Access the full text.
Sign up today, get DeepDyve free for 14 days.
Loading next page...
/lp/unpaywall/mitogen-activated-protein-kinase-kinase-2-activation-is-essential-for-Bz2kytAzAK
References
References for this paper are not available at this time. We will be adding them shortly, thank you for your patience.
- Publisher
- Unpaywall
- ISSN
- 0021-9258
- DOI
- 10.1074/jbc.274.5.2732
- Publisher site
- See Article on Publisher Site
Abstract
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 274, No. 5, Issue of January 29, pp. 2732–2742, 1999 © 1999 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Mitogen-activated Protein Kinase Kinase 2 Activation Is Essential /M Checkpoint Arrest in Cells for Progression through the G Exposed to Ionizing Radiation* (Received for publication, October 13, 1998) Derek W. Abbott‡ and Jeffrey T. Holt§ From the Vanderbilt University Departments of Cell Biology and Pathology and the Vanderbilt University Cancer Center, Nashville, Tennessee 37232 An increasing body of evidence suggests that mitogen- dsbs. For example, the cancer predisposition genes ATM and induced activation of the RAF/ERK signaling pathway is DNA-PK are kinases that are activated by dsbs. Cells with functionally separate from the stress-induced activation mutations in these two genes show a decreased ability to repair of the SEK/JNK/p38 signaling pathway. In general, dsbs, and it is thought that this inability leads to the genomic stress stimuli strongly activate the p38s and the JNKs instability that is characteristic of cancers containing ATM or while only weakly activating ERK1 and ERK2. However, DNA-PK mutations (Refs. 1–3 and reviewed in Refs. 4 and 5). a number of independent groups have now shown that Whereas ATM and DNA-PK are required for the signaling the RAF/ERK signaling pathway is strongly activated by initiated by dsbs, the cancer predisposition gene BRCA2 is ionizing radiation. In this work, we examine this para- required for the actual repair of dsbs. Cells that lack BRCA2 dox. We show that both mitogen-activated protein show decreased ability to survive ionizing radiation (6 – 8), and (MAP) kinase kinase 1 (MEK1) and MAP kinase kinase 2 this inability to repair dsbs is central to BRCA2-initiated car- (MEK2) are activated by ionizing radiation. Blockage of cinogenesis (8 –10). Several oncogenes also have similar effects this activation through the use of dominant negative on dsb signaling and dsb repair. The c-abl proto-oncogene has MEK2 increases sensitivity of the cell to ionizing radia- been shown to be activated upon the formation of dsbs via an tion and decreases the ability of a cell to recover from interaction with DNA-PK (12–15), and the retroviral oncogene, /M cell cycle checkpoint arrest. Blocking MEK2 the G FBR v-fos, has been shown to inhibit the response of the cell to activation does not affect double-strand DNA break re- ionizing radiation (16). All of these findings implicate dsb for- pair, however. Although MEK1 is activated to a lesser mation in the initiation of carcinogenesis. extent by ionizing radiation, expression of a dominant Much of the work implicating dsb formation in the initiation negative MEK1 does not affect radiation sensitivity of /M checkpoint of the cell, or double- the cell, the G of carcinogenesis has focused on the signaling pathways in- strand break repair. Because ionizing radiation leads to duced by dsbs (e.g. ATM/DNA-PK) and on the repair of those /M arrest) than that typ- a different cell cycle arrest (G 2 dsbs (e.g. BRCA2). Less is known about the signaling induced ically seen with other stress stimuli, and because we by the agent causing the dsb. The most commonly used agent to /M checkpoint ki- have shown that MEK2 can affect G induce dsbs is ionizing radiation. Whereas dsbs are the most netics, these results provide an explanation for the ob- prominent effect of ionizing radiation exposure, ionizing radi- servation that the MEKs can be strongly activated by ation also causes lipid peroxidation, glutathione depletion, and ionizing radiation and only weakly activated by other protein oxidation (reviewed in Refs. 17 and 18). Thus, ionizing stressful stimuli. radiation activates the stress response pathway of the cell in a manner that is not necessarily dependent on the formation of dsbs (19). Double-strand DNA breaks (dsbs) are recognized as key Ionizing radiation will activate JNK (20), p38, SEK (21), and components of the initiation of multi-step carcinogenesis. A NF-kB (22), and it will induce the immediate early genes, c-jun, number of cancer predisposition genes and oncogenes exert c-fos, and egr-1 (23–27). Although it is not surprising that their effects through the cell signaling