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Molecular and Cellular Biology, July 2000, p. 4543-4552, Vol. 20, No. 13
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Involvement of the MKK6-p38gamma Cascade in gamma -Radiation-Induced Cell Cycle Arrestdagger

Xiaofei Wang,1 Clare H. McGowan,2 Ming Zhao,1,Dagger Liusheng He,3 Jocelyn S. Downey,1 Colleen Fearns,1 Yibin Wang,4 Shi Huang,3 and Jiahuai Han1,*

Department of Immunology1 and Department of Molecular Biology,2 The Scripps Research Institute, and Program in Oncogenes and Tumor Suppressor Genes, The Burnham Institute,3 La Jolla, California 92037, and Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 212014

Received 10 November 1999/Returned for modification 22 December 1999/Accepted 3 April 2000


    ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

The p38 group of kinases belongs to the mitogen-activated protein (MAP) kinase superfamily with structural and functional characteristics distinguishable from those of the ERK, JNK (SAPK), and BMK (ERK5) kinases. Although there is a high degree of similarity among members of the p38 group in terms of structure and activation, each member appears to have a unique function. Here we show that activation of p38gamma (also known as ERK6 or SAPK3), but not the other p38 isoforms, is required for gamma -irradiation-induced G2 arrest. Activation of the MKK6-p38gamma cascade is sufficient to induce G2 arrest in cells, and expression of dominant negative alleles of MKK6 or p38gamma allows cells to escape the DNA damage-induce G2 delay. Activation of p38gamma is dependent on ATM and leads to activation of Cds1 (also known as Chk2). These data suggest a model in which activation of ATM by gamma  irradiation leads to the activation of MKK6, p38gamma , and Cds1 and that activation of both MKK6 and p38gamma is essential for the proper regulation of the G2 checkpoint in mammalian cells.


    INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Cell cycle arrest is a common response to stress stimuli that can cause DNA damage. It is believed that cell cycle arrest has an important role in minimizing the consequences of DNA damage to cells (22). It is generally believed that G2 arrest provides cells with time to repair DNA damage before moving into mitosis (48). DNA damage and its effect on cell cycle progression have been intensively studied in yeast and Xenopus oocyte extracts as well as in mammalian cells. Such studies show that the kinase activity of Cdc2-cyclin B complex is required for the G2-to-M-phase (G2/M) transition in the normal cell cycle and that tyrosine phosphorylation of Cdc2 inhibits its kinase activity (47). Both inhibition of Cdc2 kinase activity and enhanced phosphorylation of Cdc2 have been observed following DNA damage (6, 27, 36). The phosphorylation state of Cdc2 is maintained by the kinases Wee1 and Myt1 and by the phosphatase Cdc25 (10, 39, 41, 43). Although checkpoint regulation of both sides exists, it is thought regulation of Cdc25 activity is an important factor in the maintenance of a G2 arrest after DNA damage (53). In mammalian cells, two kinases, Chk1 and Cds1 (also known as Chk2), have been identified (5, 38, 54) and shown to phosphorylate Cdc25C and prevent it from dephosphorylating and activating Cdc2 (5, 9, 16, 38, 52, 54). It is thought that phosphorylation on Cdc25 facilitates association with 14-3-3 protein, resulting in its export from the nucleus (37); however, the mechanism of Chk1 and Cds1 activation following irradiation is not clear. The response of Cds1 to DNA damage has been shown to be dependent on the activity of ATM, the gene of which is mutated in patients with ataxia telangiectasia (AT) (5, 8, 9, 38). ATM is thus upstream of the signaling pathway. Consistent with the notion that G2 arrest has a protective effect against DNA damage, patients with an ATM defect suffer enhanced sensitivity to irradiation (32). Pharmacological agents which override the G2/M block often sensitize the cells to gamma  radiation (30, 49); gamma -radiation-induced DNA damage followed by cell death is considered to be the mechanism for cancer cell elimination by radiotherapy.

The p38 mitogen-activated protein (MAP) kinase pathway is a primary signaling pathway that is activated by stressful events such as UV irradiation. The p38 group of MAP kinases belongs to a subfamily of the MAP kinase superfamily. The prototypic member of this group, p38alpha (also known as p38, CSBP, or RK), was discovered as being tyrosine phosphorylated in macrophages upon treatment with bacterial lipopolysaccharide (20, 21). This protein was also identified as a specific target of a series of anti-inflammatory compounds of which SB203580 is the prototypic member (34). p38alpha and p38beta are sensitive to inhibition by SB203580, while the activities of p38gamma and p38delta are not affected by this compound (17). While the activation profiles of different p38 isoforms in response to stress are similar, an increasing body of evidence suggests that individual p38 isoforms have distinct biological functions. For example, p38alpha and p38beta antagonize each other in cardiomyocyte hypertrophy, and p38gamma has been implicated in muscle differentiation and in the response to hypoxia (33, 35, 40). Several proteins have been identified as substrates for p38alpha , including transcription factors such as CHOP10, MEF2C, and Sap1 in TCF, enzymes such as cPLA2, and downstream protein kinases such as MAPKAPK2/3, MNK1/2, and PRAK (for reviews, see references 46 and 50). All four members of the p38 kinase family can phosphorylate the transcription factor activating factor 2 (ATF2) in vitro (17). Like other MAP kinases, activation of p38 group MAP kinases requires specific phosphorylation by their upstream kinases. Two MAP kinase kinases, MKK3 and MKK6, have been identified as immediate upstream activators of the p38 family (13).

The involvement of MAP kinases in cell cycle arrest has been studied in a number of organisms. Perhaps due to differences in substrate specificity and regulation among the MAP kinases, their roles in cell cycle regulation appear to be different. Activation of p42MAPK is required for the G2/M transition in the maturation of Xenopus oocytes (51), and inactivation of this MAP kinase releases G2 arrest at the time of Xenopus oocyte fertilization (2). MEK2 activity was recently reported to be necessary for G2 arrest in mammalian cells (1), while MEK1 activity is required for the G2/M transition (60). BMK1 (ERK5) was reported to be required for epidermal growth factor-induced progression through S phase (29). p38alpha has been reported to be involved in Cdc42-induced G1 arrest as well as the spindle assembly checkpoint (42, 56).

