Together these experiments indicate that USP7 regulates Chk1 protein levels, an effect that is independent within the control of USP7 on Claspin stability. Open in a separate window Figure 1. USP7 depletion results in decreased Chk1 protein levels. ubiquitin hydrolase, USP7 Abbreviations CIcatalytic inactiveDDRDNA damage responseDUBdeubiquitylating enzymeUSPubiquitin specific peptidaseWTwild type Intro The DNA damage response (DDR) is essential to keep up genomic stability and functions as a first defense in the early stages of malignancy development.1 The ATR-Chk1 branch of this response is activated by single-stranded DNA that happen at stalled replication forks or through resection of DNA double strand breaks.2 Tight regulation of effector kinase Chk1 at different levels is critical for correct functioning of this response. Phosphorylation of Chk1 on Ser317 and Ser345 by ATR upon genotoxic stress activates the LRRK2-IN-1 enzyme, which in turn phosphorylates downstream substrates to mediate an arrest in cell cycle progression, stabilizes replication forks and signals to DNA restoration.3 In addition, ATR-mediated phosphorylation also regulates dissociation Tagln of Chk1 from your chromatin which is thought to facilitate the transmission of DNA damage signals to downstream targets.4,5 Finally, phosphorylation of Chk1 triggers Chk1 polyubiquitination and subsequent proteasome-mediated degradation at later times after damage induction, thereby terminating the checkpoint.6,7 Protein ubiquitination has emerged as an important regulatory mechanism in the monitoring machinery controlling genomic stability. Mono- or polyubiquitin modifications can target protein stability, localization, or activity and several ubiquitin ligases have been recognized to LRRK2-IN-1 function in the DDR. In contrast, ubiquitin removal by deubiquitylating enzymes (DUBs) is definitely less well characterized but equally important. In fact, aberrant DUB activity was recognized in human cancers.8 The DUB ubiquitin specific peptidase 7 (USP7/HAUSP) has many substrates among DDR proteins. Although in the beginning p53 was thought to be the primary substrate of USP7, 9 later on studies showed that USP7 actually has a much higher affinity for MDM2/HDM2, an E3 ligase advertising p53 degradation.10 Subsequently, many other substrates were recognized, for example phosphatase PTEN, a suppressor of the PI3K-AKT pathway, transcription factor FOXO4 and Claspin, a mediator protein in the ATR-Chk1 pathway, critical for Chk1 activation.11-13 Here we identify Chk1 like a novel substrate of USP7. Depletion or inhibition of USP7 decreases Chk1 protein levels. On the other hand, overexpression of USP7 crazy type, but not a catalytic mutant, elevates Chk1 levels and increases the half-life of Chk1 protein. Finally, USP7 can deubiquitinate Chk1 in vivo and in vitro, demonstrating that USP7 directly regulates Chk1 protein levels by cleavage of the poly-ubiquitination chain. Results USP7 depletion reduces Chk1 protein levels Recent studies have shown that Chk1 protein levels are controlled by ubiquitin-mediated proteasomal degradation. Although Cul1-Cul4A E3 ligases were described to be involved in this process,6,7 a ubiquitin hydrolase has not been recognized yet. While studying the effect LRRK2-IN-1 of USP7 within the stability of Claspin, a mediator protein in the ATR-Chk1 pathway, we noticed that depletion of this ubiquitin hydrolase not only affected the levels of Claspin but also Chk1.14 This was studied in more detail by downregulating USP7 in U2OS cells by 3 different siRNA oligos, all of which resulted in a decrease in Chk1 protein levels (Fig. 1A). Chk1 levels were reported to vary during the cell cycle.15 As USP7 knock down did not affect the cell cycle progression as determined by flow cytometry analysis (data not shown), the effect of USP7 downregulation on LRRK2-IN-1 Chk1 protein levels could not be explained by an aspecific effect on cell cycle progression. As depletion, inhibition of USP7 by small molecule inhibitor “type”:”entrez-protein”,”attrs”:”text”:”P22077″,”term_id”:”134707″,”term_text”:”P22077″P2207716 resulted in decreased levels of Chk1 in both 293T and U2OS cells (Fig. 1B). As expected, inhibition of USP7 also lowered Claspin protein levels LRRK2-IN-1 (Fig. 1B). We regarded as it therefore a possibility that the effect of knock down of USP7 on Chk1 might be indirectly due to lower Claspin levels as suggested before by others.17,18 However, although Claspin knock down has a minor impact on (reducing) Chk1 levels, depleting USP7 lead to lower Chk1 protein levels as compared to the Chk1 levels after Claspin knock down (Fig. 1C). Importantly, depletion of USP29, a recently explained regulator of Claspin stability by deubiquitination,14 resulted in decreased Claspin levels without influencing Chk1 protein (Fig. 1D). Finally, elevated Claspin protein levels by overexpressing Flag-Claspin in USP7-depleted cells did not.