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Home , Cell cycle checkpoint, DUSP2, G2 phase, PPM1D, S phase

COMMENTARY reverse -mediated checkpoints

Lawrence A. Donehower1 for the damage or dysfunction to be repaired Departments of Molecular Virology and Microbiology, Molecular, and Cellular Biology, Pediatrics without being transmitted to progeny cells. and the Sequencing Center, Baylor College of Medicine, Houston, TX 77030 Thus, p53 has been called the “guardian of the genome” (3).Giventheimportanceof p53 as a cellular failsafe mechanism, it is The Genome Atlas and other large- In the normal cell, the p53 func- not surprising that its inactivation is a highly scale cancer genome sequencing projects tions as a stress integrator that becomes selected event in cancer progression. have identified an impressive number of activated in response to myriad dysfunctional Activated p53 can halt in both states (e.g., damaged DNA, aberrant onco- significantly mutated that are likely the G1 and G2 phases of the cell division genic signaling, and hypoxia). Once activated, drivers of cancer progression (1). These stud- cycle. G1 is the preparation phase of the cell p53 can initiate stress response programs to ies have definitively confirmed that the before replication of its DNA and G2 pre- eliminate the dysfunctional state, such as encoding the p53 tumor suppressor is the pares the cell for . Arrest and repair of repair of DNA damage. If the stressed cell most frequently mutated gene in human can- cells before DNA replication or mitosis are is dividing, p53 can enact any one of several cers. A major component of the p53 antican- likely to prevent damage-induced mutational antiproliferative programs. Under low to cer response is its ability to arrest cell division events or abnormal segregation moderate stress conditions, the dividing cell events, respectively. Once repair of damage is at different stages of the cell cycle. In PNAS, may be transiently arrested. Alternatively, achieved, the cell uses pathways to relieve the a study by Shaltiel et al. provides additional under conditions of high stress, the cell p53-mediated cell cycle arrest and return to insights into the mechanisms of reversal of may be permanently arrested (senescence) a normal dividing state. the p53-mediated cell cycle arrest (2). Such or eliminated completely in an ordered Much of the p53-mediated cell cycle arrest insights may provide new cancer therapeutic manner (). In those cases where cell is effected by that phosphorylate p53 opportunities. division is merely arrested, time is allowed (Fig. 1). Therefore, phosphatases acting on the p53 phosphorylation sites are natural candidates to reverse cell cycle arrest. Unfor- DAMAGE-INDUCED CHECKPOINTS RECOVERY FROM CHECKPOINTS tunately, our knowledge of phosphatases that remove phosphate groups from p53 (about seven identified thus far) is less advanced

P than our knowledge of the kinases that phos- phorylate it (at least 33 identified) (4). In CHK2 ATM 2005, my laboratory reported that the ser- PPM1D G1 Progression ine/threonine WIP1 ( ) could dephosphorylate p53 at serine 15, the same site phosphorylated by the p53-activat- PP4 ing ATM (5), and act to reverse the G2/M cell cycle arrest caused by DNA dam- P P age. The Medema laboratory later showed that WIP1 was able to relieve G2 arrest by KAP1 p53 G1 Block KAP1 p21 interfering with the ability of p53 to repress expression of key G2/M progression factors P such as B1 (6) (Fig. 1). Interestingly, G2/M Block p53 G2/M Progression WIP1 had earlier been discovered as a p53 CCNB1 p53 transcriptional target gene (7). Thus, WIP1 is likely to operate as part of a negative feedback CCNB1 P WIP1 regulatory loop with p53 in which damage- p53 activated p53 up-regulates WIP1 that accu- WIP1 mulates and inactivates p53 once damage is repaired. The suppressor (WIP1) of a tumor Fig. 1. Phosphatases PP4 and WIP1 reverse G1 and G2/M checkpoints. Following DNA damage, ATM and CHK2 kinases phosphorylate p53 and KAP1. Phosphorylation of p53 stimulates its transcriptional activation of the p21 gene, resulting in a G1 cell cycle arrest. ATM and CHK2 phosphorylation of KAP1 prevents its normal repression of p21 RNA expression. In Author contributions: L.A.D. wrote the paper. addition, p53 directly binds to genes that mediate G2/M progression such as (CCNB1) and inhibits their ex- The author declares no conflict of interest. pression, enhancing G2/M cell cycle arrest. The phosphatase WIP1 is up-regulated by p53 and participates in a negative feedback loop that relieves the G2/M block through of p53 (facilitating degradation of p53). The G1 See companion article on page 7313. block is relieved by PP4 dephosphorylation of KAP1, resulting in its reactivation and suppression of p21 RNA transcription. 1E-mail: [email protected].

