ATM is a core regulator in the DDR and phosphorylates hundreds of substrates containing closely spaced Ser-Gln (SQ) and Thr-Gln (TQ) motifs to orchestrate the response to DNA damage through initiating the regulation of cell cycle checkpoint, DNA repair and cell apoptosis49C51. repair. test. d Working model of CtIP regulation by USP52. Discussion CtIP is required for DNA end processing and essential for the initial step of homology-directed repair in eukaryotes9,11. Therefore, the protein level, the DNA damage site recruitment and the activity of CtIP are all tightly controlled for proper DNA end resection17,38,43. It was reported that post-translational modifications such as phosphorylation, sumoylation, and ubiquitination play important roles in CtIP regulation. For example, it is clear that CDK-mediated phosphorylation of CtIP on Thr-847 modulates ssDNA generation, RPA recruitment, and phosphorylation to ensure appropriate DNA end resection during S/G2 cell phase37,38,40. In addition, CDK-phosphorylated CtIP on Ser-327 promotes the interaction with BRCA1 and facilitates the ubiquitination of CtIP by BRCA1, which is required for CtIP participated in G2/M checkpoint control15. Sumoylation of CtIP at lysine 896 by SUMO E3 ligase CBX4 is also important for the role of CtIP in regulating DNA end processing and genomic stability43. Several studies have reported that E3 ubiquitin ligases or their substrate adapter such as APC/CCdh1 and KLHL15 participate in regulating the protein stability and activity of CtIP17,25. Whether the ubiquitin on CtIP protein affects its function and how this process is controlled are still largely unknown. Here, we found that CtIP ubiquitination negatively regulates its phosphorylation at Thr-847. Furthermore, we discovered that CtIP is deubiquitinated by USP52, which removes the ubiquitin from CtIP to promote DNA end resection and HR repair (Fig.?6d). Our results suggest a critical role for ubiquitination in regulating CtIP activity and illustrate the regulatory mechanism of the USP52/CtIP pathway in the DDR. USP52, also named PAN2 (poly(A) nuclease), is a bona fide DUB, which was reported to deubiquitinate and stabilize histone chaperone ASF1A to facilitate chromatin assembly and breast carcinogenesis34. Though lacking an active-site cysteine residue, USP52 was able to hydrolyze K6-, K11-, K48-, K63-, and M1-linked ubiquitin chains through its UCH domain34. In addition, USP52 has been reported to be a key component of p-bodies which prevents the degradation of HIF1A mRNA and regulates HIF1A-mediated hypoxic response44. Here, we reveal that USP52 is also engaged in DNA end resection and HR repair through removing ubiquitin from CtIP, which dependent on the catalytic activity of the UCH domain. After DNA damage, CtIP is deubiquitinated in USP52-dependent manner, indicating that USP52/CtIP pathway plays a critical role in ensuring appropriate DNA end resection and HR repair. Based on the size of ubiquitiated CtIP, our data suggest that CtIP is not polyubiquitinated but rather is monoubiquitinated at K760 and K782. Because CtIP is a large protein, we could not detect an apparent shift caused by its ubiquitination. Ubiquitination can affect the protein stability, cellular localization, protein interactions or activity of target substrates45,46. We found that USP52-mediated CtIP deubiquitination does not affect CtIP protein level and recruitment to the DNA damage sites, but regulates the activity of CtIP through promoting the phosphorylation of CtIP at Thr-847. This increases DNA end resection and HR repair. Because the ubiquitination sites (K760 and K782) and the phosphorylation site (T847) are not the same (they are all located within the C-terminal domain of CtIP), it is possible that the ubiquitination of CtIP alters the conformation and subsequently masks the phosphorylation site. Alternatively, the ubiquitination of CtIP could be recognized by scaffold proteins or kinases/phosphatase with ubiquitin-binding domains that subsequently affect the phosphorylation of CtIP indirectly. The detailed mechanism regarding how CtIP ubiquitination affects its phosphorylation will need to be studied in further detail. DUB activity and specificity are modulated by a variety. We found that USP52-mediated CtIP deubiquitination does not affect CtIP protein level and recruitment to the DNA damage sites, but regulates the activity of CtIP through promoting the phosphorylation of CtIP at Thr-847. CtIP deubiquitination in DNA repair. test. d Working model of CtIP regulation by USP52. Discussion CtIP is required for DNA end processing and essential for the initial step of homology-directed repair in eukaryotes9,11. Therefore, the protein level, the