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In response to DNA damage eukaryotic cells activate cell cycle checkpoints - complex kinase signaling networks that prevent further progression through the cell cycle. In parallel to implementing a cell cycle arrest, checkpoint signaling also mediates the recruitment of DNA repair pathways. If the extend of damage exceeds repair capacity, additional signaling cascades are activated to ensure elimination of these damaged cells. The DNA damage response has traditionally been divided into two major kinase branches. The ATM/Chk2 module is activated after DNA double strand breaks and the ATR/Chk1 pathway responds primarily to DNA single strand breaks or bulky lesions. Both pathways converge on Cdc25, a positive regulator of cell cycle progression, which is inhibited by Chk1 or Chk2-mediated phosphorylation. Recently a third effector kinase complex consisting of p38MAPK and MK2 has emerged. This pathway is activated downstream of ATM and ATR in response to DNA damage. MK2 has been shown to share substrate homology with both Chk1 and Chk2. Here we will discuss recent advances in our understanding of the eukaryotic DNA damage response with emphasis on the Chk1, Chk2, and the newly emerged effector kinases p38MAPK and MK2.
To maintain genomic integrity and faithful transmission of fully replicated and undamaged DNA during cell division, eukaryotic organisms evolved a complex DNA surveillance program. Prior to mitosis cells progress through G1/S, intra-S and G2/M cell cycle checkpoints [1–3]. Checkpoint signaling is activated in response to incomplete DNA replication due to stalled replication forks, and damaged DNA induced by both internal and external sources such as UV light, ionizing radiation, reactive oxygen species or DNA damaging chemotherapeutic agents. Active checkpoints prevent further progression through the cell cycle. If the genotoxic insult exceeds repair capacity, additional signaling pathways, leading to cell death, presumably via apoptosis, are activated [1,4,5].
The canonical DNA damage response network has traditionally been divided into two major kinase signaling branches utilizing the upstream PI3-Kinase-like kinases ATM and ATR. These kinases control the G1/S, intra-S and G2/M checkpoints through activating their downstream effector kinases Chk2 and Chk1, respectively [1–3]. We have recently identified the p38MAPK/MAPKAP-K2 (MK2) complex as a third checkpoint effector module that operates parallel to Chk1 and is activated downstream of ATM and ATR [6,7].
How are DNA lesions, such as double strand breaks (DSBs), initially detected and transduced into signals activating ATM and/or ATR? The earliest events likely involve alterations in chromatin structure [8–10], but the biochemical details underlying this are poorly understood. Among the earliest events is recruitment of a mediator complex consisting of Mre11, Rad50 and Nbs1 (MRN), and phosphorylation of a variant H2A histone – H2AX –in the DNA near the break, extending for distances up to several megabases . Working together, MRN and phosphorylated H2AX (γH2AX) act as a signal amplifier that recruits additional signaling molecules to the DSB lesion and dramatically enhances local activation of ATM. The MRN complex serves as an initial DSB sensor, at least one component of which (Nbs1) localizes to the break in an H2AX-independent manner [12,13] and facilitates recruitment of ATM to the site of the lesion .
