Distinct phosphatases antagonize the p53 response in different phases of the cell cycle

Abstract

The basic machinery that detects DNA damage is the same throughout the cell cycle. Here, we show, in contrast, that reversal of DNA damage responses (DDRs) and recovery are fundamentally different in G1 and G2 phases of the cell cycle. We find that distinct phosphatases are required to counteract the checkpoint response in G1 vs. G2. Whereas WT p53-induced phosphatase 1 (Wip1) promotes recovery in G2-arrested cells by antagonizing p53, it is dispensable for recovery from a G1 arrest. Instead, we identify phosphoprotein phosphatase 4 catalytic subunit (PP4) to be specifically required for cell cycle restart after DNA damage in G1. PP4 dephosphorylates Krüppel-associated box domain-associated protein 1-S473 to repress p53-dependent transcriptional activation of p21 when the DDR is silenced. Taken together, our results show that PP4 and Wip1 are differentially required to counteract the p53-dependent cell cycle arrest in G1 and G2, by antagonizing early or late p53-mediated responses, respectively. A cell's genomic integrity is constantly challenged by endogenous and exogenous sources of DNA damage. Doublestrand breaks (DSBs) are particularly threatening to the genomic stability of proliferative cells and provoke a checkpoint response that coordinates repair processes with further cell cycle progression to prevent the replication and segregation of broken DNA. This DNA damage response (DDR) is orchestrated by multiple kinases that sense the DNA damage and relay this signal (1). Cellular recovery from a DNA damage insult ultimately requires the termination of the DDR once repair of the DNA is complete.PI3-kinase-related kinases (PIKKs), ataxia telangiectasia mutated (ATM), and ATM-and Rad3-related (ATR) are activated by distinct structures of damaged DNA and phosphorylate histone H2AX in the vicinity of the damaged site to recruit repair proteins (2). In addition to such local events, ATM and ATR activate a subsequent layer of checkpoint kinase 2 (Chk2) and Chk1, respectively, that disseminates from the damaged site (3, 4). ATM also activates p38 mitogen-activated protein kinase (MAPK), which coordinates the DDR outside the nucleus (5, 6). Combined, these checkpoint kinases ensure that cell cycle progression is prevented at the G1/S or G2/M boundary (7).PIKKs and checkpoint kinases commonly converge on the transcription factor p53, a key regulator of stress responses. Phosphorylation of p53 prevents its degradation by mouse double minute 2 (Mdm2)-mediated polyubiquitination, allowing p53 to accumulate and induce its target genes, including p21 (1). Both p53 and its transcriptional target p21 are sufficient to impose an arrest in both G1 and G2, and they are absolutely required for a bona fide checkpoint arrest in G1 (8-11).Recovery from a checkpoint-induced arrest requires silencing of the checkpoint machinery and coincides with the removal of phosphorylations deposited by PIKKs and other checkpoint kinases. We have previously shown that WT p53-induced phosphatase 1 (Wip1) is essential for checkpoint recovery from a...

The same, only different – DNA damage checkpoints and their reversal throughout the cell cycle

“…G2 checkpoint recovery might thus represent checkpoint adaptation, where DNA damage triggers an arrest whose duration is not necessarily conditioned by DNA repair. In support of this notion, the kinetics of recovery from DSBs in G2 in non-transformed cell lines and the mouse intestinal epithelium in vivo indeed show relatively synchronous mitotic entry after a delay of several hours (Chwalinski and Potten, 1986;Shaltiel et al, 2014). However, it is important to note that the inducible expression of a constitutively active mutant of Plk1 (T210D) fails to override an established DNA damage checkpoint (R.H.M., unpublished results), indicating that Plk1 activity is insufficient to drive mitotic entry of cells with a fully activated DDR.…”

“…Similarly, PP4 dephosphorylates cH2AX, RPA2, 53BP1, CHD4 and KAP1, but recovery in its absence is mostly affected in G1 cells (Fig. 3), and can be effectively overcome by expression of a non-phosphorylatable mutant of KAP1 (S473A) Shaltiel et al, 2014).…”

“…Chemical inhibitors of checkpoint kinases, such as caffeine, present a useful tool to separate defects in DNA repair from defects in terminating the checkpoint downstream of these kinases (Davies et al, 2004;van Vugt et al, 2005). In contrast to PP2A, loss of Wip1 or PP4 have limited effects on DSB repair, but strongly affect checkpoint silencing, cell cycle restart and survival after DNA damage, even in the presence of checkpoint kinase inhibitors (Lindqvist et al, 2009a;Moon et al, 2010;Nakada et al, 2008;Shaltiel et al, 2014). Crucial functions of these phosphatases in checkpoint reversal are limited to a select number of substrates, possibly owing to the extensive redundancy between phosphatases.…”

“…In addition, G2-arrested cells that activate APC/C Cdh1 are likely to be subjected to a G1-type DDR, owing to the lack of Cdk1/2 activity. Reversal of the G1 DNA damage checkpoint appears to be distinct from G2 as, firstly, the crucial players that promote checkpoint recovery in G2 are absent (Plk1 and Cdc25B) or do not affect recovery in G1 (Wip1; Shaltiel et al, 2014). Secondly, unlike the G2 checkpoint, the G1 checkpoint remains reversible for several days in untransformed cells and the mechanisms that cause an irreversible arrest in G2 (Fig.…”

T cell proliferation and adaptive immune responses are critically regulated by protein phosphatase 4

“…16 In contrast, knockout of PP4 by the CD4 promoter-driven Cre recombinase transgene (CD4-cre) does not significantly impact thymocyte differentiation, but partially impairs regulatory T cell functions to induce the onset of spontaneous colitis. 17 Recently, PP4 has also been implicated in DNA damage response via its ability to either permit cell cycle reentry, 18 dephosphorylate gH2AX, 19,20 regulate the activity of KAP1, 21 or control cell cycles in Drosophila 22 or yeast 23 ; however, its role in regulating mammalian cell proliferation has not been thoroughly investigated. Finally, it is worth noting that okadaic acid (OA), which is generally recognized as a specific inhibitor of PP2A, actually also suppresses PP4 activity with equal 24 or better 25 efficacy; these results then raise the possibility that many biological processes, such as IL-2 signaling modulation, 26,27 AMPK activation 28 and the regulation of T cell proliferation, 29 that have been linked to PP2A via OA treatments may actually be attributed to the functions of PP4.…”