To address the role of p38 and its isoforms in response to gamma  radiation, we examined activation of the components of the p38 pathway in gamma -irradiated cells. The function of these signaling molecules was determined by introducing constitutively active and/or dominant negative alleles of these molecules. We report here that MKK6 and all p38 isoforms are activated by gamma  irradiation, that the active form of MKK6 is sufficient to arrest cells in G2, and that inhibiting MKK6 or a specific p38 isoform, p38gamma , disrupts the DNA damage checkpoint. Furthermore, we find that the activation of p38gamma in gamma -irradiated cells is ATM dependent and that the MKK6-p38gamma cascade is involved in regulating Cds1 activity. Activation of the p38 pathway ultimately leads to Cdc2 phosphorylation and subsequent G2 arrest. These data support an important interplay between the p38 pathway and G2 cell cycle checkpoint control.


    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Cell culture. Normal (GM637G; ATM+/+) and AT-deficient (GM5849C; ATM-/-) simian virus 40-transformed human fibroblasts were obtained from Coriell Institute for Medical Research, Camden, N.J. Other cell lines were obtained from the American Type Culture Collection. Cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 50 U of penicillin/ml, 50 mg of streptomycin/ml, and 1% nonessential amino acids.

gamma irradiation. Cells in six-well plates were irradiated with a single dose from a 137Cs source in a Gamma Cell 40 at a rate of 65 rads/min (18).

Adenovirus infection. Recombinant adenoviruses were generated as described elsewhere (25). Cells were seeded into six-well plates at 2 × 105 cells per well 24 h before infection with recombinant adenovirus (103 inclusion-forming units [IFU]/cell). After the indicated time postinfection, the cells were harvested and subjected to further analysis.

Cell cycle analysis. Cells were harvested, washed, resuspended in phosphate-buffered saline (PBS), and fixed for >18 h after the stepwise addition of ice-cold absolute ethanol. Cells were resuspended in RNase A (1 mg/ml) for 30 min at 37°C and then stained with propidium iodide (PI; 0.05 mg/ml) for 1 h on ice. Fluorescence-activated cell sorting (FACS) analysis was performed with a FACS IV flow cytometer (Becton Dickinson, San Jose, Calif.). Cells were excited at 488 nm, and the emission was collected above 590 nm.

Mitototic index assay. Immediately after gamma  irradiation or adenovirus infection, cells were treated with nocodazole (100 ng/ml), Calbiochem, Calif. for 14 h. Then cells were fixed in 70% ethanol and stored at 4°C for 24 h before staining with Hoechst DNA stain (Molecular Probes). Mitotic index was scored under a fluorescence microscope.

Pulse-chase labeling. Ninety percent confluent cultures of HeLa and U2OS cells in six-well plates were labeled with Trans-[35S]Label ([35S]Met-Cys; Amersham) for 1 h before exposure to radiation. The cells were then chased for the times indicated by culture in medium containing excess unlabeled Met-Cys but no [35S]Met-Cys, and lysed Cdc25C was immunoprecipitated with anti-Cdc25C antibody (Santa Cruz Biotechnology, Santa Cruz, Calif.) and protein G resin and analyzed by polyacrylamide gel electrophoresis (PAGE).

Clonogenic survival assay. Cells were detached, suspended as single-cell suspensions, and counted. Clonogenic survival was determined by plating cells in growth medium plus 0.3% Noble agar. After 2 weeks, plates were fixed and stained with 0.01% crystal violet in 1.5% acetic acid, and colonies larger than 50 cells were counted. The number of survival colonies in nonirradiated samples is taken as 100%.

In vitro kinase assay. Kinase reactions were performed in vitro, using [gamma -32P]ATP as described previously (13, 26). Recombinant fusion proteins of activating transcription factor 2 (glutathione S-transferase [GST]-ATF2) and an inactive form of p38 [GST-p38(KM)] were used as the substrates for p38 and MKK6, respectively. A synthetic peptide corresponding to residues 208 to 225 of Cdc25C was used as the substrate for human Cds1. The levels of peptide phosphorylation were determined as follows. The reaction mixture was applied onto Whatman P-81 phosphocellulose paper. The filters were washed twice in 1 mM acetic acid followed by 4 mM sodium pyrophosphate. The remaining radioactivity was quantified by liquid scintillation counting. The phosphorylation of protein substrates was analyzed by sodium dodecyl sulfate (SDS)-PAGE as described elsewhere (13); enzyme activity was determined by autoradiography or phosphorimaging.

Immunocomplex kinase assay. Kinases were immunoprecipitated from cells lysed in buffer containing 20 mM Tris-HCl, 120 mM NaCl, 10% glycerol, 1 mM Na3VO4, 2 mM EDTA, 1% Triton X-100, and 1 mM phenylmethylsulfonyl fluoride (pH 7.5) for 15 min at 4°C (13, 26). Insoluble material was removed by centrifugation at 13,000 × g for 10 min at 4°C. Then 3 to 5 µl of antibody for MAPK or MKK or 1 µl for Cds1 was added, and the mixture was incubated overnight at 4°C; 40 µl of protein A-Sepharose (1:1 slurry in PBS) was added, and the tube was rotated for 2 h at 4°C to allow the protein A to bind to the antibody. The immunoprecipitate was then washed five times with PBS containing 1% Triton X-100 and used as enzyme in the in vitro kinase assay described above.

Recombinant proteins. GST-ATF2 and GST-Cds1 fusion proteins were expressed in Escherichia coli BL21(DE3) and purified using glutathione-Sepharose 4B beads (Pharmacia Biotech, Uppsala, Sweden). All of the His6-tagged recombinant proteins were expressed in E. coli BL21(DE3) and purified using a Ni-nitrilotriacetic acid purification system (Qiagen). His6-Cds1 generated from recombinant baculovirus-infected Sf9 cells has been described previously (5). Western blotting was performed as described elsewhere (20). Anti-Cdc2 and anti-phospho-Cdc2 antibodies were from New England Biolabs (NEB), Beverly, Mass. Anti-Cdc25C antibody was from Santa Cruz Biotechnology. Antiactin antibody was from Sigma, St. Louis, Mo.