Inactive ATM appears to exist as a multimer that dissociates into active monomers upon activation . Bakkenist and Kastan showed that human ATM underwent a rapid phosphorylation on Ser-1981 following as little as 0.5 Gy of IR and that phosphorylation at this site appeared to correlate with dissociation of ATM into active monomers based on cross-linking studies and co-immunoprecipitation experiments. Furthermore, mutation of Ser-1981 to Ala was shown to block human ATM dissociation in 293 cells, and had a dominant inhibitory effect on both the IR-induced G2/M and S-phase checkpoints when transfected into HeLa cells . However, Pellegrini et al.  generated a murine Ser-1987 to Ala knock-in mouse (corresponding to human Ser-1981) and showed that ATM activation and checkpoint responses after IR appeared to be normal. Thus, the precise role of Ser-1981 phosphorylation in ATM activation remains somewhat unclear. Intriguingly, Price and colleagues recently reported that acetylation of Lys-3016 in the FATC domain of human ATM appears to be required for ATM activation following IR, and acetylation at this site shows identical kinetics as Ser-1981 phosphorylation [17,18]. The acetyl transferase likely responsible for ATM acetylation at Ser-3016 is Tip60, a recently described haploinsufficient tumor suppressor that in addition to forming complexes with ATM, also binds to the MRN complex at double strand breaks via its-cofactor Trrap and is required for efficient loading of Brca1 and 53BP1 onto damaged chromatin [19–21]. RNAi-mediated Tip60 depletion or expression of a dominant negative Tip60 mutant was shown to result in diminished ATM activation and reduced cell survival following DNA damage, suggesting that acetylation of ATM, rather than auto- or cross-phosphorylation, may be the critical activating event .
Active ATM then phosphorylates the variant histone H2AX on a critical Ser residue, Ser-139, yielding γ-H2AX [3,11]. The phosphorylation site on H2AX, corresponding to the sequence S-Q-E-Y, is subsequently recognized by the phosphopeptide-binding BRCT domains of another mediator protein, MDC1 [22–24], which in turn gets phosphorylated by ATM at multiple sites, allowing MDC1 to recruit an E3 ubiquitin ligase complex through binding to the ring-finger protein RNF8. The E3 ubiquitin ligase RNF8, together with the E2 enzyme Ubc13, in turn mediates ubiquitination of H2AX, and possibly H2A or other chromatin-associated proteins, ultimately recruiting a complex consisting of Rap80, Abraxas and BRCA1 via the ubiquitin interaction motif in Rap80 [25–28]. BRCA1, along with 53BP1, which is also recruited downstream of RNF8, play important roles in DNA repair via homologous recombination (HR) and non-homologous end joining (NHEJ), respectively [29,30]. In addition, MDC1 also undergoes phosphorylation by CK2 after DNA damage to generate a phospho-motif on MDC1, which binds directly to Nbs1. This CK2-dependent recruitment of Nbs1 seems to be important for stabilizing MRN at the DSB [31,32]. In addition, Nbs1 can also immobilize ATM at the site of the DSB via direct binding of ATM to a C-terminal ATM interaction motif on Nbs1 . Thus, MDC1, through ATM- and CK2-directed phosphorylation, promotes tighter binding of the MRN complex and ATM at the site of the DSB, forming a powerful ATM auto-amplification loop.
When replication forks stall during S-phase, MCM helicases continue to unwind the DNA template, resulting in exposure of stretches of ssDNA, which are rapidly coated by the ssDNA-binding protein RPA . RPA bound to ssDNA recruits ATR through direct binding of its regulatory subunit ATRIP to RPA . The precise biochemical mechanism responsible for activating ATR following its recruitment to ssDNA lesions is largely unknown, although the process appears to involve ATR-ATRIP interaction with an additional DNA-associated protein complex composed of Rad9-Rad1-Hus1 (9-1-1) and TopBP1. The 9-1-1 complex functions as a clamp, encircling the DNA, and recruits the BRCT domain-containing protein TopBP1 in a phospho-dependent manner (for a detailed review see ). Dunphy and colleagues have shown in Xenopus extracts that TopBP1 is required for proper ATR activation and replication checkpoint control . Although ATR is thought to be activated primarily by single stranded DNA lesions, under certain circumstances it can also be activated downstream of, DSBs. If cells encounter DSBs in late S-phase or G2, ATR is activated in an ATM-dependent manner likely through the generation of RPA coated ssDNA by the endo- and/or exonuclease activity of Mre11/CtIP complexes [37,38].
