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The serine threonine kinase checkpoint kinase 2 (CHK2) is a DNA damage checkpoint protein important for the ATM-p53 signaling pathway. In addition to its phosphorylation, CHK2 is also ubiquitylated, and both post-translational modifications are important for its function. However, although the mechanisms that regulate CHK2 phosphorylation are well established, those that control its ubiquitylation are not fully understood. In this study, we demonstrate that the ubiquitin E3 ligase PIRH2 (p53-induced protein with a RING (Really Interesting New Gene)-H2 domain) interacts with CHK2 and mediates its polyubiquitylation and proteasomal degradation. We show that the deubiquitylating enzyme USP28 forms a complex with PIRH2 and CHK2 and antagonizes PIRH2-mediated polyubiquitylation and proteasomal degradation of CHK2. We also provide evidence that CHK2 ubiquitylation by PIRH2 is dependent on its phosphorylation status. Cells deficient in Pirh2 displayed accumulation of Chk2 and enhanced hyperactivation of G1/S and G2/M cell-cycle checkpoints. This hyperactivation was, however, no longer observed in Pirh2 PIRH2 (p53-induced protein with a RING (Really Interesting New Gene)-H2 domain), also known as RCHY1, encodes for an ubiquitin (Ub) ligase that is transcriptionally regulated by p53.1 PIRH2 interacts with the active tetrameric form of p53, mediates its ubiquitylation and regulates its turnover via Ubproteasome mechanisms.1,2 PIRH2 exerts its ubiquitylation function in cooperation with the E2 Ub-conjugating enzyme UbcH5b.1 A recent study indicated that PIRH2 negative regulation of p53 is enhanced in the presence of the ubiquitylation factor E4B.3 In addition to p53, other ubiquitylation substrates have been also reported for PIRH2, including c-Myc, 4 p27kip1, 5 e-COP, 6 the signal recognition particle receptor b subunit, 7 the DNA polymerase eta 8 and p73. 9,10The serine threonine kinase CHK2 (checkpoint kinase 2) is a checkpoint effector important for the signaling of DNA double-strand breaks and the activation of cell-cycle checkpoints.11 In response to DNA damage, ataxia telangiectasia mutated (ATM) phosphorylates CHK2 on its threonine 68 (T68) and allows it to interact with the FHA domain of another CHK2.12-14 This dimerization of CHK2 leads to its autophosphorylation on T383 and T387 and promotes its full activation.15-17 CHK2 phosphorylates p53 on Serine 20 (S23 for mouse) in response to DNA damage and has also other phosphorylation substrates, including PML, E2F1, CDC25C and BRCA1, highlighting its importance in processes such as DNA repair, cell-cycle arrest, apoptosis and senescence.18 CHK2 is also involved in DNA repair via the phosphorylation of Forkhead Box M1. 19 In addition, in collaboration with BRCA1, and independently of p53, CHK2 is required for the normal progression of mitosis and chromosomal stability. 20,21Although Chk2 is phosphorylated and activated primarily by the kinase ATM, it is also ubiquitylated; however, the mechanism of this ubiquitylation remains poorly understood.11 In this study, we rep...

PerSPeCtive U biquitylation is currently recognized as a major posttranslational modification that regulates diverse cellular processes. Pirh2 is a ubiquitin E3 ligase that regulates the turnover and functionality of several proteins involved in cell proliferation and differentiation, cell cycle checkpoints, and cell death. Here we review the role of Pirh2 as a regulator of the DNA damage response through the ubiquitylation of p53, Chk2, p73, and PolH. By ubiquitylating these proteins, Pirh2 regulates cell cycle checkpoints and cell death in response to DNA double-strand breaks or the formation of bulky DNA lesions. We also discuss how Pirh2 affects cell proliferation and differentiation in unstressed conditions through ubiquitylation and degradation of c-Myc, p63, and p27 kip1 . Finally, we link these different functions of Pirh2 to its role as a tumor suppressor in mice and as a prognosis marker in various human cancer subtypes.

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Rnf8 is an E3 ubiquitin ligase that plays a key role in the DNA damage response as well as in the maintenance of telomeres and chromatin remodeling. Rnf8−/− mice exhibit developmental defects and increased susceptibility to tumorigenesis. We observed that levels of p53, a central regulator of the cellular response to DNA damage, increased in Rnf8−/− mice in a tissue- and cell type–specific manner. To investigate the role of the p53-pathway inactivation on the phenotype observed in Rnf8−/− mice, we have generated Rnf8−/−p53−/− mice. Double-knockout mice showed similar growth retardation defects and impaired class switch recombination compared to Rnf8−/− mice. In contrast, loss of p53 fully rescued the increased apoptosis and reduced number of thymocytes and splenocytes in Rnf8−/− mice. Similarly, the senescence phenotype of Rnf8−/− mouse embryonic fibroblasts was rescued in p53 null background. Rnf8−/−p53−/− cells displayed defective cell cycle checkpoints and DNA double-strand break repair. In addition, Rnf8−/−p53−/− mice had increased levels of genomic instability and a remarkably elevated tumor incidence compared to either Rnf8−/− or p53−/− mice. Altogether, the data in this study highlight the importance of p53-pathway activation upon loss of Rnf8, suggesting that Rnf8 and p53 functionally interact to protect against genomic instability and tumorigenesis.

BLM is a DNA helicase important for the restart of stalled replication forks and for homologous recombination (HR) repair. Mutations of BLM lead to Bloom Syndrome, a rare autosomal recessive disorder characterized by elevated levels of sister chromatid exchanges (SCEs), dwarfism, immunodeficiency, infertility and increased cancer predisposition. BLM physically interacts with MUS81, an endonuclease involved in the restart of stalled replication forks and HR repair. Herein we report that loss of Mus81 in Blm hypomorph mutant mice leads to infertility, and growth and developmental defects that are not observed in single mutants. Double mutant cells and mice were hypersensitive to Mitomycin C and γ-irradiation (IR) compared with controls and their repair of DNA double-strand breaks (DSBs) mediated by HR pathway was significantly defective, whereas their non-homologous-end-joining repair was elevated compared with controls. We also demonstrate the importance of the loss of the nuclease activity of Mus81 in the defects observed in Mus81(-/-) and double mutant cells. Exacerbated IR-induced chromosomal aberration was observed in double mutant mice and despite their reduced SCE levels, these mutants showed increased tumorigenesis risks. Our data highlight the importance of Mus81 and Blm in DNA DSB repair pathways, fertility, development and cancer.

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