Northern blotting. Total RNA was extracted from U2OS cells using Trizol reagent from Gibco BRL, Gaithersburg, Md. Northern blotting and hybridization were performed by standard methods. cDNA against human Cdc25C mRNA was generated from the coding region of human Cdc25C cDNA by PCR.

Western blotting. The cell lysates or immunoprecipitates were resolved by SDS-PAGE and transferred to a nitrocellulose membrane. Polyclonal antibodies raised in rabbits were used. Dilutions of the first antibodies are as follow: anti-p38alpha /beta , 1:5,000; anti-p38gamma , 1:2,000; anti-p38delta , 1:1,000; antiactin (Sigma), 1:1,000; anti-phospho-Cdc2 (NEB, Beverly, Mass.), 1:1,000; and anti-Cdc2 (NEB), 1:1,000.

Reporter gene assay. The GAL4-responsive plasmid pG5E1bLuc contains five GAL4 sites cloned upstream of a minimal promoter driving a luciferase gene. Expression plasmids encoding the GAL4 DNA binding domain fused with the activation domain of MEF2C, ATF2, ELK1, or c-Jun were described previously (61). Cells were grown on 35-mm-diameter multiwell plates and transiently transfected with 1 µg of total plasmid DNA using Lipofectamine reagent (Gibco BRL). A beta -galactosidase expression plasmid (pCMV-beta -gal; Clontech, Palo Alto, Calif.) was used to control for transfection efficiency. The total amount of DNA for each transfection was kept constant by using the empty vector pcDNA3. Cell extracts were prepared 36 h later, and beta -galactosidase and luciferase activities were measured. In the experiments to evaluate the inhibitory effect of inactive MKKs, the cells were infected with recombinant adenovirus 12 h before the transfection. A stimulation with serum or tumor necrosis factor was applied 24 h after transfection for 12 h.


    RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Activation of the p38 MAP kinase pathway by gamma  irradiation. To evaluate the role of the p38 pathway in gamma -irradiation-stimulated cells, we initially examined the activation of different components of the p38 pathway. The activation of the MAP kinase kinases in this cascade, MKK3 and MKK6, was determined in U2OS cells (a human osteosarcoma cell line) exposed to gamma  radiation. The cells were treated with 6 Gy of gamma  radiation and harvested at different time points postirradiation. MKK3 or MKK6 was immunoprecipitated by specific antibodies as described elsewhere (25). The immunoprecipitates were used to phosphorylate p38(KM) in in vitro kinase assays. As shown in Fig. 1A, the activation of MKK6 was detected 1 h following irradiation and was maintained for 16 h. In contrast, only moderate activation of MKK3 was observed under the same conditions, and the activation was much delayed. A similar approach was used to study the activation of the p38 isoforms by gamma  radiation. p38 isoforms were immunoprecipitated with isoform-specific antibodies after irradiation, and a kinase assay was performed with ATF2 as substrate. Figure 1B shows thatp38alpha /beta , p38gamma , and p38delta are activated by gamma  radiation in similar time frames. The activation was seen 1 h following irradiation and remained elevated for at least 16 h, correlating with the kinetics of MKK6 activation. The specificity of the antibodies used in the experiments is shown in Fig. 1C. Similar activation of MKK6 and p38 isoforms was seen when HeLa cells were used (data not shown). The activation of p38 isoforms observed here is most likely to be downstream of MKK6 and MKK3, while MKK6 appears to be primarily responsible for the activation. The prolonged activation of p38s seen in response to gamma  radiation contrasts with that produced by other stimuli, such as cytokines, in which case p38 activation is transient (17, 46).


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  		FIG. 1.   gamma irradiation activates the MKK6-p38 pathway. (A) U2OS cells were treated with 6 Gy of gamma  irradiation, and MKK3 and MKK6 were immunoprecipitated at the indicated times postirradiation. Kinase activities of MKK3 and MKK6 were determined by kinase assay using GST-p38(KM) as substrate. Western blotting (WB) was used to ensure that equivalent amounts of kinase were used in all samples (lower panel). (B) Time course of gamma -irradiation-induced activation of p38alpha /beta , -gamma , and -delta by immunocomplex kinase assay. The kinase activities of p38s isolated by immunoprecipitation with p38 isoform-specific antiserum were measured with GST-ATF2(1-109) as substrate. Again, equal loading of kinase was ensured by Western blotting (lower panel). Fold activation was calculated by dividing the radioactive intensity of phosphorylated p38(KM) (A) or ATF2 (B) at different time points by that at time zero. Similar results were obtained in two experiments. (C) Specificity of isoform-specific antibodies was determined as follows. Flag-tagged p38alpha , p38beta , p38gamma , and p38delta were expressed in 293 cells. The expression of these epitope-tagged proteins was determined by Western blotting using anti-Flag monoclonal antibody M2 (top panel). p38alpha /beta , p38gamma , or p38delta was immunoprecipitated using the isoform-specific antibodies from the cell lysates of 293 cells that had been transiently transfected with different Flag-tagged p38 isoforms. The immunoprecipitates were analyzed by Western blotting using anti-Flag monoclonal antibody M2.

Constitutive activation of MKK6 leads to G2 arrest. Since the p38 pathway is activated by gamma  irradiating cells and gamma  radiation can cause G2 arrest of many different cells, it is possible that gamma  radiation induced activation of the p38 pathway contributes to G2/M checkpoint control. We therefore tested whether activation of the p38 pathway can mimic gamma  radiation in inducing a G2 arrest. We used Ad.MKK6(E), the constitutively active form of MKK6 encoded by recombinant adenovirus, to activate the p38 pathway without extracellular stimuli (25). The adenovirus gene delivery system reached >95% infection efficiency without influencing cell viability or the cell cycle in our experimental cell lines (data not shown). U2OS cells were cultured in six-well plates and treated with gamma  radiation, Ad.MKK6(E), or Ad.MKK6(A) (adenovirus-encoded inactive MKK6). The cell cycle phase distribution of cells was determined by FACS analysis; as shown in Fig. 2A, cells accumulated with a 4N DNA content after gamma  irradiation. Cells infected with Ad.MKK6(E) also accumulated with a 4N DNA content (Fig. 2B). The number of cells with 4N DNA started to increase at 24 h after Ad.MKK6(E) infection, and concomitant reduction of cells in G1 phase was seen. The increase in cells with a 4N DNA content followed expression of MKK6(E), which was detectable at 16 h postinfection (data not shown). Cell morphology was examined by light microscopy. Cells infected with Ad.MKK6(E) showed morphological changes similar to those of cells gamma  irradiated and were typically enlarged and flattened with almost no dividing cells. Thus, the accumulation of cells with 4N DNA is unlikely to be due to an increase in M-phase cells. This was further confirmed using the mitotic index assay (data not shown). Infection with Ad.MKK6(A) (Fig. 2B) or adenoviruses encoding green fluorescent protein (Ad.GFP) (data not shown) had no effect on the cell cycle profile or on cell morphology (data not shown). Thus, both gamma  radiation and MKK6(E) expression induce G2 cell cycle arrest.