The locally increased ATM activity is believed to be important for efficient phosphorylation of ATM substrates including the downstream effector kinase Chk2 and the prominent tumor suppressor protein p53. Several groups have reported that within the nucleus ATM phosphorylates Chk2 on Thr-68 in a Ser/Thr cluster following IR in vitro and in vivo [39–41]. This phosphorylation is thought to be involved in Chk2 activation, since Thr-68-Ala mutation results in impaired Chk2 activation. However, phosphorylation on Thr-68 does not seem to be essential for Chk2 activation, since Thr-68-Ala mutation does not completely abolish IR-induced Chk2 activation, when the mutant is expressed on a wildtype background . In fact, it has been proposed that phosphorylation on Thr-68 triggers a chain of additional autophosphorylation events on Thr-383 and Thr-387 in the activation loop, which ultimately result in Chk2 activation [39,42]. In addition, oligomerization of Chk2 is believed to increase the kinase activity of Chk2 . Piwnica-Worms and colleagues recently confirmed Thr-68, 383 and 387 as Chk2 phosphorylation sites and also identified Ser-516 as an additional Chk2 phosphorylation site . Using Chk2-proficient cells transfected with a kinase dead Chk2 expression construct, Piwnica-Worms and colleagues demonstrated that exogenous Chk2 gets phosphorylated on Thr-68, 383 and 387. Ser-516 was not phosphorylated, suggesting autophosphorylation in cis at this site. However, when Chk2-deficient cells were transfected, only Thr-68 was found to be phosphorylated, suggesting that Thr-383 and 387 are indeed Chk2 autophosphorylation sites that can be phosphorylated in trans. Interestingly, Thr-68 was also found to be phosphorylated in response to IR even in ATM-deficient cells and in cells pretreated with caffeine, suggesting that kinases other than ATM, ATR or DNA-PKcs might be involved. Chk2 expressed in E. coli was found to be phosphorylated on Thr-68, 383, 387 and Ser-516, which correlated with increased activity even in the absence of IR. Similar findings were made when Chk2 was overexpressed in 293T cells or Chk2-deficient MEFs. These observations are in agreement with data from Gilbert et al., who suggested that locally increased concentrations of Rad53, the budding yeast homologue of Chk2, results in increased Rad53 kinase activity, as a result of trans-phosphorylation . Moreover, Chk2 oligomerization was found to be impaired when Thr-157 was mutated to Ile, a mutation in the FHA domain of Chk2, found in a subset of Li-Fraumeni syndrome patients . Notably, when either wildtype or Chk2-Thr157Ile were co-expressed in Chk2-deficient MEFs, Chk2-Thr157Ile displayed markedly reduced phosphorylation on Thr-68, 383 and 387 in the absence of IR. Whether this lack of phosphorylation is a reflection of impaired oligomerization or merely the result of gross structural irregularity remains elusive.
The effector kinase Chk1 is one of the best-studied ATR substrates and its effective activation by ATR-mediated phosphorylation requires the presence of the mediator protein claspin [45–47]. ATR phosphorylates Chk1 on two serine residues that lie outside the kinase domain, Ser-317 and 345, resulting in a marked increase in Chk1 activity . It remains unclear whether ATR kinase activity is required to recruit claspin to RPA-coated ssDNA. However, in the context of ssDNA generated during S-phase progression, ATR-mediated phosphorylation of the ssDNA binding protein Rad17 is required for claspin recruitment .
Besides activation of Chk1 and Chk2 following DNA damage, proteins involved in many different cellular processes, such as DNA repair and replication, RNA metabolism, nonsense mediated decay, regulation of translation, chromatin remodeling, circadian rhythm, insulin signaling, mitosis, regulation of transcription and MAPK signaling have been identified as putative ATM and ATR substrates by screening approaches over recent years [3,50]. Modulation of most of these events by ATM and ATR remain largely unexplored.
