the MRE11-RAD50-NBS1 complex, exhibit reduced binding to the ATRIP SUMOylation mutant in cells and display affinity for SUMO2 chains in vitro, suggesting that they bind not only ATRIP but also SUMO. Fusion of a SUMO2 chain to the ATRIP SUMOylation mutant enhances its interaction with the protein group and partially suppresses its localization and functional defects, revealing that ATRIP SUMOylation promotes ATR activation by providing a unique type of protein glue that boosts multiple protein interactions along the ATR pathway.[Keywords: ATR; ATRIP; checkpoint; SUMOylation] Supplemental material is available for this article.Received January 18, 2014; revised version accepted June 2, 2014.The maintenance of genomic stability requires not only DNA repair machineries but also signal transduction pathways that regulate and coordinate the DNA damage response (DDR) (Ciccia and Elledge 2010). In human cells, DNA damage signaling is primarily initiated by the ataxia telangiectasia-mutated (ATM) and the ATMand Rad3-related (ATR) kinases. Whereas ATM is activated by DNA double-stranded breaks (DSBs), ATR is elicited by a much broader spectrum of DNA damage and replication stress (Cimprich and Cortez 2008;Marechal and Zou 2013;Shiloh and Ziv 2013). Once activated, ATM and ATR phosphorylate and activate their effector kinases, Chk2 and Chk1, respectively. Together, the ATMChk2 and ATR-Chk1 kinase cascades phosphorylate a number of substrates involved in DNA repair, DNA replication, and cell cycle transitions, coordinating these processes to suppress genomic instability. In addition to the phosphorylation events mediated by the ATM and ATR pathways, several other types of post-translational modifications (PTMs), such as ubiquitylation, SUMOylation, methylation, acetylation, and poly-ADP ribosylation, are also implicated in the DDR (Bekker-Jensen and Mailand 2010;Huen et al. 2010;Lukas et al. 2011;Jackson and Durocher 2013). We recently found that the efficient activation of ATR relies on a ubiquitylation circuitry mediated by RPA-ssDNA (RPA-coated ssDNA) and PRP19 (Marechal et al. 2014). This new finding raises a question as to whether other PTMs also participate in DNA damage signaling through the ATR path...

Protein Kinase C-Mediated Phosphorylation of BCL11B at Serine 2 Negatively Regulates Its Interaction with NuRD Complexes during CD4+ T-Cell Activation

“…The ratio of interacting P300 relative to BCL11B was measured as described above for panel C. of endogenous BCL11B proteins under denaturing conditions allowed the detection of SUMOylated BCL11B proteins in activated Jurkat cells (Fig. 6H) (37). As a whole, these data identify phosphorylation of BCL11B Ser2 by PKC as a new posttranslational modification of BCL11B during human CD4 ϩ T-cell activation, preceding and acting independently of its SUMOylation.…”

“…These cell extracts were immediately boiled for 10 min and diluted in 9 volumes of radioimmunoprecipitation assay (RIPA) buffer (20 mM Tris-HCl [pH 8], 0.5 mM EDTA, 150 mM NaCl, 0.5% NP-40, 10% glycerol) supplemented with 10 mM NEM and protease inhibitor cocktail for immunoprecipitation with anti-BCL11B antibodies (37).…”

The SUMO (Small Ubiquitin-like Modifier) Ligase PIAS3 Primes ATR for Checkpoint Activation

“…Immunoprecipitation-Immunoprecipitation of endogenous ATRIP and analysis of ATRIP SUMOylation were previously described (20). For immunoprecipitation of endogenous ATR, cell extracts were prepared in the NETN buffer (20 mM TrisHCl pH 8.0, 0.5 mM EDTA, 150 mM NaCl, 0.5% Igepal CA-630, and 10% glycerol) supplemented with protease inhibitor mixture.…”

“…To test if PIAS3 localizes to laser-induced DNA damage stripes, U2OS cells were micro-irradiated with UV laser as previously described (20). The pre-extraction step of immunostaining was skipped to avoid potential loss of PIAS3 from DNA damage stripes.…”

“…We recently showed that depletion of UBC9, the sole SUMO E2 in human cells, compromised the activation of the ATR-Chk1 DNA damagesignaling pathway (20). Furthermore, we showed that ATRIP, the regulatory partner of ATR, is SUMOylated at two specific lysine residues, which are important for ATRIP to interact with multiple proteins in the ATR-Chk1 pathway efficiently (20). Although our previous work has linked protein SUMOylation to DNA damage signaling, which SUMO ligases are involved in this process and how they function are yet to be determined.…”