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  		FIG. 2.   Both gamma  irradiation and MKK6(E) expression induce G2 arrest. (A) U2OS cells were collected and fixed at the indicated times after 6 Gy of gamma  irradiation (IR), and the cell cycle profile was determined by FACS analysis using PI staining. The graph was generated using CellQuest software. (B) U2OS cells were seeded in six-well plates at a density of 2 × 105 cells/well and infected with Ad.MKK6(E) or Ad.MKK6(A) at 1,000 IFU/cell 24 h after plating. Cells were then collected for cell cycle analysis at the indicated times postinfection. (C) HeLa, U2OS, and SW480 cells were infected with either Ad.GFP (control) or Ad.MKK6(E), and cell cycle analysis was performed 48 h postinfection. The percentage of cells in the G2/M stage was calculated using MetLight software. Data are means ± standard errors of three independent observations. (D) Western blotting analysis of adenovirus-mediated MKK6 gene expression. HeLa, U2OS, or SW480 cells were infected with Ad.MKK6(E) at 1,000 IFU/cell 24 h after plating. The cells were lysed 16 h postinfection, and a Western analysis was performed using anti-MKK6 polyclonal antibody. The expression of adenovirus-encoded MKK6 was evidenced by the increase of total MKK6 protein. Equal loading of samples was shown by staining the membrane with Ponceau S. A portion of the membrane is shown at the bottom.

Ad.MKK6(E) infection of HeLa cells and SW480 cells (Fig. 2C) also leads to an increase in cells with a 4N DNA content. Figure 2D shows the expression of Ad.MKK6(E) detected by Western blotting. The tendency of cells to arrest in G2 differed slightly between cell lines, although all three lines demonstrated significant G2 arrest after MKK6(E) expression. While U2OS cells express wild-type p53 (24), HeLa cells and SW480 colon cancer cells are p53 deficient. Curiously, U2OS showed the highest percentage of G2-arrested cells. Whether p53 status plays a role in determining the level of MKK6-mediated G2 arrest is unknown. Similar experiments were performed with Ad.MKK3(E), which can cause G2 arrest but to a much lesser extent compared to MKK6(E)-treated cells (data not shown). Since gamma  irradiation and MKK6(E) expression induce G2 arrest (Fig. 2), and since the p38 pathway is activated by gamma  radiation (Fig. 1), it is likely that MKK6 is an intermediate component in the signal transduction pathway by which gamma  irradiation induces a G2 arrest.

The MKK6-p38gamma cascade is required for G2 arrest in response to DNA damage. Activation of the p38 pathway is sufficient for G2 arrest (Fig. 2). We therefore tested whether activation of the p38 pathway is required for the G2 arrest induced by gamma  irradiation. We first determined whether MKK6 activation is required for G2 arrest. U2OS or HeLa cells were infected with Ad.MKK6(A) and treated 24 h later with 6 Gy of gamma  radiation. The cell cycle profile was analyzed at different time points as shown in Fig. 3A. gamma  radiation induces a time-dependent increase of cells with a 4N content of DNA in U2OS, SW480, and HeLa cultures. Expression of MKK6(A) prior to gamma  irradiation significantly reduced the extent of G2 arrest seen in U2OS cells (left panel); the inhibitory effect of MKK6(A) on G2 arrest was also seen in HeLa cells (middle panel) but less than in U2OS cells. A similar experiment was performed using U2OS, HeLa, and SW480 cells, except that triplicate samples were analyzed at a single time point (right panel). MKK6(A) expression interfered with gamma -irradiation-induced G2 arrest in all three cell lines. The inhibitory effect of MKK6(A) on gamma -irradiation-induced G2 arrest was further supported by the observation that MKK6(A) expression did not have a noticeable effect on the normal cell cycle without gamma  irradiation (Fig. 3A), and infection of cells with Ad.GFP had no influence on the cell cycle progress of either gamma -irradiation-treated or untreated cells. The effect of MKK3(A) expression on gamma -irradiation-induced G2 arrest was tested. Despite comparable expression of MKK3(A) and MKK6(A), MKK3(A) expression had no detectable effect on gamma -irradiation-induced G2 arrest (data not shown). These data suggest that MKK6 activity is necessary for G2 arrest induced by gamma  irradiation.


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  		FIG. 3.   MKK6 is necessary for gamma -irradiation-induced G2 arrest. (A) U2OS or HeLa cells were seeded at 2 × 105/well in six-well plates and infected with Ad.GFP (control) or Ad.MKK6(A) for 24 h before being treated with 6 Gy of gamma  radiation. Cells were then collected at the indicated time points, and the cycle profile was analyzed using PI staining and flow cytometry (left and middle panels). U2OS, HeLa, or SW480 cells were seeded at 2 × 105/well in six-well plates and infected with Ad.GFP (control) or Ad.MKK6(A) for 24 h before being treated with 6 Gy of gamma  radiation (IR). Cells were then collected at 16 h after gamma  irradiation, and the cycle profile was analyzed using PI staining and flow cytometry (right panel). Data with error bars are means ± standard errors of triplicate samples. (B) U2OS cells were infected with the indicated forms of Ad.MKK(A) for 24 h and then treated with 6 Gy of gamma  radiation; 24 h after irradiation, G2 arrest was analyzed. (C) Inhibitory effects of different inactive MKK(A)s on endogenous MKKs. U2OS cells were infected with recombinant adenovirus encoding GFP (control), MEK1(A), MEK5(A), MKK6(A), or MKK7(A). The cells were replaced with serum-free medium 12 h after infection. The infected cells were then cotransfected with beta -galactosidase expression vector pCMVbeta , reporter plasmid pG5E1bluc, or an expression vector for the GAL4 DNA binding domain fused with the transactivation domain of ELK1, MEF2C, ATF2, or c-Jun as indicated. Empty vector pcDNA3 was used to normalize the total DNA to 1 µg per transfection. Cells were stimulated with 20% fetal bovine serum or tumor necrosis factor alpha (100 ng/ml) 24 h following transfection for 12 h. Luciferase and beta -galactosidase activities in the cell lysates were measured. The ratio of luciferase activity to beta -galactosidase activity is presented as mean ± standard deviation (n = 3).

