(A) U2OS cells were infected with lentiviruses delivering luciferase-, MK2- or Chk1- specific shRNAs (see also Figures S1). The ability of these cells to engage and maintain a functional cell cycle checkpoint following genotoxic stress was analyzed using a FACS-based nocodazole trap experiment (Methods). Knockdown of MK2 or Chk1 did not grossly affect the cell cycle distribution of untreated cells (top panel). In response to 1 μM doxorubicin treatment for 1 hr, luciferase control shRNA-expressing cells mounted an intra-S and G₂/M checkpoint response that remained stable for the 30 hr course of the experiment, as evidenced by the accumulation of a largely pHH3-negative 4N population (bottom panel, left and panel B). MK2 shRNA-expressing cells initiated a functional intra-S, G₂/M cell cycle checkpoint that remained intact for at least 18 hrs. At the 24 hour measurement this checkpoint response started to decline with an increasing number of pHH3-positive cells showing a 4N DNA content, reflecting mitotically trapped cells (bottom panel, middle and panel B). In contrast to the MK2 knockdown cells which retained the ability to initiate a functional checkpoint response, cells that were depleted of Chk1 failed to initiate or maintain a checkpoint response within 12 hrs following doxorubicin treatment (bottom panel, right and panel B). (B) Quantification of pHH3 staining of the samples shown in A (n=7 experiments).

(A) GFP, GFP.MK2 and GFP.Chk1 fusion constructs were expressed in U2OS cells. Following treatment with 10 μM doxorubicin, the same set of cells were imaged over time real-time imaging. GFP-MK2 relocalized to the cytoplasm within 1 hr following addition of doxorubicin and remained largely cytoplasmic for 24 hr following genotoxic stress (top panel). GFP.Chk1 remained largely nuclear through the 24 hr course of the experiment (middle panel). Unfused GFP localized diffusely throughout the cytoplasm and the nucleus. (See also Figure S3).
(B) The GFP.MK2 fusion protein is activated with the same kinetics, as endogenous wildtype MK2. Stably transfected U2OS cells were either mock-treated or exposed to 10μM doxorubicin as indicated. Following treatment cells were lysed, proteins separated on SDS PAGE and visualized by immunoblot. MK2 activity was monitored with phospho-specific antibodies detecting p38-mediated activation/phosphorylation of Thr-334, located between the kinase domain and the C-terminal regulatory domain.
(C) The GFP.Chk1 fusion protein is activated with the same kinetics, as endogenous wildtype Chk1. Cells were treated as in B) and ATR-dependent phosphorylation on Ser-345 in the C-terminal regulatory region was monitored using immunoblotting.
(D) In response to doxorubicin, endogenously expressed MK2 and Chk1 show a biochemical re-localization pattern that is identical to their exogenously expressed GFP-fused counterparts (shown in A). Nuclear and cytoplasmic fractions were isolated using hypotonic lysis and MK2 and Chk1 protein levels were determined using immunoblotting. Staining for tubulin (a cytoplasmic marker) and histone H1 (a nuclear marker) was performed to assess the purity of the isolated fractions.
(E) Indirect immunofluorescence staining was performed to verify the subcellular localization of endogenous MK2 and Chk1 in response to doxorubicin. Hoechst stain was used as a counterstain to provide a nuclear reference point. MK2 was predominantly localized in the cytoplasmic compartment 6 hr following doxorubicin exposure (top panel), while Chk1 remained nuclear (bottom panel).
(F) The doxorubicin-induced cytoplasmic relocalization of MK2 depends on a caffeine-sensitive kinase(s), but is independent of Chk1. U2OS cells were either left untreated or incubated with 10 μM doxorubicin for 6 hr and the subcellular localization of MK2 was assessed by immunofluorescence as in (E). Doxorubicin treatment induced a robust translocation from the nucleus to the cytoplasm (upper two panels). This relocalization was completely prevented when cells were pretreated with 10mM caffeine 30 min prior to doxorubicin application (middle panel). Inhibition of Chk1 with AZD-7762 (200nM) or PF-477736 (5μM) 30 min prior to doxorubicin failed to prevent cytoplasmic accumulation of MK2 (lower two panels).
