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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...