To test whether accumulation of G2-arrested cells is a general functional feature of all MAP kinase pathways, we examined the effect of inhibiting the ERK, JNK, and ERK5/BMK pathways on gamma -radiation-induced G2 arrest. Adenoviruses encoding inactive forms of MEK1, MEK5, and MKK7 were used to impede the ERK, BMK, and JNK pathways, respectively. U2OS cells were irradiated with 6 Gy of gamma  radiation 24 h after virus infection and harvested 24 h later for cell cycle analysis. As shown in Fig. 3B, expression of MEK1(A), MKK7(A), or MEK5(A) did not influence the G2 arrest induced by gamma  radiation. The expression of these mutants and their ability to interfere with their respective MAP kinase pathways has previously been demonstrated (12, 28). Analysis of reporter genes transcription was used to demonstrate that ~50% inhibition of endogenous genes was obtained in these experiments (Fig. 3D). Thus, our data suggested no requirement for MEK1, MKK7, or MEK5 in gamma -radiation-induced G2 cell cycle arrest and support the hypothesis that the inhibitory effect of MKK6(A) on G2 arrest is specific.

Four isoforms of p38 were activated by gamma  irradiation (Fig. 1). We therefore examined which p38 isoforms are required for gamma -irradiation-induced G2 arrest. Inactive forms of the different p38 isoforms, in which the phosphorylation sites TGY were mutated to AGF (AF mutation), were expressed as described above using an adenovirus-mediated delivery system. Expression of these proteins was determined to be comparable by Western blotting (Fig. 4A, top panel). The ability of these mutants to inhibit endogenous p38 MAP kinases was determined by immunocomplex kinase assay as shown in Fig. 4A. Approximately 50% inhibition of endogenous p38s activity was observed in U2OS cells. Cells were irradiated with 6 Gy of gamma  radiation 24 h after virus infection, and the DNA contents were analyzed at different times after gamma  irradiation. As shown in Fig. 4B, only Ad.p38gamma (AF) was able to attenuate the gamma -irradiation-induced G2 arrest. Infection with control virus expressing GFP or virus expressing the AF mutant of p38alpha , -beta , or -delta had no effect. We also used SB203580, an inhibitor of p38alpha /beta , to test the role of these two p38 isoforms in gamma -irradiation-induced G2 arrest. SB203580 had no influence on G2 arrest even at a very high concentration (50 µM). Collectively, our data place p38gamma as a downstream kinase of MKK6 in mediating the G2 arrest.


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  		FIG. 4.   p38gamma is involved in gamma -radiation-induced G2 arrest. (A) U2OS cells were infected with Ad.GFP (control) or adenoviruses expressing an inactive form of p38 as indicated and were gamma  irradiated 24 h later. The percentage of cells in the G2/M stage was analyzed at the indicated times postirradiation. Data with error bars are means ± standard errors of triplicate observations; data from two experiments with similar results were obtained. (B) Inhibitory effect of p38alpha (AF), p38beta (AF), p38gamma (AF), or p38delta (AF) expression on endogenous p38 activity. U2OS cells were infected with Ad.GFP (control) or Ad.p38 as indicated; 24 h later, the cells were treated with gamma  irradiation (IR; 6 Gy). The expression of p38(AF) mutant was determined by a Western blotting analysis using anti-Flag antibody M2 (top panel). The kinase activities of p38alpha /beta in Ad.GFP (control)- or Ad.p38alpha (AF)-infected cells, p38alpha /beta in Ad.GFP (control)- or Ad.p38beta (AF)-infected cells, p38gamma in Ad.GFP (control)- or Ad.p38gamma (AF)-infected cells, and p38delta in Ad.GFP (control)- or Ad.p38delta (AF)-infected cells were determined by immunocomplex kinase assay 6 h after irradiation using GST-ATF2(1-109) as substrate.

Activation of p38gamma is ATM dependent. It is believed that DNA damage is a key initiator of gamma -irradiation-induced cellular responses. ATM is thought to promote cell survival in the face of DNA damage by activating a number of cellular functions including a G2 cell cycle arrest. Therefore, we tested whether the activation of p38gamma has any relationship with ATM. ATM+/+ or ATM-/- human fibroblasts were irradiated with 6 Gy of gamma  radiation, and the activity of p38gamma was measured by immunocomplex kinase assay as described above. The p38gamma isoform was activated in ATM+/+ cells but not in ATM-/- cells (Fig. 5A). UV-induced activation of p38gamma was normal in ATM-/- cells (data not shown), which is similar to the JNK activation by gamma  irradiation and UV observed in ATM+/+ and ATM-/- cells (55). Thus, activation of the p38 pathway by gamma  irradiation is downstream of ATM.


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  		FIG. 5.   p38gamma activation by gamma  irradiation is ATM dependent. (A) Both ATM+/+ and ATM-/- human fibroblasts were seeded into 6-cm-diameter dishes and treated with 6 Gy of gamma  radiation. p38gamma was immunoprecipitated from the cell lysates collected at the indicated time points after irradiation. p38gamma activities were measured in kinase assays using GST-ATF2(1-109) as substrate. Equivalent loading of p38gamma in the gamma -irradiated samples was confirmed by Western blotting using anti-p38gamma antibody. (B) Both ATM+/+ and ATM-/- human fibroblasts were seeded into 6-cm-diameter dishes and infected with Ad.GFP (control) or Ad.MKK6(E) at 10,000 IFU/cell 24 h after plating. The cells were treated with or without gamma  radiation (IR) 24 h after infection. Mitotic index was determined 24 h postirradiation as described in Materials and Methods. Data with error bars are means ± standard errors of triplicate samples.