(G) MK2 is activated in human tumor samples. Sections from human squamous cell head and neck cancer (T) and the surrounding stroma (S) were stained with antibodies against total MK2 (left panel), the activated/phosphorylated form of MK2 (middle panel), and its downstream substrate phospho-hsp27 (right panel). Of note, these tumors show spontaneous DNA damage as indicated by positive γH2AX staining (right panel inset) which correlates with MK2 activation and cytoplasmic accumulation of MK2 (left panel inset, arrowheads). In contrast, the stroma shows predominantly nuclear staining of MK2 (left panel inset, arrows), and minimal phospho-MK2 and phospho-hsp27 staining.

(A) MK2 and Chk1 localization mutants were generated as indicated. Live cell images obtained before and 24 hrs following treatment with10 μM doxorubicin are shown below each construct. Of note, inactivation of the NES in MK2 results in a mutant with abolished ability to localize to the cytoplasm following genotoxic stress (A iii). Fusion of the MK2 NES between GFP and Chk1 produces a Chk1 mutant that is localized primarily to the cytoplasm (A iv).
(B, C) Functional assessment of the ability of the localization mutants to establish and maintain cell cycle checkpoints. U2OS cells were infected with lentiviruses expressing luciferase control shRNA, MK2-specific shRNA or shRNA targeting Chk1. Knockdown cells were complemented with the localization mutants as indicated (see also Figure S2). Cells were treated with doxorubicin in a 30 hr nocodazole trap experiment and cell cycle profiles were assessed by FACS using DNA content profiles (B) and phospho-histone H3 staining (C). Of note, loss of nuclear Chk1 could be functionally compensated by expression of the GFP.MK2.ΔNES mutant that was re-localized to the nucleus, while loss of cytoplasmic MK2 could be rescued by expression of the GFP.NES.Chk1 mutant that was re-localized to the cytoplasm. (See also Figure S4).

(A) Loss of MK2 precludes doxorubicin-induced Gadd45α mRNA and protein upregulation. HeLa cells were infected with lentiviruses expressing luciferase control or MK2-specific shRNA. Cells were treated with 10 μM doxorubicin and Gadd45α mRNA levels were examined by RT-PCR 18 hrs later, Control cells robustly induced Gadd45α after doxorubicin exposure, while MK2-depleted cells failed to upregulate Gadd45α mRNA and protein in response to doxorubicin (left and middle panel). Of note, co-expression of the GFP.NES.Chk1 mutant that re-localized to the cytoplasm rescued the MK2 RNAi phenotype (right panel).
(B) Gadd45α depletion in functionally p53-deficient HeLa cells prevents the engagement of a functional intra-S, G₂/M checkpoint following doxorubicin. HeLa cells expressing luciferase control shRNA or Gadd45α-specific hairpins were treated with doxorubicin (10 μM) in a 30 hr nocodazole trap experiment and cell cycle profiles were assessed by FACS. Control cells mounted a robust intra-S, G₂/M arrest in response to doxorubicin, as evidenced by an accumulation of 4N cells (monitored by PI staining) and a lack of pHH3 staining. In contrast, ~23% of Gadd45α-depleted cells entered mitosis throughout the 30 hr course of the experiment, indicating a bypass of the doxorubicin-induced cell cycle arrest in these cells.
