In our bioinformatics analysis of

In our bioinformatics analysis of proteins with increased SUMOylation upon treatment with MMC and HU, we found clusters of co-regulated proteins that are known to function together in the RS response. In addition to the ATR activation proteins, BRCA1 and BARD1, we also found Fanconi anemia proteins and DSB response proteins, like MDC1, NBN, and CtIP. This is particularly interesting in light of the recent idea that SUMO functions as a molecular glue to mediate protein complex formation under specific cellular states and that this modification takes form of a “SUMO spray” (Jentsch and Psakhye, 2013). A consequence of this N-Nonyldeoxynojirimycin is that SUMOylation should occur on functionally related proteins to promote cooperation and interaction in protein networks, and this is precisely what we observed in our dataset. Interestingly, we found that proteins co-modified by SUMOylation and phosphorylation generally have many regulated sites in response to RS. This poses a challenge for functional studies because site-directed mutagenesis of specific SUMOylation acceptor sites has been shown to result in little effect on overall protein SUMOylation or function (Jentsch and Psakhye, 2013). Here, we present an integrated analysis of global protein phosphorylation and SUMOylation in RS responses and the largest resource to date of regulated SUMOylation targets under these conditions. We propose that increased SUMOylation occurs on specific and relevant factors in response to distinct DNA lesions, as illustrated by the SUMOylation dynamics upon RS and RS-induced DSBs. Our data suggest that these SUMOylation responses are orchestrated by the apical kinases ATR and ATM in parallel with or as part of their phosphorylation signaling. These findings, and further investigations of the co-regulation of these two modifications, are currently of great interest because the induction of RS-provoked DSBs is increasingly used in chemotherapy to induce cancer cell killing (Li and Heyer, 2008). In light of the essential role of SUMO in the maintenance of genomic integrity (Bergink and Jentsch, 2009, Jackson and Durocher, 2013), the increasing interest in this system as a druggable target (Kessler et al., 2012) will require the understanding of how its perturbation affects global signaling networks.
Experimental Procedures Further details and an outline of the resources used in diploid work can be found in the Supplemental Experimental Procedures.
Author Contributions J.O.S. performed the experiments described in Figures 1, 2, 3, and 4. Z.X. performed the experiments described in Figure 5. T.S.B. provided help and performed part of the dataset presented in Figure 2. G.F. validated the experiments presented in Figure S5. L.v.S. and A.J.L.-C. supervised J.O.S. and S.M. and gave critical input on the manuscript. A.C.O.V. supervised Z.X., J.V.O. supervised J.O.S., and S.M. generated and analyzed the data shown in the remaining figures. S.M., A.C.O.V., and J.V.O. conceived the study, designed the experiments, critically evaluated the results, and wrote the manuscript.
Acknowledgments Work at The Novo Nordisk Foundation Center for Protein Research (CPR) is funded in part by a generous donation from the Novo Nordisk Foundation (grant NNF14CC0001). The proteomics technology developments applied were part of a project that has received funding from the European Union Horizon 2020 Research and Innovation Programme under grant agreement 686547. We would like to thank the PRO-MS Danish National MS Platform for Functional Proteomics and the CPR MS Platform for instrument support and assistance. L.v.S. was supported by the Danish Research Council (Research Career Program FSS Sapere Aude). J.V.O. was supported by the Danish Cancer Society (Project Grant KBVU R90-A5844) and Lundbeckfonden (R191-2015-703). A.C.O.V. was supported by the European Research Council (310913) and by the Netherlands Organisation for Scientific Research (NWO 93511037). J.O.S., Z.X., A.C.O.V., and J.V.O. were supported by the Marie Curie Initial Training Networks Program of the European Union (290257-UPStream). A.J.L.-C. and S.M. were supported by grants from the Danish Cancer Society (KBVU-2014) and the European Research Council (ERC-2015-STG-294679068). A.J.L.-C. was also supported by the Danish National Research Foundation (DNRF115) and the Danish Council for Independent Research (Sapere Aude, DFF Starting Grant 2014, 4004-00185B).