Both, the ATM/Chk2 and the ATR/Chk1 pathway converge to inactivate members of the Cdc25 family of dual-specificity phosphatases, which play an important role in driving dividing cells through the cell cycle . At the G1/S and G2/M transition these enzymes remove critical inhibitory phosphorylations on Cdk/Cyclin complexes, the most important of which is dephosphorylation of Tyr-15 in the ATP-binding loop of Cdks 1 and 2. Of the three known Cdc25 family members Cdc25A is believed to be a critical Chk1 substrate for the intra-S phase checkpoint, marking Cdc25A for rapid destruction by the 26S proteasome [51,52]. Recent data suggest a role for Cdc25A at the G2/M boundary, as well . The role of Chk2 in the regulation of Cdc25A is less clear. One report suggests that Chk2 may perform similar functions under certain conditions , although Harper and colleagues have recently brought this into question . Following intra S-phase checkpoint activation, Cdc25A is phosphorylated on the known Chk1 site Ser-76 and subsequently undergoes SCFβ-TRCP-dependent ubiquitination and proteasomal degradation . Recognition of Cdc25A by β-TRCP occurs via a noncanonical phosphodegron in Cdc25A that, besides pSer-76, involves pSer-79 and pSer-82, sites that do not match the basophilic Chk1 substrate motif . In addition to the intra S-phase checkpoint, which is governed by Chk1, Cdc25A is also targeted for ubiquitin-mediated degradation during G1. Piwnica-Worms and colleagues recently demonstrated that GSK3β phosphorylates Cdc25A on Ser-76 in G1 after priming of Cdc25A by Plk3-mediated phosphorylation of Thr-80 . Ser-76 phosphorylation by Chk1 or GSK3β appears to be a requirement for subsequent Ser 82 phosphorylation, which in turn is essential for Cdc25A recognition by SCFβ-TRCP [2,52,56–58]. At the G2/M boundary Cdc25B has been reported to be the starting phosphatase that initially activates Cdk1/CyclinB, which activates Cdc25C, creating an autoactivation loop . Cdc25B-1/2 and Cdc25C are functionally inactivated upon phosphorylation on Ser-309/323 and Ser-216, respectively, leading to 14-3-3 binding and nuclear exclusion [51,59]. In addition, Chk1 has also been shown to directly target and activate Wee1, the kinase directly responsible for the inhibitory phosphorylation of Cdk1 and 2 on Tyr-15. Dunphy’s group, using Xenopus extracts, and Piwnica-Worms’ group, using HeLa cells, showed that Chk1-dependent Wee1 phosphorylation on Ser-549 (Xenopus) and Ser-642 (human) promotes Wee1 association with 14-3-3, directly increasing its catalytic activity [60,61]. Mutation of Ser-549/642 to Ala prevented 14-3-3 binding and substantially reduced the ability of Wee1 to phosphorylate Cdk1. Furthermore, replacement of endogenous Wee1 with Wee1 carrying a Ser-549 Ala mutation in Xenopus egg extracts, or expression of the Ser-642 Ala mutant in HeLa cells, attenuated a Chk1-induced cell cycle delay . Similar observations were reported by Nurse and colleagues using S. pombe as a model system .
It has become increasingly clear that the DNA damage response network extends beyond the canonical ATM/Chk2 and ATR/Chk1 signaling modules, to include connections to pathways as diverse as those involving PI 3-kinase/AKT, IKK/NFκB, and various MAP Kinases (MAPKs) [63–66]. Multiple lines of evidence suggest an increasingly important role for the p38MAPK pathway and its downstream effector kinase MK2.
The p38MAPK/MK2 signaling complex is considered to be a general stress response pathway, which is activated in response to a variety of extrinsic and intrinsic stimuli including osmotic stress, heat shock, various toxins, UV and ionizing radiation, reactive oxygen species (ROS), cytokines, loss of centrosome integrity and DNA damage . p38MAPK activation drives a plethora of changes in transcription, protein synthesis, cell surface receptor expression, and cytoskeletal structure, ultimately affecting cell survival and apoptosis.