(C) Fusion of the Gadd45α mRNA 3'UTR to GFP confers MK2-dependent sensitivity to genotoxic stress to GFP protein expression. HeLa cells expressing luciferase control shRNA or MK2-specific hairpins were co-transfected with vectors encoding unfused eGFP or eGFP fused to the Gadd45α 3'UTR. In these experiments, the GFP-3'UTR protein is ~3 kD smaller than eGFP due to a deletion of 23 amino acids at the C-terminus (Lal et al., 2006). Cells were mock treated, or exposed to cisplatin (10μM), doxorubicin (1μM) or UV (20J/m²), harvested 36 hours later and GFP expression levels were monitored by immunoblot. Expression levels of unfused GFP did not change following genotoxic stress, however, fusion of the Gadd45α 3'UTR to GFP resulted in repression of GFP expression that could be relieved upon genotoxic stress in a MK2-dependent manner. The right panel schematically depicts the endogenous Gadd45α transcript (top), the GFP-3'UTR fusion and unfused GFP constructs.
(D) Relative GFP expression levels as shown in (C) were quantified from 3 independent experiments using ImageQuant software. Mean values are shown with error bars indicating standard deviation. Note the expanded y-axis scale in panels 2–4.

(A) Immunoprecipitation followed by Western blotting for the RNA binding proteins that were investigated.
(B) HeLa cells were either treated with doxorubicin (1μM) for 12 hrs or left untreated, lysed, and the binding of endogenous ARE-binding RBPs (HuR, TIAR, TTP, and hnRNP A0) to Gadd45α mRNA assessed using RNA-IP as described in Methods.
(C) hnRNP A0 interacts with the Gadd45a 3'UTR following genotoxic stress. HeLa cells were co-transfected with HA-tagged hnRNP A0 and either GFP fused to the Gadd45α 3'UTR or unfused GFP. Cells were treated with doxorubicin (10μM) or vehicle for 12 hrs, lysed and HA.hnRNP A0 was immunoprecipitated followed by GFP RT-PCR. hnRNP A0 strongly boung to Gadd45α 3'UTR-fused GFP mRNA following doxorubicin. However, no interaction between hnRNP A0 and unfused GFP mRNA was detected, indicating that hnRNP A0 directly binds to the 3'UTR of Gadd45a mRNA.
(D) hnRNP A0 depletion in functionally p53-deficient HeLa cells prevents the engagement of a functional intra-S, G₂/M checkpoint following doxorubicin. HeLa cells expressing luciferase control shRNA or hnRNP A0-specific hairpins were treated with 10 μM doxorubicin in a 30 hr. nocodazole trap experiment and cell cycle profiles were assessed by FACS. Control cells mounted a robust intra-S, G₂/M arrest in response to doxorubicin, as evidenced by an accumulation of 4N cells (monitored by PI staining), which were largely staining negative for pHH3. In contrast, ~15% of hnRNP A0-depleted cells entered mitosis throughout the 30 hr course of the experiment, indicating a bypass of the doxorubicin-induced cell cycle arrest in these cells.
(E) In vitro kinase assays with bacterially purified recombinant MK2 and GST.hnRNP A0 wildtype or hnRNP A0 in which Ser-84 was mutated to Ala. GST served as a control. Following completion of the kinase assay, reaction mixtures were separated on SDS-PAGE and ³²P incorporation was visualized by autoradiography. GST.hnRNP A0 wildtype was readily phosphorylated by MK2 in vitro, while mutation of Ser-84 to Ala completely abolished hnRNP A0 phosphorylation.
(F) MK2-mediated hnRNP A0 phosphorylation on Ser-84 is essential for hnRNP A0 binding to Gadd45α mRNA. HeLa cells expressing luciferase control shRNA or MK2-specific shRNA were transfected with HA-tagged hnRNP A0 wildtype or the Ser-84 to Ala mutant. Cells were treated with doxorubicin (10μM) or vehicle, lysed 12 hrs later, and hnRNP A0 was immunoprecipitated with anti HA-antibodies. While HA.hnRNP A0 readily co-precipitaed with Gadd45α mRNA following genotoxic stress in control cells, the Ser-84 to Ala mutant failed to interact with Gadd45a mRNA (left panel). This interaction was MK2-dependent, since hnRNP A0: Gadd45a mRNA complex formation was abolished in MK2 depleted cells (middle panel). Loss of MK2 could be rescued by expression of the activatable, cytoplasmic Chk1 mutant.
