IndraLab

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UCHL3 is phosphorylated. 59 / 59
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"To further demonstrate a potential function of UCHL3 phosphorylation, we reconstituted UCHL3-deficient cells with wild-type UCHL3 or the UCHL3 S75A mutant."
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sparser
"Because S75 is located in the catalytic domain of UCHL3, we next examined whether UCHL3 phosphorylation regulates its activation."
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sparser
"Following the recruitment of XRCC4-LIG4 and ligation of the broken DNA ends, UCHL3 is phosphorylated and destabilized, which tilts the ubiquitylation balance of Ku80 in favour of ubiquitylation by exposing Ku80 to E3 ligases."
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"As shown in xref C, S75 was phosphorylated after DNA damage, while the S75A mutation inhibited DNA damage-induced UCHL3 phosphorylation."
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sparser
"Wild-type UCHL3 (UCHL3 WT ) and simulated phosphorylated UCHL3 (UCHL3 S75E ) were recombinantly expressed and purified using affinity chromatography, ion exchange chromatography, and gel filtration chromatography."
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rlimsp
"Our data supports the idea that Ku80 ubiquitylation is induced by loss of UCHL3 triggered by ATM-mediated phosphorylation, and E3 ligases targeting Ku80 might be constitutively activated."
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sparser
"To further establish that the signal detected with the anti-phospho S/TQ antibody indicates phosphorylated UCHL3, immunoblotting was performed with a mutant UCHL3 in which Ser75 was substituted with Ala (S75A)."
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rlimsp
"Lastly, we provide evidence showing that DNA damage-induced UCHL3 phosphorylation (which is dependent on UCHL3 catalytic activity, ATM kinase and downstream NHEJ factors) promotes destabilization of UCHL3, suggesting that UCHL3 may dissociate from Ku80 to allow Ku ubiquitylation and subsequent removal from DNA."
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rlimsp
"Following the recruitment of XRCC4-LIG4 and ligation of the broken DNA ends, UCHL3 is phosphorylated and destabilized, which tilts the ubiquitylation balance of Ku80 in favour of ubiquitylation by exposing Ku80 to E3 ligases."
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sparser
"To test whether UCHL3 could be phosphorylated by ATM/ATR, we examined UCHL3 phosphorylation using an antibody against the consensus of ATM/ATR phosphorylation sites (anti-phospho-SQ/TQ [anti-pSQ/TQ]) following DNA damage."
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sparser
"We also sought to establish the function of this UCHL3 phosphorylation."
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rlimsp
"UCHL3 is phosphorylated in a manner dependent on ATM and its catalytic activity."
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sparser
"As shown in xref A, UCHL3 was phosphorylated at the SQ/TQ motif following IR treatment, and this phosphorylation was blocked by an ATM-specific inhibitor (KU55933) and λ phosphatase treatment."
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sparser
"Furthermore, UCHL3 was phosphorylated at the SQ/TQ motif in ATM +/+ but not ATM −/− cells ( xref B)."
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sparser
"In 2016, Luo et al. first found that phosphorylated UCHL3 at Ser75 can cleave single or polyubiquitin chains on the substrate RAD51 and play an important role in DNA damage repair and tumor occurrence."
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sparser
"Phosphorylated UCHL3 showed stronger ubiquitin chain cleavage activity against RAD51 in vivo [ xref ]."
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sparser
"Accordingly, the interaction of Ku80 and UCHL3, which requires UCHL3 catalytic activity (Fig.  xref ), was not markedly affected when FLAG-tagged phospho-mutants of UCHL3 were examined by immunoprecipitation (Supplementary Fig.  xref )."
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rlimsp
"UCHL3 phosphorylation requiring its catalytic activity and downstream NHEJ factors regulates UCHL3 stability."
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sparser
"Luo et al. found that phosphorylated UCHL3 can cleave polyubiquitin chains in vivo, but in vitro evidence is still lacking [ xref ]."
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sparser
"Together, these results suggest that UCHL3 phosphorylation by ATM is important for RAD51 focus formation, HR, and radiosensitivity."
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sparser
"Next, we further investigated how UCHL3 phosphorylation affects its function."
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sparser
"In our previous study, we reported elevated phosphorylation of Uch-L3 in BMP2-induced osteoblast differentiation [27] ."
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sparser
"These results demonstrate that UCHL3 phosphorylation by ATM is important for UCHL3 activation following DNA damage."
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sparser
"In response to DNA damage, UCHL3 is phosphorylated and activated by ATM, and activated UCHL3 positively regulates HR by deubiquitinating RAD51 to promote the BRCA2-RAD51 interaction xref ."
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sparser
"We know that phosphorylated UCHL3 by ATM can deubiquitinate RAD51 in vivo; however, our studies showed that mimetic phosphorylated UCHL3 can only cleave K48 tri-Ub or longer K48 tetra-Ub with a slower rate in vitro."
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sparser
"In addition, phosphorylated UCHL3 by ATM may show excellent hydrolytic properties toward K48 ubiquitin chains compared to wild-type UCHL3."
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sparser
"These data indicate that phosphorylation of UCHL3 at sites of DNA damage requires downstream c-NHEJ factors."
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sparser
"We first analyzed the structures of the simulated phosphorylation of UCHL3 S75E and the complex of UCHL3 S75E with Ub-PA."
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sparser
"Mechanistically, in response to DNA damage, the deubiquitinase UCHL3 is phosphorylated and activated by ATM."
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sparser
"Therefore, inhibition of UCHL3 activity or UCHL3 phosphorylation may provide new insights in cancer therapy."
