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. 2009 Jan 12;184(1):13-9.
doi: 10.1083/jcb.200807137. Epub 2009 Jan 5.

USP7 counteracts SCFbetaTrCP- but not APCCdh1-mediated proteolysis of Claspin

Helene Faustrup  1 , Simon Bekker-Jensen, Jiri Bartek, Jiri Lukas, Niels Mailand
Affiliations

USP7 counteracts SCFbetaTrCP- but not APCCdh1-mediated proteolysis of Claspin

Helene Faustrup et al. J Cell Biol. .

Abstract

Claspin is an adaptor protein that facilitates the ataxia telangiectasia and Rad3-related (ATR)-mediated phosphorylation and activation of Chk1, a key effector kinase in the DNA damage response. Efficient termination of Chk1 signaling in mitosis and during checkpoint recovery requires SCF(betaTrCP)-dependent destruction of Claspin. Here, we identify the deubiquitylating enzyme ubiquitin-specific protease 7 (USP7) as a novel regulator of Claspin stability. Claspin and USP7 interact in vivo, and USP7 is required to maintain steady-state levels of Claspin. Furthermore, USP7-mediated deubiquitylation markedly prolongs the half-life of Claspin, which in turn increases the magnitude and duration of Chk1 phosphorylation in response to genotoxic stress. Finally, we find that in addition to the M phase-specific, SCF(betaTrCP)-mediated degradation, Claspin is destabilized by the anaphase-promoting complex (APC) and thus remains unstable in G1. Importantly, we demonstrate that USP7 specifically opposes the SCF(betaTrCP)- but not APC(Cdh1)-mediated degradation of Claspin. Thus, Claspin turnover is controlled by multiple ubiquitylation and deubiquitylation activities, which together provide a flexible means to regulate the ATR-Chk1 pathway.

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Figures

Figure 1.
Figure 1.
Claspin interacts with USP7. (A) U2OS cells were transfected with indicated siRNAs for 72 h and analyzed by IB with the indicated antibodies. The functionality of the USP1 siRNA has been demonstrated previously (Huang et al., 2006). (B) Lysates of 293T cells transfected with indicated plasmids for 24 h were subjected to Myc IP followed by IB. (C) Lysates of 293T cells were subjected to IP with USP7 antibody or preimmune rabbit serum (IgG) followed by IB. (D) 293T cells were transfected with WT or CI forms of Myc-USP7, and processed as in B. The extra band in the USP7 CI lane corresponds to ubiquitylated USP7 (unpublished data).
Figure 2.
Figure 2.
USP7 deubiquitylates and stabilizes Claspin. (A) U2OS/Myc-USP7 cell lines were induced with doxycycline (DOX), harvested at the indicated times, and processed for IB with the indicated antibodies. (B) U2OS/Myc-USP7 WT cells were induced or not induced with DOX for 30 h, after which cycloheximide (CHX) was added to the cultures for the indicated times. The half-life of Claspin was estimated by IB of total cell extracts. (C) U2OS/Myc-USP7 CI cells were treated as in B. (D) U2OS/Myc-USP7 WT or CI cells were induced or not induced for 18 h with DOX, and left untreated (Exp) or incubated with nocodazole for an additional 12 h to synchronize cells in mitosis (M). The cell extracts were then analyzed by IB. Mobility-shifted Wee1 served as a marker for mitotically synchronized cells. (E) [35S]-labeled Claspin (amino acids 1–380) was incubated in ubiquitylation reaction mix, then supplemented with extracts of exponentially growing or mitotic U2OS cells and in vitro–translated βTrCP1. Where indicated, bacterially purified GST-USP7 WT was added to the reaction. Claspin ubiquitylation was visualized by autoradiography. Numbers to the right of the gel blots indicate molecular mass standards in kD. (F) U2OS/Myc-USP7 cell lines were transfected with indicated constructs for 24 h, and lysates were processed for IP with FLAG antibody and IB.
Figure 3.
Figure 3.
Degradation of Claspin by APCCdh1 in G1 is not opposed by USP7. (A) U2OS/Myc-USP7 WT cells were induced or not induced with DOX for 18 h, and left untreated or synchronized in mitosis by nocodazole treatment for an additional 12 h. Mitotic cells were washed, plated in fresh medium, and collected at the indicated times after release. Lysates of these and asynchronous control cells (−) were analyzed by IB. (B) U2OS/Myc-Cdh1 cells were induced by removal of tetracycline (Tet) for the indicated times and processed for IB. (C) U2OS cells were treated with MG132 for 4 h or transfected with βTrCP or Cdh1 siRNAs for 48 h. The cells were then fixed and immunostained with indicated antibodies. DNA was visualized by counterstaining with ToPro3. Arrows indicate G1 cells. Bars, 10 μm. (D) U2OS/Myc-Cdh1 cells were induced or not induced by tetracycline withdrawal for 48 h, exposed to UV (20 J/m2), and harvested 1 h later. Total cell extracts were processed for IB. (E) U2OS/Myc-USP7 WT cells were induced or not induced with DOX for 48 h, and processed for immunofluorescence as in C. Arrows indicate G1 cells. Bars, 10 μm. (F) U2OS cells were transfected with the indicated plasmids for 24 h and processed for IB.
Figure 4.
Figure 4.
USP7 controls the timing of Chk1 phosphorylation through regulation of Claspin stability. (A) U2OS cells were transfected with indicated siRNAs for 72 h, then subsequently treated with UV (20 J/m2) and harvested 1 h later. Cells were then processed for IB. (B) U2OS/Myc-USP7 WT cells were induced or not induced with DOX for 12 h, and treated with HU for an additional 24 h. Cells were then released from the HU block, harvested at the indicated times, and processed for IB. (C) U2OS/Myc-USP7 CI cells were treated as in B. (D) U2OS/Myc-USP7 WT cells were induced or not induced with DOX for 24 h, and left untreated or synchronized in mitosis with nocodazole for an additional 12 h. Cells were then treated with etoposide for 2 h where indicated, and processed for IB.
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References

    1. Bartek, J., and J. Lukas. 2007. DNA damage checkpoints: from initiation to recovery or adaptation. Curr. Opin. Cell Biol. 19:238–245. - PubMed
    1. Bassermann, F., D. Frescas, D. Guardavaccaro, L. Busino, A. Peschiaroli, and M. Pagano. 2008. The Cdc14B-Cdh1-Plk1 axis controls the G2 DNA-damage-response checkpoint. Cell. 134:256–267. - PMC - PubMed
    1. Bekker-Jensen, S., C. Lukas, R. Kitagawa, F. Melander, M.B. Kastan, J. Bartek, and J. Lukas. 2006. Spatial organization of the mammalian genome surveillance machinery in response to DNA strand breaks. J. Cell Biol. 173:195–206. - PMC - PubMed
    1. Busino, L., M. Donzelli, M. Chiesa, D. Guardavaccaro, D. Ganoth, N.V. Dorrello, A. Hershko, M. Pagano, and G.F. Draetta. 2003. Degradation of Cdc25A by beta-TrCP during S phase and in response to DNA damage. Nature. 426:87–91. - PubMed
    1. Canning, M., C. Boutell, J. Parkinson, and R.D. Everett. 2004. A RING finger ubiquitin ligase is protected from autocatalyzed ubiquitination and degradation by binding to ubiquitin-specific protease USP7. J. Biol. Chem. 279:38160–38168. - PubMed

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