There are 4 p38MAPK isoforms denoted α, β, γ, and δ . Splice variants exist for p38α and β, giving rise to a total of six p38MAPK isoforms . p38α was first identified as a tyrosine-phosphorylated protein in extracts of LPS-treated macrophages and was subsequently shown to have significant homology with the yeast stress kinase HOG1 . Additional members of the p38MAPK family were cloned by homology. p38α appears to be ubiquitously expressed, with high levels in leukocytes, liver, spleen, bone marrow, thyroid, and placenta. In contrast, p38β shows some specificity for brain and heart, while p38γ is expressed at highest levels in skeletal muscle. p38δ has been reported to be expressed at highest levels in the lung, kidney, gut, salivary gland and endocrine organs such as the testis, ovary, adrenal-, and pituitary gland . Intriguingly, p38α exists in a stable complex with its downstream substrate, the kinase MK2 [69–72], and the stability of p38α has been reported to depend on the presence of MK2. Gaestel and colleagues reported modestly reduced levels of p38MAPK in MK2-deficient mice . Whether similar complexes exist with p38β, γ, or δ is less clear. As with all known MAPKs, p38MAPKs are activated by dual phosphorylation on a T-X-Y motif in the activation loop through the action of MAP kinase kinases (MAPKKs, or MKKs). MAPKKs in turn are activated by a plethora of different MAP kinase kinase kinases (MAPKKKs, or MEKKs).
Many upstream MAPKKKs are known to participate in activation of the p38MAPK cascade including MEKK1/4, ASK1, and TAO kinases, depending on the particular type of initiating stimulus, which the cell is responding to. Using functionally p53-deficient HeLa cells, Cobb and colleagues showed that the MAPKKK TAO, activated p38α in an ATM-dependent manner in response to UV, IR and hydroxyurea treatment . Davis and colleagues, on the basis of overexpression studies, mouse knock-outs, and siRNA experiments, have shown that the MAPKKs MKK3 and 6 are the major upstream activators of p38α, β2, and γ in response to hyperosmolar stress and signaling by the pro-inflammatory cytokines TNFα and IL-1, while MKK4 has a redundant role along with MKK3 and 6 in p38MAPK activation by UV-irradiation [75,76]. MKK4 is a known JNK MAPKK, suggesting that it may function as a signaling hub to integrate JNK and p38MAPK signaling . The upstream MAPKK activators of p38MAPK in response to genotoxic agents other than UV has not been reported.
Among the known substrates of p38MAPK are a number of transcription factors, including ATF1/2/6, MEF2A/C, p53, SAP1, STAT1, Gadd153 and Max, as well as the MAPK activated protein kinases MSK1 and 2, MNK1 and 2 and MK2, 3, and 5 (for detailed reviews see [67,78,79]).
Over the last decade a number of observations have been published that point to a critical role for the p38MAPK module as an integral part of the DNA damage response network. Besides UV, p38α and β have been shown to be activated by other, more DNA damage-specific agents, such as cisplatin, doxorubicin and temozolomide [6,7,74,80,81]. Hirose et al.  observed a p38MAPK-dependent G2/M arrest following temozolomiode exposure in a mismatch repair-proficient human glioma cell line. p38MAPK signaling was associated with nuclear inactivation of Cdc25C, and RNAi-mediated knock down of p38α or pharmacologic inhibition of p38α and β reversed these effects. Mikhailov et al.  were able to show that p38MAPK signaling is activated upon treatment of PtK1 cells with topoisomerse II inhibitors, and resulted in a late G2/early prophase (so-called ‘antephase’) delay prior to mitotic entry. This delay could be overridden upon pharmacological inhibition of p38α and β. In this cell type, p38α and β were not required for normal mitotic progression in the absence of topoisomerase inhibition, or for the spindle assembly checkpoint. Although not a direct genotoxin, Sun et al., examined H-rasV12 oncogene-induced replicative stress, and found that p38MAPK mediated activation of MK5 is required for H-rasV12-induced senescence in a murine model of DMBA-induced skin carcinogenesis. Induction of p38MAPK-dependent senescence was mediated by MK5-induced phosphorylation of p53 on Ser-37 , and suggests that the p38MAPK pathway may function as a tumor suppressor in this context. Further insight into the DSB-mediated activation of p38MAPK came from the study of VDJ recombination in DN3 thymocytes. During T-lymphocyte maturation, cells undergo physiological induction of DSBs by RAG recombinase, followed by DSB repair in a DNA-PKcs-dependent manner. Pedraza-Alva et al.  used wild-type (WT) DN3 