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"For example, UCHL3 is phosphorylated and activated by ATM following DNA damage, which in turn deubiquitinates RAD51 and promotes the interaction between RAD51 and BRCA2 ( xref )."
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"Obviously, the biological role and mechanism of UCHL3 phosphorylation has not been fully understood yet, and this requires further research ( xref )."
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"UCHL3 is phosphorylated in a manner dependent on ATM and its catalytic activity."
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"Since UCHL3 was previously listed as a potentially phosphorylated protein in response to IR xref , and harbours a single SQ site (Ser75), the consensus sequence of ATM, ATR and DNA-PKcs kinases, we tested whether UCHL3 is phosphorylated upon phleomycin treatment."
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sparser
"Mechanistically, we show that DNA damage induces UCHL3 phosphorylation, which is dependent on ATM, downstream NHEJ factors and UCHL3 catalytic activity."
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sparser
"The phosphorylation of UCHL3 was also induced by IR treatment (data not shown)."
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rlimsp
"Mechanistically, we show that DNA damage induces UCHL3 phosphorylation, which is dependent on ATM, downstream NHEJ factors and UCHL3 catalytic activity."
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rlimsp
"Mechanistically, we show that DNA damage induces UCHL3 phosphorylation, which is dependent on ATM, downstream NHEJ factors and UCHL3 catalytic activity. Furthermore, this phosphorylation destabilizes UCHL3, despite having no effect on its catalytic activity."
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sparser
"Indeed, DNA damage-induced UCHL3 phosphorylation was not detected with enzymatically inactive UCHL3 (Fig.  xref ), suggesting that UCHL3 could be phosphorylated at sites of DNA damage."
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rlimsp
"Interestingly, we found that UCHL3 phosphorylation peaked at 1 hour after phleomycin removal even though the canonical DSB-induced phosphorylation of CHK2 was detected immediately after phleomycin treatment (Fig. 6C)."
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sparser
"Here, we first analyzed the structure of simulated phosphorylated UCHL3 S75E and the complex of UCHL3 S75E with Ub-PA and preliminarily explained the structural mechanism of phosphorylation-enhanced UCHL3 deubiquitinating activity."
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rlimsp
"We also sought to establish the function of this UCHL3 phosphorylation."
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sparser
"Interestingly, we found that UCHL3 phosphorylation peaked at 1 hour after phleomycin removal even though the canonical DSB-induced phosphorylation of CHK2 was detected immediately after phleomycin treatment (Fig.  xref )."
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rlimsp
"Furthermore, to define which kinase is responsible for UCHL3 phosphorylation, identical experiments were carried out with specific inhibitors targeting ATM or DNA-PKcs kinase activities."
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sparser
"This phenotype prompted us to investigate whether the completion of c-NHEJ or recruitment of downstream factors to DSB sites is prerequisite for UCHL3 phosphorylation."
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rlimsp
"Given the findings that UCHL3 interacted with Ku80 and was recruited to sites of damage in a catalytic activity dependent manner (Fig. 1B–E, Supplementary Fig. S1E,F), we investigated whether catalytic activity is required for its phosphorylation."
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rlimsp
"In line with this idea, siRNA mediated depletion of XRCC4 or LIG4, which obstructs NHEJ completion, resulted in attenuated UCHL3 phosphorylation (Fig. 6D and data not shown)."
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sparser
"In line with this idea, siRNA mediated depletion of XRCC4 or LIG4, which obstructs NHEJ completion, resulted in attenuated UCHL3 phosphorylation (Fig.  xref and data not shown)."
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rlimsp
"Indeed, DNA damage-induced UCHL3 phosphorylation was not detected with enzymatically inactive UCHL3 (Fig. 6B), suggesting that UCHL3 could be phosphorylated at sites of DNA damage."
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rlimsp
"These data indicate that phosphorylation of UCHL3 at sites of DNA damage requires downstream c-NHEJ factors."
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rlimsp
"The phosphorylation of UCHL3 was also induced by IR treatment (data not shown)."
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sparser
"Furthermore, to define which kinase is responsible for UCHL3 phosphorylation, identical experiments were carried out with specific inhibitors targeting ATM or DNA-PKcs kinase activities."
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sparser
"Lastly, we provide evidence showing that DNA damage-induced UCHL3 phosphorylation (which is dependent on UCHL3 catalytic activity, ATM kinase and downstream NHEJ factors) promotes destabilization of UCHL3, suggesting that UCHL3 may dissociate from Ku80 to allow Ku ubiquitylation and subsequent removal from DNA."
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sparser
"As shown in xref H, RAD51 ubiquitination was dramatically decreased following IR treatment in cells reconstituted with wild-type UCHL3 but not in those reconstituted with the S75A mutant, suggesting that UCHL3 phosphorylation is important for UCHL3's deubiquitination of RAD51 following DNA damage."
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trips
"Mechanistically, in response to DNA damage, the deubiquitinase UCHL3 is phosphorylated and activated by ATM."
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sparser
"Therefore, the mechanism of phosphorylation-induced UCHL3 activity changes belong to the conformational changes caused by phosphorylation of the noncatalytic center, and phosphorylated UCHL3 has a greater degree of structural changes when bound to the Ub substrate."
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"To further test whether UCHL3 phosphorylation is able to activate UCHL3, we performed a two-step assay."
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"In addition, the polyubiquitin chains on the substrate RAD51 can be cleaved by ATM-phosphorylated UCHL3 instead of simulated phosphorylated UCHL3 in vivo."
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"Both wild-type UCHL3 and the S75A mutant could deubiquitinate RAD51 in vitro, again suggesting a basal deubiquitination activity of UCHL3 that is not regulated by UCHL3 phosphorylation."
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