Since it appears that the MKK6-p38gamma cascade is downstream of ATM following gamma  irradiation, we tested whether G2 arrest can be induced in ATM-/- cells by MKK6(E) expression. ATM+/+ and ATM-/- human fibroblasts were irradiated with 6 Gy of gamma  radiation with or without preinfection with Ad.MKK6(E). G2 arrest was determined by mitotic index assay as shown in Fig. 5B. gamma  irradiation induced G2 arrest in ATM+/+ cells but not ATM-/- cells, while MKK6(E) caused cell cycle arrest in both cell types. This result supported the idea that MKK6-p38gamma is downstream of ATM in mediating a G2 arrest.

Activation of MKK6-p38gamma cascade influences Cds1 activity indirectly. There are several reports regarding the function of p38gamma (11, 23, 33, 35, 40). However, available data on p38gamma provide little information regarding the mechanisms by which p38gamma regulates G2 arrest. Genetic studies on yeast cell cycle checkpoint control identified Chk1 as a necessary component of gamma -irradiation-induced G2 arrest (57), and Cds1 is required for a DNA replication checkpoint (44). In mammalian cells, both Chk1 and Cds1 are thought to be involved in G2 arrest after gamma  irradiation (5, 38, 54). To determine whether Cds1 is a downstream target of activated p38gamma , we examined the activation of Cds1 in cells that had been infected with constitutively active MKK6 or dominant inactive p38gamma . Cds1 autophosphorylation is activated in response to gamma  irradiation (5, 38), and we used this assay system to determine if Cds1 is activated by MKK6(E). Cds1 was immunoprecipitated from U2OS cells 24 h after infection with either Ad.MKK6(E) or control virus Ad.GFP, or from cells 1 h after gamma  irradiation. As shown in Fig. 6A, enhanced autophosphorylation of Cds1 shows that both gamma  irradiation and MKK6(E) activate Cds1. Similar results were obtained from HeLa cells (data not shown). When U2OS cells were preinfected with Ad.p38gamma (AF) for 24 h prior to gamma  irradiation, Cds1 activation was significantly attenuated (Fig. 6B). Control virus expressing GFP had no effect. These data suggest that the MKK6-p38gamma cascade participates in Cds1 activation by gamma  irradiation.


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  		FIG. 6.   MKK6(E) activates Cds1 in vivo. (A) U2OS cells were collected and lysed 24 h after adenovirus infection as indicated or 1 h after treatment with 6 Gy of gamma  irradiation (IR). (B) U2OS cells were first infected with adenovirus as indicated for 24 h before gamma  irradiation. Human Cds1 (hCds1) was immunoprecipitated, autophosphorylated in vitro using [gamma -32P]ATP, and visualized by SDS-PAGE. Similar results were obtained from three independent experiments.

gamma irradiation induces a modification of human Chk1 that can be detected as an increase in electrophoretic mobility on SDS-PAGE (54). To determine if Chk1 is also regulated by MKK6(E) expression, U2OS cells were transfected with hemagglutinin-tagged human Chk1 cDNA and then infected with Ad.MKK6(E) or Ad.GFP. Recombinant Chk1 protein was visualized by Western blotting. No Chk1 band shift was observed on MKK6(E) expression (data not shown), suggesting that Chk1 may not be regulated by the MKK6-p38gamma cascade.

Since dominant active MKK6 expression produces upregulation of Cds1 activity, it is possible that Cds1 lies directly downstream of the MKK6-p38gamma pathway. To examine this possibility, we tested whether p38gamma can directly regulate Cds1 activity. Recombinant His-tagged human Cds1, p38gamma , nd MKK6(E) were prepared, and coupled kinase assays with MKK6(E), p38gamma , Cds1 and a synthetic peptide corresponding to residues 208 to 225 of Cdc25C were performed (7). MKK6(E) was included in the reaction mixture to activate p38gamma , with the peptide being a substrate for Cds1. No significant increase in Cds1 activity was detected despite strong p38gamma activity (measured using GST-ATF2 as a substrate [data not shown]). Similar results were obtained both with bacterially and baculovirally expressed Cds1. Since recombinant Cds1 had a measurable basal activity toward Cdc25C, the negative result was probably not due to misfolding of recombinant protein. Thus, in vivo activation of Cds1 by p38gamma may be indirect.

MKK6 activation leads to enhanced phosphorylation on Cdc2. Activation of the Cdc2-cyclin B complex is a key event controlling the G2/M transition. G2 arrest requires inhibitory tyrosine phosphorylation of Cdc2 (6, 27). Therefore, the phosphorylation state of Cdc2 in the cells subject to gamma  irradiation or Ad.MKK6(A) or Ad.MKK6(E) infection was determined. Both gamma  irradiation (Fig. 7A) and MKK6(E) (Fig. 7B) increased the phosphorylation levels of Cdc2 as determined by antibody specific to phosphorylated Cdc2. Constitutive activity of MKK6(E) can maintain the phosphorylation stage of Cdc2 for several days (Fig. 7B). In contrast, no increase in Cdc2 phosphorylation was observed upon expression of MKK6(A) (Fig. 7B).