thymocytes, as well as Rag−/− and Scid DN3s. WT cells were found to activate p38MAPK when DSBs were generated during VDJ recombination, and concomitantly underwent a G2/M arrest. While Rag−/− cells do not accumulate unresolved DSBs, Scid cells do so, due to a lack of DNA-PK activity, which is mutated on the Scid background. Since DNA-PK is a key component of the NHEJ repair machinery, Scid thymocytes are unable to repair the Rag induced DSBs. When the authors compared levels of activated p38MAPK in Rag−/− and Scid thymocytes, they found increased levels of active p38MAPK only in Scid cells. The authors further describe the accumulation of both phosho-p38MAPK and phospho-p53 in Rag−/− thymocytes expressing constitutively active MKK6, and suggested that p38MAPK, acting through p53, was responsible for the G2/M checkpoint observed in these cells. Phosphorylation of p53 on Ser-18 and Ser-389 (corresponding to human Ser-15 and Ser-392) depended on p38MAPK and was abolished by the addition of the p38MAPK inhibitor SB203580. This study indicated that the p38MAPK pathway is activated in response to the accumulation of DSBs independently of DNA-PK, and may be involved in a p53-dependent G2/M arrest.
Kurosu and colleagues examined the role of p38MAPK in Burkitt’s lymphoma cells treated with the topoisomerase II inhibitor etoposide. This DNA damage-specific approach revealed that p38MAPK is activated following induction of DNA DSBs after topoisomerase II inhibition. In subsequent experiments using pharmacological inhibition of p38MAPK, as well as inducible expression of a dominant negative p38MAPK mutant, they were able to demonstrate that p38MAPK is required for an etoposide induced G2/M arrest and that abrogation of this checkpoint resulted in increased apoptosis .
Fornace and colleagues made the important observation that p38α and β signaling is necessary for the initiation of a G2/M arrest after low-dose UV-irradiation, with p38α playing a more prominent role. Application of the specific p38MAPK inhibitor SB202190, or reduction of p38α and/or β levels using antisense oligonucleotides reversed this effect resulting in sustained mitotic activity for the first several hours after irradiation, although the mitotic index fell to similar levels as those observed in control cells 8 hrs later . This group further reported that p38MAPK-containing immunoprecipitates contained a kinase activity capable of directly phosphorylating Cdc25B and C to generate critical 14-3-3 binding sites, an effect which was interpreted as evidence that p38MAPK could directly generate 14-3-3-binding sites on Cdc25B/C. Ben-Levi et al. , however, demonstrated that p38α forms a tight nuclear complex with its downstream substrate MK2 and that upon activation of p38MAPK in this complex, p38MAPK phosphorylates and activates MK2. This phosphorylation is essential for the nuclear export of the p38MAPK/MK2 complex in response to arsenite, and presumably other activating stimuli [69,70,78]. Based on a long–standing interest in p38MAPK and its substrates, we determined the optimal sequence motifs phosphorylated by p38α and its downstream effector kinase MK2  using oriented peptide library screening. The optimal p38MAPK motif requires the presence of a Pro immediately C-terminal to the phospho-acceptor Ser or Thr residue, and does not conform to the critical 14-3-3- binding sites on Cdc25B or C. In contrast, the optimal phosphorylation motif found for MK2, ([L/I/F]-X-R-[Q/M/S/T]-X-[S/T]-, where [S/T] denotes the phosphoacceptor residue and indicates a hydrophobic amino acid, perfectly matches the known pSer-323 14-3-3 binding site in Cdc25B, as well as pSer-216 14-3-3 binding site in Cdc25C . Manke et al. went on to demonstrate that recombinant MK2 from bacteria directly phosphorylated Cdc25B on Ser-323, thereby generating a 14-3-3 binding site on this molecule, and that RNAi-mediated knockdown of MK2 abolished the increase in 14-3-3 binding to cdc25B and C upon UV irradiation. Ducommun’s lab recently mapped the p38MAPK and MK2 phosphorylation sites on Cdc25B by mass spectrometry, providing independent confirmation that MK2 generates the Ser-323 binding site for 14-3-3 . Manke et al. found that MK2-depleted cells were defective in both the G1/S and G2/M checkpoints after irradiation, rendering them more sensitive to UV-induced cell death as a consequence of mitotic catastrophe following checkpoint dysfunction. Together, these observations indicated that MK2 is the critical checkpoint effector that functions downstream of p38MAPK to arrest cell cycle progression in response to UV irradiation.