pathways initiated by ionizing radiation activates the stress response pathway, it is unexpected that the typically growth-responsive MAP kinase cascade (reviewed in Refs. 28 –30) is also activated by ionizing * This work was supported in part by National Institutes of Health radiation. Although exceptions exist (31), an increasing body of Public Service Grant R01CA51735. The costs of publication of this evidence suggests that the stress response pathway is function- article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance ally separate from the growth-responsive MAP kinase pathway with 18 U.S.C. Section 1734 solely to indicate this fact. (11, 32–36). The stress response pathway (SEK, JNK, and p38) ‡Supported by National Institutes of Health Medical Scientist Train- is poorly activated by mitogens, whereas the MAP kinase path- ing Program Grant 5T32GM07347 from the National Institutes of way is generally poorly activated by stressful stimuli (UV ra- Health. § To whom correspondence should be addressed: 2220 Pierce Ave. diation, osmotic stress, etc.) (11, 32–36). However, c-ras,c-RAF, South, Rm. 659 MRB II, Vanderbilt University Cancer Center, Nash- ERK1, and ERK2 have all been shown by independent groups ville, TN 37232. Tel.: 615-936-3114; Fax: 615-936-1790; E-mail: to be activated by ionizing radiation (37– 41). It is unknown [email protected]. how activation of the MAP kinase pathway affects cellular The abbreviations used are: dsb, double-strand DNA break; ATM, ataxia telangectasia gene; DNA-PK, DNA-dependent protein kinase; survival in response to ionizing radiation, but the fact that BRCA2, breast cancer 2 gene; MEK, MAP kinase kinase; SEK, stress- activation has been seen so consistently suggests that activa- activated protein kinase kinase; MAP kinase, mitogen-activated protein tion of the MAP kinase cascade could be important for response kinase; JNK, Jun kinase; Gy, gray; bp, base pair; PAGE, polyacryl- of the cell to ionizing radiation. amide gel electrophoresis; PBS, phosphate-buffered saline; IP, immunoprecipitation. The activation of the MAP kinase pathway by the stress 2732 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. Role of MEK2 in G /M Checkpoint Recovery 2733 establishment or within 3 weeks following liquid nitrogen thawing. stimulus of ionizing radiation could be due to the fact that dsbs Plasmids—The plasmids used in this work were the gifts of Edwin lead to different cell cycle checkpoint control than what is Krebs, University of Washington, Seattle (K97A MEK1, S222A MEK1); normally seen for DNA-damaging agents (reviewed in Refs. 42 Natalie Ahn, University of Colorado, Boulder (pCML-MEK2); and Kun- and 43). Repair of dsbs follows two pathways, nonhomologous Liang Guan, University of Michigan, Ann Arbor (pGEX-MEK2). The recombinational repair (occurring in G ) and homologous re- 1 K97A MEK and S222A MEK1 plasmids were supplied in the vector combination repair (44 – 47). Homologous recombinational re- pCDNA 3.1 (Invitrogen). To subclone these genes into the pREP4 vector (Invitrogen), pCDNA-K97A MEK1, pCDNA-S222A MEK1, and pREP4 pair occurs in G , the time point when homologous, undamaged were digested with XhoI and HindIII (Life Technologies, Inc.). The double-strand DNA is present to serve as a template for correct K97A MEK1 fragment and the S222A MEK1 fragment was then ligated repair. Because the damaged DNA strand has a template for into pREP4. To generate the dominant negative K101A MEK2 con- repair, homologous recombinational repair leads to fewer mu- struct, pCML-MEK2 was digested with BamHI (Life Technologies, Inc.) tations than nonhomologous repair (44, 47, 48). Therefore, the and ligated into pGEM4Z (Promega). Pgem4Z-MEK2 was then digested G /M checkpoint is essential for the proper repair of DNA with PstI (Life Technologies, Inc.) to generate a 400-bp fragment con- taining the region to be mutated. This 400-bp PstI fragment was then damaged by ionizing radiation. Recently, the MAP kinase path- ligated into pGEM7Z (Promega). This pGEM7Z-400-bp MEK2 construct way has been implicated in G /M cell cycle regulation. In Xe- was then digested with BalI and NaeI (Life Technologies, Inc.), and an nopus oocytes, MAP kinase activity has been shown to be oligonucleotide containing the sequence 59-CCAGGGCGCTGATCCAC- necessary for progression through G (49 –51). The MEK1 and CTC GAGATCAAGCC-39 was ligated into the digested MEK2. This MEK2 activator, c-mos, has also been shown to be necessary for oligonucleotide sequence contains a 2-base pair replacement (in bold) progression through G (52), and in mouse oocytes, MAP kinase designed to mutate Lys-101 to alanine and a single base pair replace- ment (in italics) designed to