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  		FIG. 7.   Both gamma  radiation and MKK6(E) lead to Cdc2 phosphorylation. U2OS cells were subjected to 6 Gy of gamma  radiation (A) or infected with either Ad.MKK(E) or Ad.MKK6(A) (B). Cells were collected at the indicated times after treatment, and the level of phosphorylation on Cdc2 was detected by Western blotting with anti-phospho (p)-Cdc2 antibody. Blots were stripped and reprobed with anti-Cdc2 antibody to confirm equal protein loading.

gamma irradiation and MKK6 activation downregulate Cdc25C levels. To evaluate any relationship between MKK6 activation and the mitotic inducer Cdc25C, Cdc25C protein levels were measured by Western blotting. Following gamma  irradiation, a significant reduction in Cdc25C protein level was observed (Fig. 8A and B). The kinetics of Cdc25C reduction following gamma  irradiation correlated with the enhanced phosphorylation of Cdc2 (Fig. 7A) and the G2 arrest (Fig. 2A). This was also true for cells expressing MKK6(E). Expression of MKK6(A) had no effect on the Cdc25C level. Therefore, the reduction of the Cdc25C level is linked to activation of MKK6-p38 or gamma  radiation and is not a consequence of nonspecific metabolic changes in infected cells. To determine the cause of the reduction of Cdc25C, we measured Cdc25C mRNA levels and protein stability. As shown in Fig. 8C, Northern blotting revealed a time-dependent reduction of steady-state Cdc25C mRNA levels after gamma  irradiation, which may account for the change seen in protein expression. The stability of Cdc25C protein was also decreased in cells treated with gamma  radiation based on the pulse-chase labeling experiments (Fig. 8D), suggesting that a decrease in Cdc25C stability may also contribute to the reduction of Cdc25C caused by activation of the MKK6-p38gamma cascade. The decrease in Cdc25C levels was seen 4 h postirradiation. It may therefore contribute to the maintenance of the G2 arrest in cells which have arrested initially due to inactivation or sequestration of Cdc25 (5, 52).


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  		FIG. 8.   Reduction of Cdc25C level after gamma  irradiation or MKK6(E) expression. U2OS cells were treated with 6 Gy of gamma  radiation (A) or Ad.MKK6(E) (B), and Cdc25C levels were analyzed by Western blotting at the indicated time points after treatment. The blots were stripped and reprobed with antiactin antibody to show even loading. (C) A 20-µg aliquot of total RNA from U2OS cells treated with 6 Gy of gamma  radiation was separated on an agarose gel, and Cdc25C mRNA levels were detected by Northern blotting. Levels of 28S rRNA were shown to indicate even loading. (D) U2OS cells were prelabeled with [35S]Cys-Met for 1 h and gamma  irradiated. Cells were then chased with excess unlabeled Met-Cys for the indicated times before labeled Cdc25C was immunoprecipitated from the cell lysate and visualized by SDS-PAGE. A band that was nonspecifically pulled down was used as a loading control. The experiment was repeated three times with similar results.

Blocking the MKK6-p38gamma cascade sensitizes cells to radiation-induced cell killing. Since we have shown that blocking the MKK6-p38gamma cascade impedes cell cycle arrest induced by gamma  irradiation, we tested whether inhibiting MKK6 or p38gamma would sensitize cells to gamma  radiation. Cells that fail to repair damaged DNA before going into mitosis may have chromosomal strand breaks and encounter disruption in subsequent cell cycles. This is reflected by their inability to form colonies. A colony survival assay was performed using cells infected with either Ad.GFP, Ad.MKK6(A), or Ad.MEK1(A) before gamma  irradiation. Serial dilutions of cells were cultured until colonies were large enough to be counted (>50 cells per colony). gamma  radiation reduced the number of colonies formed in a dose-dependent manner, demonstrating its cytotoxic effect. However, cells infected with Ad.MKK6(A) dramatically reduced the number of colonies formed after gamma  irradiation in comparison with mock-infected cells or cells with Ad.GFP or Ad.MEK1(A) (Fig. 9A). No effect of adenovirus infection was observed on colony formation without gamma  irradiation. Blocking p38gamma activity in infecting cells with Ad.p38gamma (AF) also sensitized cells to the killing effects of gamma  radiation, as shown in Fig. 9B; 4 Gy of gamma  radiation had a significantly higher cytotoxic effect on Ad.p38gamma (AF)-infected cells than on cells infected with Ad.p38alpha (AF), Ad.p38beta (AF), Ad.p38delta (AF), or Ad.GFP. These data clearly demonstrate that interfering with the MKK6-p38gamma cascade sensitizes tumor cells to gamma  radiation.


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  		FIG. 9.   Inhibition of the MKK6-p38gamma pathway enhances gamma -radiation-induced cell death. U2OS cells were seeded in serial dilution and treated with nothing (-), Ad.GFP (control), Ad.MKK6(A), or Ad.MEK1(A) (A) or with Ad.p38alpha (AF), Ad.p38beta (AF), Ad.p38gamma (AF), or Ad.p38delta (AF) (B) for 24 h before being exposed to the indicated doses of gamma  radiation (4 Gy in panel B). Cells were cultured, and colonies larger than 50 cells were counted. Survival was calculated by comparison to the survival of nonirradiated cells (taken as 100%). Data shown are means ± standard errors of triplicate observations.


    DISCUSSION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Essential role of the MKK6-p38gamma cascade in signaling G2 arrest. In this report we have shown that the MKK6-p38gamma cascade is activated by gamma  irradiation (Fig. 1) and that this activation is not only necessary (Fig. 3 and 4) but also sufficient (Fig. 2) for G2 arrest following DNA damage. Thus, the MKK6-p38gamma cascade forms an essential part of the signaling pathway leading to G2 arrest. Although MKK6 appears to be the primary MKK for activating p38s in gamma -irradiated cells, the modest activation of MKK3 may have an additive effect on the activation of p38 group members. It was recently reported that expression of dominant negative MEK2, but not MEK1, leads to slightly delayed G2/M arrest upon gamma  irradiation and a substantial inability to progress through the G2 arrest upon recovery from the radiation exposure (1). We have also shown that MEK1 is not required for G2 arrest (Fig. 3B). However, MEK1 was reported to be required for the G2/M transition in normal cell cycle progression (60). MEK1 and MEK2 are closely related homologues. Functional differences between MEK1 and MEK2 in cell cycle regulation are analogous to the respective roles of p38alpha and p38gamma in spindle assembly and G2/M checkpoint control. Although there are no data indicating whether MEK2 activation is sufficient for G2 arrest, the influence of dominant negative MEK2 on gamma -radiation-induced G2 arrest suggests the involvement of other MAP kinases in this process. It would be useful to investigate the relationship between the MKK6-p38gamma cascade and MEK2. The ERK cascade is independent of the p38 pathway; therefore, it is unlikely that MKK6-p38gamma and MEK2 are in the same cascade. However, the possibility of cross talk cannot be formally excluded.