This role as a cell cycle regulator for MK2 appears to be highly conserved among eukaryotes. Lopez-Avilez et al.  showed that overexpression of Srk1, the S. pombe homologue of human MK2, caused a delay in mitotic entry while cells lacking Srk1 were shown to enter mitosis prematurely. These cell cycle regulatory effects resulted from Srk1 interaction with, and phosphorylation of, Cdc25 at sites at least partially identical to those phosphorylated by Chk1 and Cds1, the S. pombe homologue of Chk2. Following Srk1-dependent phosphorylation, Cdc25 bound to Rad24, one of two S. pombe 14-3-3 proteins, leading to Cdc25 nuclear exclusion, stabilization, and catalytic inhibition, essentially recapitulating the identical MK2-Cdc25-14-3-3 pathway seen in mammalian cells. In contrast to mammalian cells, however, Srk1-deficient S. pombe cells did not display increased sensitivity to UV irradiation, although depletion of Chk1 rendered them highly sensitive. Thus, in fission yeast, it appears that Srk1 functions during the normal G2 phase of an unperturbed cell cycle, but is not part of the UV-induced DNA damage cell cycle checkpoint response. Srk1 activation towards Cdc25 is controlled by Stk1, the S. pombe homologue of p38MAPK, with which Srk1 forms a stable complex in the resting state .
The budding yeast S. cerevisiae contains two potential MK2 homologues, rck1 and rck2. In an unbiased global screen of nearly 5,000 haploid deletion strains, Begley et al. observed that loss of rck2, resulted in increased UV sensitivity, indicating that this MK2 homologue is physiologically important for the cellular response to UV-induced damage in budding yeast .
Mammalian MK2, like the fission yeast homologue, may also control cell cycle progression in response to stimuli other than direct DNA damage. Huard et al.  recently reported that the viral protein R (VPR) of HIV, which induces a G2 arrest in lymphocytes to block clonal T-cell expansion, does so by activating MK2. Similar to our observations on UV-activation of MK2, this VPR-activated form of MK2 was found to mediate phosphorylation of Cdc25C on the known Ser-216 14-3-3-binding site. Okamoto et al. reported that HIV gp120, which induces a G1 arrest in neuronal stem cells, and may contribute to HIV-induced dementia, also functions through the activation of the p38MAPK/MK2 pathway. These HIV-related findings suggest that global cellular stresses from viral infection events trigger MK2-mediated cell cycle arrest, likely independently of damage to the DNA backbone or bases .