7172–7173 | PNAS | May 20, 2014 | vol. 111 | no. 20 www.pnas.org/cgi/doi/10.1073/pnas.1405663111 Downloaded by guest on September 30, 2021 suppressor (p53) is a strong candidate to damage response pathways (12). When the importance of multiple phosphatases in pro- COMMENTARY have oncogenic properties. Indeed, the gene authors depleted PP4 or its regulatory sub- moting recovery from the checkpoint. They encoding WIP1, PPM1D, has been shown to unit PP4R2 from irradiated test cells, recov- identified a phosphatase (PP4) involved in be amplified and overexpressed in a number ery from G1 arrest was dramatically reduced. p53 checkpoint recovery and mechanistically of human (8). However, depletion of both PP4 and PP4R2 showed how PP4 and WIP1 act in check- A second phosphatase shown to act on had no effect on G2 arrest recovery, indi- point relief. WIP1 acts primarily in G2 by p53 was the dual-specificity phosphatase 26 cating a critical role for PP4 only in regulat- preventing p53 suppression of key G2/M (DUSP26). It is an inhibitor of p53-mediated ing the G1 checkpoint. Importantly, it was drivers, whereas PP4 acts in G1 by promot- functions through dephosphorylation of ser- demonstrated that the PP4-mediated check- ing down-regulation of p21CIP1 (Fig. 1). ine 20 and 37, reversing the effects of the point regulation was p53 dependent, as cells Naturally, a study such as this raises damage-induced kinases CHK1/2 and ATR codepleted for PP4 and p53 showed a com- additional questions. Are there additional (9). DUSP26 was shown to be overexpressed plete checkpoint recovery. mechanisms of p53 checkpoint relief? Are in primary neuroblastomas and neuroblas- PP4 regulation of the p53-mediated G1 there undiscovered phosphatases that act toma cell lines, implicating it as a potential checkpoint was shown to be related to the on p53 to relieve the checkpoint? As of this oncogene. Yet another phosphatase that activities of p53 transcriptional target gene study, four phosphatases, WIP1, DUSP26, inhibits p53 signaling is PP1, which has been p21CIP1, a key inhibitor of the cyclin–CDK PP1, and PP4, have been definitively shown shown to dephosphorylate serine 15 on p53 complexes that drive cell cycle progression. to dephosphorylate p53 or its effectors in the and repress p53 activity through dephos- KRAB domain-associated protein 1 (KAP1) regulation of p53 checkpoint functions. Ad- phorylation and stabilization of the p53 in- is a corepressor associated with the promoter ditional screening approaches should pro- hibitor MDM4 (10). The catalytic subunit of of the p21 gene (13). In unstressed cells, it vide new candidates and new mechanisms PP1, PP1CA, has also been demonstrated to inhibits p21 expression. However, on DNA of checkpoint relief in the coming years. be amplified and overexpressed in oral squa- damage, ATM and CHK2 phosphorylate Of the four phosphatases that have been mous cell carcinomas (11). KAP1 at serines 824 and 473, respectively. shown to inhibit p53 checkpoint functions, The key role of WIP1 in relieving G2 arrest KAP1 phosphorylation deactivates KAP1 all four have been shown to be overexpressed raised the question of whether WIP1 also suppressive function and p21 expression or amplified in some tumor types, implicat- functions in regulating the p53-mediated G1 is increased (Fig. 1). The authors decided ing them as oncogenic cancer drivers (8, 9, arrest. Shaltiel et al. address this question in to examine whether PP4 might reactivate 11, 14, 15). A number of laboratories and an elegant set of experiments using immor- KAP1 activity through dephosphorylation pharmaceutical companies have developed talized retinal pigment epithelial cells express- of S824 and S473. Indeed, they found that small molecule inhibitors to WIP1 that have ing G1 and -specific fluorescent PP4 or PP4R2 depletion produced an en- shown significant efficacy in preclinical stud- fusion in cells transfected with hanced and prolonged phosphorylation of ies in cancer cells with intact p53 (16, 17). WIP1 and control siRNAs (2). When these S473, associated with increased p21 expres- With the discovery of PP4 as a p53 G1 check- cells were DNA damaged with 1 Gy of ion- sion. The authors concluded that PP4 is an point regulator, it may be worth considering izing radiation during , siRNA de- essential component of the recovery from G1 combinatorial approaches with PP4 and pletion of WIP1 reduced entry of cells into