DNA damage site recruitment and the activity of CtIP are all tightly controlled for proper DNA end resection17,38,43. It was reported that post-translational modifications such as phosphorylation, sumoylation, and ubiquitination play important roles in CtIP regulation. For example, it is clear that CDK-mediated phosphorylation of CtIP on Thr-847 modulates ssDNA generation, RPA recruitment, and phosphorylation to ensure appropriate DNA end resection during S/G2 cell phase37,38,40. In addition, CDK-phosphorylated CtIP on Ser-327 promotes the interaction with BRCA1 and facilitates the ubiquitination of CtIP by BRCA1, which is required for CtIP participated in G2/M checkpoint control15. Sumoylation of CtIP at lysine 896 by SUMO E3 ligase CBX4 is also important for the role of CtIP in regulating DNA end processing and genomic stability43. Rabbit Polyclonal to GALR3 Several studies have reported that E3 ubiquitin ligases or their substrate adapter such as APC/CCdh1 and KLHL15 participate in regulating the protein stability and activity of CtIP17,25. Whether the ubiquitin on CtIP protein affects its function and how this process is controlled are still largely unknown. Here, we found that CtIP ubiquitination negatively regulates its phosphorylation at Thr-847. Furthermore, we discovered that CtIP is deubiquitinated by USP52, which removes the ubiquitin from CtIP to promote DNA end resection and HR repair (Fig.?6d). Our results D-(-)-Quinic acid suggest a critical role for ubiquitination in regulating CtIP activity and illustrate the regulatory mechanism of the USP52/CtIP pathway in the DDR. USP52, also named PAN2 (poly(A) nuclease), is a bona fide D-(-)-Quinic acid DUB, which was reported to deubiquitinate and stabilize histone chaperone ASF1A to facilitate chromatin assembly and breast carcinogenesis34. Though lacking an active-site cysteine residue, USP52 was able to hydrolyze K6-, K11-, K48-, K63-, and M1-linked ubiquitin chains through its UCH domain34. In addition, USP52 has been reported to be a key component of p-bodies which prevents the degradation of HIF1A mRNA and regulates HIF1A-mediated hypoxic response44. Here, we reveal that USP52 is also engaged in DNA end resection and HR repair through removing ubiquitin from CtIP, which dependent on the catalytic activity of the UCH domain. After DNA damage, CtIP is deubiquitinated in USP52-dependent manner, indicating that USP52/CtIP pathway plays a critical role in ensuring appropriate DNA end resection and HR repair. Based on the size of ubiquitiated CtIP, our data suggest that CtIP is not polyubiquitinated but rather is monoubiquitinated at K760 and K782. Because CtIP is a large protein, we could not detect an apparent shift caused by its ubiquitination. Ubiquitination can affect the protein stability, cellular localization, protein interactions or activity of target substrates45,46. We found that USP52-mediated CtIP deubiquitination does not affect CtIP protein level and recruitment to the DNA damage sites, but regulates the activity of D-(-)-Quinic acid CtIP through promoting the phosphorylation of CtIP at Thr-847. This increases DNA end resection and HR repair. Because the ubiquitination sites (K760 and K782) and the phosphorylation site (T847) are not the same (they are all located within the C-terminal domain of CtIP), it is possible that the ubiquitination of CtIP alters the conformation and subsequently masks the phosphorylation site. Alternatively, the ubiquitination of CtIP could be identified by scaffold proteins or kinases/phosphatase with ubiquitin-binding domains that consequently impact the phosphorylation of CtIP indirectly. The detailed mechanism concerning how CtIP ubiquitination affects its phosphorylation will need to be analyzed in further fine detail. DUB activity and specificity are modulated by a variety of mechanisms including transcriptional and translational rules, proteins relationships, and post-translational modifications to avoid inadvertent cleavage of non-substrate proteins46,47. Many DUBs activities are regulated from the phosphorylation on serine, threonine and tyrosine residues46,47. It was reported that a quantity of DUBs such as USP13, USP15, and UCHL3 were phosphorylated and triggered by ATM in response to DNA damage to participate in the DNA restoration process29,30,48. ATM is definitely a core regulator in the DDR and phosphorylates hundreds of substrates comprising closely spaced Ser-Gln (SQ) and Thr-Gln (TQ) motifs to orchestrate the response to DNA damage through initiating the rules of cell cycle checkpoint, DNA restoration and cell apoptosis49C51. Our results suggest that the phosphorylation of USP52 on Ser-1003 by ATM increases the activity of USP52 to.