The E3 ligase PIRH2 polyubiquitylates CHK2 and regulates its turnover

Abstract

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...

Pirh2

“…36 Our most recent study of Pirh2 −/− mice revealed an increase in Chk2 levels both under unstressed conditions and following DNA damage. 7 Despite the similar level of expression and activation of Atm in the absence or presence of Pirh2, γ-irradiation resulted in higher phosphorylation levels of p53 and E2F1, 2 Chk2 substrates in Pirh2 −/− cells compared with wild-type controls. This finding suggested the possibility that accumulated Chk2 in Pirh2 −/− cells might be due to defective ubiquitylation of this kinase.…”

“…Interestingly, concomitant loss of Chk2 completely rescued this phenotype suggesting that increased Chk2 levels and activity were responsible for this hyperactivation of the cell cycle checkpoints. 7 …”

“…This suggests a strong role for Chk2 phosphorylation for the regulation of its stability, especially following DNA damage. 7 USP28, a deubiquitylating enzyme that is a downstream target of ATM in response to DNA damage, is needed to stabilize Chk2 in response to DNA damage. 40 We found that USP28 counteracts Pirh2 ubiquitylation of Chk2.…”

“…40 We found that USP28 counteracts Pirh2 ubiquitylation of Chk2. 7 To examine the effect of Pirh2 deficiency on Chk2 physiological activity we examined cell cycle checkpoint responses of Pirh2 was observed in WT cells. Interestingly, concomitant loss of Chk2 completely rescued this phenotype suggesting that increased Chk2 levels and activity were responsible for this hyperactivation of the cell cycle checkpoints.…”

“…[3][4][5][6] We have recently identified the tumor suppressor and cell cycle regulator Chk2 as an additional target of Pirh2. 7 In what follows we show that through its E3 ligase activity Pirh2 acts as a key regulator of cell proliferation and cell cycle progression under both normal and stressed conditions.…”