(G) Reduced binding of Gadd45α mRNA by TIAR following genotoxic stress depends on p38 activity. HeLa cells were treated with the p38 inhibitor SB203580 (10μM) or vehicle 1 h before treatment with doxorubicin as described in 5F. TIAR was immunoprecipitated followed by Gadd45α RT-PCR. TIAR binding to the Gadd45a mRNA that was abolished following genotoxic stress could be restored by inhibition of p38.
(H) In vitro kinase assays with bacterially purified recombinant His.MK2 or His.p38 and GST.TIAR. GST served as a control, and GST.Hsp25-peptide (AS 71–100) served as a positive control for MK2. Following completion of the kinase assay, reaction mixtures were separated on SDS-PAGE and ³²P incorporation was visualized by autoradiography. GST.TIAR was readily phosphorylated by p38 in vitro after 20 min, but it was not phosphorylated by MK2.

(A) In vitro kinase assays using bacterially purified recombinant MK2 and 6xHis-tagged PARN wildtype or a PARN mutant in which Ser-557 was mutated to Ala. Following completion of the kinase assay, reaction mixtures were separated on SDS-PAGE and ³²P incorporation was visualized by autoradiography. Equal loading was confirmed by coomassie staining.. The top panel shows a schematic representation of the modular domain structure of PARN. Ser-557 lies within an optimal MK2 consensus phosphorylation motif located C-terminal to the RNA recognition motif (RRM).
(B) MK2 mediates doxorubicin-induced phosphorylation of PARN on Ser-557 within cells. U2OS cells were infected with lentiviruses expressing luciferase or an MK2-specific shRNA. Following selection cells were treated with 10μM doxorubicin for 4 hr and endogenous PARN was affinity purified from cell lysates. The immunoprecipitated material was analyzed by mass spectrometry. Insets: only non-phosphorylated Ser-557 PARN peptides (shown in red) could be detected in untreated U2OS cells expressing the luciferase control shRNA (shLuci, co). In contrast, Ser-557 phosphorylated peptides (shown in black) were readily detected when luciferase control cells were exposed to doxorubicin (shLuci, dox). DNA damage-induced phosphorylation of PARN on Ser-557 was completely abolished in MK2-depleted cells (shMK2 co and dox panels).
(C, D) PARN Ser-557 phosphorylation is critical for maintenance of a doxorubicin-induced cell cycle arrest. HeLa cells were infected with lentiviruses expessing packaged from empty transfer vector or PARN shRNA expessing vectors. PARN shRNA-expressing cells were also co-transfected with shRNA-resistant PARN wildtype or a Ser-557 to Ala mutant. Cells were treated with 0.1μM doxorubicin for 1 hr and cell cycle profiles (phospho-histone H3 and DNA content) were assessed in a nocodazole trap experiment using FACS to monitor mitotic entry and cell cycle progression. After 24 hrs, 5mM caffeine was added to abrogate checkpoint signaling and analyze the ability of damaged cells to exit the checkpoint. Empty vector, PARN shRNA and PARN shRNA expressing cells that were complemented with shRNA-resistant wildtype PARN showed the induction of a stable cell cycle arrest, as evidenced by an accumulation of S and G2/pHH3-negative cells. PARN shRNA-expressing cells that were co-expressing the shRNA-resistant, non-phosphorylatable PARN S557A mutant failed to maintain a functional cell cycle arrest, indicated by the accumulation of ~12% of pHH3 positive cells at 24 hrs following addition of low-dose doxorubicin.