arrest through its effects on p21 expression. WIP1 inhibitors in which both p53-mediated mitosis by half relative to control cells, con- That PP4 may also inhibit p53 signaling in G1 and G2 checkpoints can be simulta- sistent with a role for WIP1 in regulating a cancer context is suggested by findings that neously enhanced, perhaps leading to more the G2 . In contrast, PP4 is overexpressed in breast, lung, and robust antiproliferative or apoptotic responses WIP1 siRNA had no apparent effect on pancreatic cancers (14, 15). in cancer cells. At the very least, this exciting cells irradiated in G1 in their subsequent This study by Shaltiel et al. represents study should lead to increased interest in entry into S phase. Experiments in another a major advance in our understanding of phosphatases as key regulators of stress cell type confirmed that WIP1 has no direct p53-mediated checkpoint functions and the responses and cancer signaling pathways. role in regulation of G1 cell cycle arrest. Because the phosphorylation of p53 is so closely linked to its effect on the G1 check- 1 Lawrence MS, et al. (2013) Mutational heterogeneity in functions in human neuroblastoma. Oncogene 29(35): point, the authors initiated a screen for novel cancer and the search for new cancer-associated genes. Nature 4938–4946. 499(7457):214–218. 10 Lu Z, Wan G, Guo H, Zhang X, Lu X (2013) phosphatases that might reverse the p53- 2 Shaltiel IA, et al. (2014) Distinct phosphatases antagonize the 1 inhibits p53 signaling by dephosphorylating and stabilizing Mdmx. mediated G1 arrest. They screened an siRNA p53 response in different phases of the cell cycle. Proc Natl Acad Sci Cell Signal 25(4):796–804. library containing 224 phosphatases and USA 111:7313–7318. 11 Hsu LC, Huang X, Seasholtz S, Potter DM, Gollin SM (2006) Gene 3 Lane DP (1992) Cancer. p53, guardian of the genome. Nature amplification and overexpression of protein phosphatase 1alpha in oral phosphatase regulators for those phospha- 358(6381):15–16. squamous cell carcinoma cell lines. Oncogene 25(40):5517–5526. tases that affected the kinetics of S phase 4 Meek DW, Anderson CW (2009) Posttranslational modification of 12 Lee DH, et al. (2012) Phosphoproteomic analysis reveals that PP4 entry following ionizing radiation exposure p53: Cooperative integrators of function. Cold Spring Harb Perspect dephosphorylates KAP-1 impacting the DNA damage response. Biol 1(6):a000950. EMBO J 31(10):2403–2415. of siRNA-treated cells. A number of candi- 5 Lu X, Nannenga B, Donehower LA (2005) PPM1D 13 Li X, et al. (2007) Role for KAP1 serine 824 phosphorylation and date phosphatases were identified as potential dephosphorylates Chk1 and p53 and abrogates cell cycle sumoylation/desumoylation switch in regulating KAP1-mediated checkpoints. Genes Dev 19(10):1162–1174. transcriptional repression. J Biol Chem 282(50):36177–36189. regulators of G1 checkpoint recovery, and 6 Lindqvist A, et al. (2009) Wip1 confers G2 checkpoint recovery 14 Wang B, et al. (2008) Protein phosphatase PP4 is overexpressed PP4, PTPRN2, PTPN6, and DUSP2 were competence by counteracting p53-dependent transcriptional in human breast and lung tumors. Cell Res 18(9):974–977. confirmed with additional siRNA experi- repression. EMBO J 28(20):3196–3206. 15 Weng S, et al. (2012) Overexpression of protein phosphatase 4 7 Fiscella M, et al. (1997) Wip1, a novel human protein phosphatase correlates with poor prognosis in patients with stage II pancreatic ductal ments. The authors chose to further investi- that is induced in response to ionizing radiation in a p53-dependent adenocarcinoma. Cancer Epidemiol Biomarkers Prev 21(8):1336–1343. gate the serine/threonine phosphatase PP4. manner. Proc Natl Acad Sci USA 94(12):6048–6053. 16 Rayter S, et al. (2008) A chemical inhibitor of PPM1D that selectively PP4 is a type 2A phosphatase previously 8 Bulavin DV, et al. (2002) Amplification of PPM1D in human tumors kills cells overexpressing PPM1D. Oncogene 27(8):1036–1044. abrogates p53 tumor-suppressor activity. Nat Genet 31(2):210–215. 17 Gilmartin AG, et al. (2014) Allosteric Wip1 phosphatase implicated in reversing phosphorylation 9 Shang X, et al. (2010) Dual-specificity phosphatase 26 is inhibition through flap-subdomain interaction. Nat Chem Biol 10(3): of proteins involved in ATM-driven DNA a novel p53 phosphatase and inhibits p53 tumor suppressor 181–187.

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