insert an XhoI site without changing the becomes activated at metaphase and localizes to the microtu- amino acid sequence. The altered 400-bp fragment was then sequenced bule-organizing centers (53). Because the MAP kinase pathway to ensure that the desired mutation was present and in frame. The is required for G /M progression in a number of systems and pGEM7Z-400-bp K101A MEK2 construct was then digested with PstI, because ionizing radiation leads both to activation of the MAP and the mutated 400-bp fragment was reinserted into pGEM4Z-MEK2. kinase pathway and to G /M arrest, it is possible that ionizing 2 This plasmid was then sequenced to ensure that the mutation was radiation activates the MAP kinase cascade to exert an effect present and in frame. In addition, in vitro translation of the protein gave a 46-kDa fragment that could be immunoprecipitated with an on G /M checkpoint control. antibody to MEK2. The pGEM4Z-K101A MEK2 fragment was then To address these questions, we focused on MEK1 and MEK2, digested with BamHI and inserted into the pREP4 vector. All pREP4 two components of the MAP kinase cascade. These two proteins plasmids used for transfections were purified by ultracentrifugation are phosphorylated in vitro by RAF (54 –55) and can both using CsCl gradients prior to transfection. phosphorylate ERK1 and ERK2 (30). MEK1 and MEK2 are Immunoprecipitations and Kinase Assays—Immunoprecipitations approximately 80% homologous and are very similar in size and kinase assays were performed using a method described previously with slight modifications (58). Cells were washed twice in phosphate- (MEK1 5 45 kDa; MEK2 5 46 kDa). They differ in the first 30 buffered saline (PBS) and harvested by scraping. Cells were pelleted by amino acids of the N terminus and in a proline-rich region that centrifugation at 2000 rpm and rewashed with PBS followed by another is only found in MEK1 (56, 57). In this work, we find that both 2000 rpm centrifugation. Cells were then lysed in Lysis Buffer (20 mM MEK1 and MEK2 are specifically activated by ionizing radia- Tris (pH 7.5), 0.27 M sucrose, 1 mM sodium orthovanadate (Sigma), 10 tion in a variety of cell lines. We show that cells that express mM sodium b-glycerophosphate (Sigma), 0.5 mM okadaic acid (Life Tech- dominant negative MEK2 show radiation hypersensitivity that nologies, Inc.), 50 mM NaF (Sigma), 5 mM sodium pyrophosphate (Sig- ma), 1% Triton X-100, 0.1% b-mercaptoethanol, 1 mM benzamidine is not seen in cells which equivalently express dominant neg- (Sigma), 0.2 mM phenylmethylsulfonyl fluoride (Sigma), 5 mg/ml leu- ative forms of MEK1. Expression of dominant negative MEK2 peptin (Sigma), and 2 mg/ml aprotinin (Sigma)) for 10 min at 4 °C. The leads to a slightly delayed G /M arrest upon ionizing radiation suspension was then sonicated briefly and centrifuged at 14,000 rpm at exposure and a substantial inability to progress through the 4 °C for 10 min. The supernatant was then extracted. Protein concen- G /M arrest upon recovery from that radiation exposure. Fi- 2 tration was standardized using the Bio-Rad protein assay. 100 mgof nally, we show that the effect of MEK2 on radiation sensitivity total protein was then incubated with 10 ml of the given antibody (anti-N-terminal MEK1 or anti-N-terminal MEK2, and total volume can be reversed by forcing G progression through the use was adjusted to 700 ml using Lysis Buffer) for2hat4 °C. Protein pharmacological agents. These data imply that ionizing radia- A/G-Sepharose (Santa Cruz Biotechnology) was then added for 1 h. The tion activates the MAP kinase cascade in a specific manner to suspension was then centrifuged, and the precipitate was washed five maintain G /M checkpoint fidelity. times with Lysis Buffer (the 2nd and 3rd lysis buffer contained 0.5 M NaCl). For the metabolic labeling experiment, 23 SDS-PAGE buffer EXPERIMENTAL PROCEDURES was added. The suspension was boiled and electrophoresed on a 10% Cell Culture and Transfection—HeLa cells, NIH 3T3-L1 cells, and SDS-PAGE gel. The gel was then dried and subjected to autoradiogra- BxPC-3 cells were obtained from the American Type Tissue Collection phy. For the kinase assays, the precipitate was then washed an addi- (ATCC) and grown in Dulbecco’s modified Eagle’s media supplemented tional three times in Kinase Buffer (50 mM Tris (pH 7.5), 0.03% Brij 35 with 10% fetal bovine serum (Sigma), 2 mML-glutamine (Sigma), and (Sigma), 0.1% b-mercaptoethanol, 0.5 mM okadaic acid (Life Technolo- 13 antimycotic/antibiotic (Sigma). HBL100 cells were obtained from gies, Inc.), 0.27 mM sodium orthovanadate, and 10 mM magnesium the ATCC and grown in McCoy’s medium (Life Technologies, Inc.) chloride). After the washes, Kinase Buffer