(E) PARN Ser-557 is critical for long-lasting expression of Gadd45α mRNA and protein following doxorubicin-induced genotoxic stress. Cells were transfected and treated with doxorubicin as in panel C. Gadd45α mRNA levels were monitored by RT-PCR and protein levels were assessed by immunoblotting. Of note, cells expressing the non-phosphorylatable PARN S557A mutant showed up-regulation of Gadd45α mRNA and protein levels at 12 hrs, but could not sustain the stabilization this inherently unstable mRNA for longer times. This loss of Gadd45α expression at 24hrs coincided with the premature cell cycle checkpoint collapse shown in panel C.

(A) Gadd45α interacts with p38. Cells were transfected with HA-tagged Gadd45α or HA-tagged 14-3-3ζ as a negative control. Lysates and anti-HA IPs were analyzed by SDS-PAGE and western blotting using anti-p38 and anti-HA antibodies.
(B) Gadd45α is required to maintain long-term MK2 activity following doxorubicin-induced DNA damage. HeLa cells were infected with lentiviruses expressing either luciferase control shRNA or MK2-specific hairpins, treated with 1μM doxorubicin for the indicated times, lysed, and proteins separated on SDS-PAGE. MK2 and Gadd45α levels were monitored by immunoblot, β-actin staining served as a loading control. Both Gadd45α and control shRNA-expressing cells showed a robust induction of MK2 activity 18 hr following doxorubicin treatment (monitored by phosphorylation-induced gel shift to a slower migrating isoform on SDS-PAGE). However, control cells maintained MK2 activity for at least 30 hours, while Gadd45α-depleted cells were unable to maintain MK2 activity for more than 18 hours.
(C) The activation-induced MK2 gel shift is blocked by the p38 inhibitor SB203580. Doxorubicin treatment (10μM) of HeLa cells induces a phosphorylation-dependent gel shift of MK2 (middle lane). Pretreatment of HeLa cells with 10μM of the p38 inhibitor SB203580 abolished the activation-induced MK2 gel shift (right lane).
(D) A simplified model depicting the early, Chk1-dependent nuclear checkpoint (left box) and the late MK2-dependent cytoplasmic checkpoint (right box). Dashed arrows between ATM/ATR and p38/MK2 indicate intermediate steps that are not shown. The MK2-mediated cytoplasmic checkpoint is sustained through a positive feedback loop. Following nuclear activation, the p38/MK2 signaling complex re-localizes to the cytoplasm through a Crm1 dependent transport mechanism. MK2-mediated hnRNP A0 and PARN phosphorylation, as well as p38-dependent TIAR phosphorylation are required to stabilize Gadd45α mRNA, resulting in increased Gadd45α protein levels. Gadd45α itself is then required to maintain MK2 activity in the cytoplasm.

(A) HeLa cells were infected with retroviruses encoding GFP fused Cdc25B (upper panels) or Cdc25C (lower panels). GFP.Cdc25B/C expressing cells were subsequently infected with lentiviruses expressing luciferase-, MK2- or Chk1-specific shRNA, treated with 0.1μM doxorubicin for 1 hr, and GFP localization was monitored by live cell imaging. Control cells mounted a cell cycle checkpoint response and resumed mitotic cell division after ~30 hr with the production of two intact daughter cells (top panels and top panel in B). MK2-depleted cells initiated a checkpoint response, which collapsed after ~24 hr invariably followed by a catastrophic mitotic event (middle panels and B middle panel). Chk1-depleted cells entered a premature, catastrophic mitotic event at ~16 hr following doxorubicin (middle panels and B middle panel) Red frames indicate the time at which nuclear relocalization of Cdc25B/C could first be observed.
(B) Detailed view of the mitotic events in the three different GFP.Cdc25B labeled cells lines. Red dashed line highlights the nuclear border.
(C) Quantitative analysis of the data shown in panels A and B. Data are shown as box and whisker plots, and represent 18 independent experiments for each cell line. The time of the first appearance of a mitotic figure was recorded. Asterisk indicates statistical significance.