The ability of MKK6 activation to produce G2 arrest in p53-deficient cells suggests that the p53 is not essential for the MKK6-p38gamma -mediated G2 arrest. However, the possibility that p53 contributes to the MKK6-p38gamma G2 response cannot be excluded. Although there is a paucity of information concerning activation of the MKK6-p38gamma cascade under different physiological conditions, there is evidence that this cascade can be activated by a variety of stimuli. However, not all of the stimuli that activate MKK6-p38gamma can cause cells to arrest at G2/M phase. Prolonged activation of the MKK6-p38gamma cascade may be a feature that distinguishes gamma -radiation-induced activation from other stimuli.

Interaction of the MKK6-p38gamma cascade with the DNA damage checkpoint machinery. Several proteins are required for the DNA damage checkpoint control machinery in mammalian cells. ATM is required for G2 arrest after gamma  irradiation and other checkpoint components, such as Cds1, lie downstream of ATM. Since p38gamma activation is ATM dependent and the activation of the MKK6-p38gamma cascade leads to Cds1 activation, we propose that the MKK6-p38gamma response is located between ATM and Cds1. A schematic diagram is shown in Fig. 10. MKK6-p38gamma lies downstream of ATM in response to gamma -radiation-induced DNA damage. Cds1 is activated in a MKK6-p38gamma - and ATM-dependent manner and, together with Chk1, downregulates Cdc25C activity by direct phosphorylation. Reduced activity of Cdc25C and a subsequent increase in Cdc2 phosphorylation lead to cell cycle arrest at G2. Although our data suggest that MKK6-p38gamma lies upstream of Cds1 activation, this model does not exclude the possibility that other pathways also regulate Cds1 activity.


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  		FIG. 10.   The MKK6-p38gamma pathway participates in the DNA damage checkpoint. See text for details.

Cdc25C, as a core regulator in DNA damage checkpoint control, is a target of both Cds1 and Chk1 (5, 16, 38, 52, 54). The current model for Cdc25C regulation in response to DNA damage is that phosphorylation of Cdc25C by Chk1 and/or Cds1 allows 14-3-3 binding, which facilitates export of Cdc25C from the nucleus (37). Inactivation of Cdc25C by Cds1 and Chk1 phosphorylation has also been reported (5, 15). We have found a decrease in Cdc25C levels after gamma  irradiation or MKK6-p38gamma activation (Fig. 7). This change in Cdc25C protein levels correlated with increased Cdc2 phosphorylation (Fig. 5), suggesting that loss of this protein also contributes to the control of Cdc2 activity. Cdc25 degradation has been reported in mammalian cells and fission yeast (4, 45) and implicated as an important regulatory mechanism for cell cycle progression. A role for changes in Cdc25C protein level in G2/M arrest has not previously been reported. Although the immediate events that lead to Cdc2 inactivation would be Cdc25C phosphorylation and inactivation, it is possible that protein reduction of Cdc25C is an additive mechanism that reinforces the cell cycle checkpoint.

Different functions of p38 group MAP kinase members in cell cycle regulation. Published data have implicated a member of the p38 group in cell cycle progression. p38alpha has been reported to be important in the spindle assembly checkpoint of somatic cell cycles (56). Microinjection of p38alpha has also been reported to prevent the G1/S transition (42). Here we provide evidence for a role for p38gamma in gamma -radiation-induced G2 arrest. Although all p38 isoforms were activated in gamma -irradiated cells, the effect of p38gamma appears to be significant because only activation of p38gamma was required to induce a G2-phase arrest independent of the other p38 group members. However, p38alpha 's effects in the G1 and/or spindle assembling checkpoint cannot be excluded from gamma -irradiated cells because some cells inevitably escape the G2 checkpoint. Therefore, other p38 group members may also participate in gamma -irradiated cells to protect cells at multiple checkpoints. The different members of the p38 kinase group may function differently in normal or stressful situations. It was observed that inhibition of p38alpha /beta by SB203580 significantly retarded cell proliferation (unpublished observation) and overexpression of p38alpha inhibited cell growth (21), suggesting an involvement of p38alpha in normal cell cycle progression. However, there is no evidence to suggest a role for p38gamma in the normal progression of the cell cycle. Inhibiting p38gamma activity by overexpression of p38gamma (AF) had no detectable effect on cell proliferation determined by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay (data not shown), indicating that p38gamma may be primarily involved in transmitting stressful stimuli to cell cycle machinery.

The MKK6-p38gamma cascade is a potential target for sensitizers of radiotherapy. Radiotherapy is widely used in cancer treatment but is often unsuccessful because of tumor cell radioresistance (58, 59). Cell death induced by ionizing radiation has been studied in many different types of cells, and its mechanism and time course appear to be variable. The length of G2/M arrest positively correlates with radioresistance of the cell (3). Pharmacological agents such as caffeine that force cells to pass the G2/M block after DNA damage are useful because they increase cell death (31). We have shown in this report that interfering with the MKK6-p38gamma cascade dramatically enhances gamma -irradiation-mediated cell killing (Fig. 9). Because p38gamma is inactive under normal conditions and inhibition of p38gamma did not influence normal cell proliferation, the molecules in this pathway can be considered potential targets for radiation sensitization. Since there has been great success in finding specific pharmacological inhibitors for different kinases (14, 34), the development of specific inhibitor for p38gamma or another kinase in this pathway is possible. Thus, understanding the mechanism by which p38gamma regulates G2 arrest may help to develop new strategies for the improvement of radiation therapy.


    ACKNOWLEDGMENTS

We thank J. V. Kuhns for excellent secretarial assistance.

This work was supported by grants from the California Cancer Research Program (J. Han) and the PEW Scholar Program in Biomedical Science (S. Huang) and by a California Breast Cancer Research Program postdoctoral fellowship (X. Wang).


    FOOTNOTES

* Corresponding author. Mailing address: Department of Immunology, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037. Phone: (858) 784-8704. Fax: (858) 784-8227. E-mail: jhan{at}scripps.edu.

dagger Publication no. 12814-IMM from the Department of Immunology, The Scripps Research Institute, La Jolla, Calif.

Dagger Present address: Department of Pathology, Linkoping University, S-581 85 Linkoping, Sweden.


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Abstract
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Materials and Methods
Results
Discussion
References

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