When DNA is intentionally damaged by anti-cancer chemotherapeutic agents in mammalian cells, activation of the p38MAPK pathway appears to require ATM and ATR. Using ATM and ATR-deficient human cells as well as pharmacological inhibition of ATM and ATR, Reinhardt et al. recently showed that the p38MAPK/MK2 module operates downstream of ATM and ATR in response to cisplatin, camptothecin and doxorubicin . Interestingly, p38MAPK/MK2 activation in response to UV was shown to be independent of ATM and ATR, suggesting that UV likely causes cellular lesions other than DNA damage that result in p38MAPK activation. Importantly, cisplatin- and doxorubicin-mediated activation of the p38MAPK/MK2 module was independent of Chk1, and conversely, Chk1 activation was independent of MK2 activity, indicating that the ATR/ATM-p38MAPK-MK2 pathway functions in parallel with the ATR-Chk1 pathway. The notion that MK2 operates in a synergistic parallel pathway to the classical checkpoint effectors Chk1 and Chk2 is further supported by the observation that S. cerevisiae MK2 homologues rck1 and rck2 can function as extragenic suppressors of S. pombe cell cycle checkpoint mutations in chk1, as well as mutations in rad1, rad9, rad17, and rad26 , reversing the sensitivity of these strains to UV and IR, and to the replication blocker hydroxyurea. The dependence on the canonical DNA damage response kinases ATM and ATR for p38MAPK/MK2 activation following genotoxic stress has now been confirmed independently [74,93].
To further investigate whether there was a context-dependent role for MK2 in cell survival following genotoxic stress, Reinhardt et al. recently investigated the effects of MK2 depletion in p53-proficient and p53-deficient MEFs . MK2 activity appeared to be dispensable for cellular survival following cisplatin or doxorubicin in p53-proficient cells. However, MK2 depletion in p53-deficient MEFs resulted in an increased sensitivity to both cisplatin and doxorubicin. This synthetic lethality between the prominent tumor suppressor p53 and MK2 was due to the inability of p53-deficient cells expressing MK2 shRNA to execute functional G1/S and G2 cell cycle checkpoints following doxorubicin or cisplatin. Similar results were obtained when allograft tumors derived from p53-deficient, H-rasV12-transformed MEFs were examined for chemosensitivity in a nude mouse model in vivo. Knockdown of MK2 in these tumors resulted in dramatically increased sensitivity to systemic chemotherapeutic treatment with cisplatin or doxorubicin. Intriguingly, MK2-depleted tumors grew more rapidly than control tumors, indicating a potential role of MK2 as a negative regulator of the unperturbed cell cycle in tumor cells, and a potential tumor suppressor.
Although several groups have now confirmed members of the Cdc25 family as physiological MK2 substrates following genotoxic stress [6,7,86,90], the question remains whether other checkpoint-relevant substrates for MK2 exist in vivo. It is intriguing to speculate that a pool of substrates exists that is not shared by Chk1 and MK2. For example, although both kinases recognize and phosphorylate the same optimal amino acid motif sequence, and both are present in the nucleus, active MK2 has been shown to translocate to the cytoplasm, in response to osmostic stress, arsenite exposure, and cytokine stimulation [69,70]. It is therefore intriguing to speculate that MK2 might control a late cytoplasmic component of the DNA damage response. Among the known targets of MK2 following cytokine stimulation are a number of proteins involved in post-transcriptional regulation of mRNA and protein translation, including Ago2, a protein critical for the RNAi pathway [94–98]. Could MK2-imposed control of mRNA metabolism, microRNA function, and protein translation also play a critical role in the DNA damage response? One important piece of evidence that hints at such a control mechanism is the result of a large-scale mass-spectrometry screen that sought to identify putative ATM and ATR substrates. This screen revealed that a substantial fraction of the putative substrates were proteins known to be involved in RNA metabolism . It is therefore tempting to speculate that the p38MAPK/MK2 complex might function by modulating the translation efficiency of a subset of critical mRNAs, which ultimately dictate cell fate following genotoxic stress. Hopefully future experiments will be able to support or refute this intriguing concept.
This work was supported by the National Institutes of Health (GM68762, CA112967, ES015339 to M.B.Y.), the Deutsche Forschungsgemeinschaft (RE2246/1-1 to H.C.R.), the Deutsche Nierenstiftung (to H.C.R.) and the David H. Koch Fund.
We apologize to our colleagues for the omission of many seminal contributions to the field, and their references, owing to space constraints.
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