was added to the immuno- supplemented with 10% fetal bovine serum. Serum starvation was precipitate such that the total volume was 50 ml. 25 ml of this solution performed in Dulbecco’s modified Eagle’s media supplemented with was added to a microcentrifuge tube containing 2.5 mg of GST-K71A 0.5% fetal bovine serum, 2 mML-glutamine, and 13 antimycotic/anti- ERK1 (Upstate Biotechology, Inc.) and incubated at 30 °C for 5 min. biotic for 24 h followed by incubation in 0.1% fetal bovine serum for The kinase reaction was then initiated by the addition of 10 ml of 0.5 mM 24 h. Metabolic labeling was performed by incubating the cells in ATP supplemented with 1 mlof[g- P]ATP (NEN Life Science Products, methionine-free media (Life Technologies, Inc.) for 30 min, followed by 3000 mCi/ml) and allowed to incubate for 10 min at 30 °C. The reaction the addition of methionine-free media supplemented with 50 mCi of was terminated by the addition of 23 SDS-PAGE Sample Buffer fol- [ S]methionine for 3 h. Stably transfected cell lines were generated by lowed by boiling. 10 ml of the kinase reaction was then electrophoresed transfecting HeLa cells at 80% confluence with 20 mg of indicated on a 7% polyacrylamide gel, dried, and subjected to autoradiography. construct (pREP4 (Invitrogen), pREP4-K97A MEK1, pREP4-S222A Counts/min phosphate transferred per mg of substrate were quantitated MEK1, pREP4-K101A MEK2, pREP4-wt MEK2, pREP4-S222E MEK1) using the Packard Electronic Autoradiography Instant Imager and using the calcium phosphate precipitation. Cells were split 1:3 the comparing activity of the sample with the activity of the unstimulated following day. Two days after transfection, selection was begun in 500 sample. mg/ml hygromycin B (Life Technologies, Inc.). Three weeks later, ap- Western Blotting—Cells were washed twice with PBS and then har- proximately 1000 colonies were pooled and shown to express the desired vested by scraping. Cells were centrifuged at 2000 rpm, rinsed again protein by Western blotting. All cell lines were used within 2 weeks of with PBS, and centrifuged at 2000 rpm. The pellet was then resus- 2734 Role of MEK2 in G /M Checkpoint Recovery pended in Lysis Buffer and allowed to sit on ice for 10 min. The was collected at 585/42 nm band pass filter at a flow rate of 12 6 3 suspension was then sonicated. The suspension was centrifuged at ml/min. 10000 events were measured per experiment. 14,000 rpm for 10 min, and the pellet was discarded. Western blotting was then performed as described previously (59). Briefly, protein con- RESULTS centration was standardized using the Bio-Rad Protein Assay. Equal MEK1 and MEK2 Are Activated by Ionizing Radiation—The amounts of lysate were subjected to polyacrylamide gel electrophoresis MAP kinase kinases (MEKs) are a central component of the (10% gel). Lysates were transferred to nitrocellulose filters (Amersham growth-response signaling pathway (reviewed in Refs. 29 and Pharmacia Biotech) for1hat40mV.To ensure equal loading of protein and equal transfer efficiency, the membrane was stained with Ponceau 30). Whereas a great deal of work has centered on their role in S prior to blocking. The membrane was blocked using 5% nonfat dry transmitting mitogenic signals, there is an increasing body of milk, 0.3% Tween 20 in Tris-buffered saline. After blocking and subse- evidence that the MEKs could play a role in the response of the quent washing, the blot was exposed to a 1:1000 dilution of the given cell to the stress of ionizing radiation (37–39). Activation of antibody overnight at 4 °C. The blot was then washed extensively with MEK1 and MEK2 involves phosphorylation upon conserved Tris-buffered saline, 0.3% Tween 20 before being exposed to a horse- serine residues (Ser-218 and Ser-222 on MEK1, Ser-222 and radish peroxidase-conjugated secondary antibody (Santa Cruz Biotech- nology) at a dilution of 1:2500 at room temperature for 1 h. Bands were Ser-226 on MEK2; see Refs. 55 and 60). To test the activation visualized using the ECL detection system (Amersham Pharmacia Bio- of MEK1 and MEK2 upon ionizing radiation exposure, an an- tech) using the manufacturer’s instructions. Antibodies used were anti- tibody that specifically recognizes phosphorylated MEK1 and phosphorylated MEK1/2 (New England Biolabs), anti-nonphosphoryl- MEK2 was obtained. HeLa cells were serum-starved and ex- ated MEK1/2 (New England Biolabs), anti-N-terminal MEK1 (Santa posed to increasing doses of ionizing radiation. As a control, one Cruz Biotechnology), and anti-N-terminal MEK2 (Santa Cruz plate of cells was mock-irradiated (0 Gy). 10 min after expo- Biotechnology). Irradiation and Cell Survival Assays—Cells were irradiated using a sure, lysates were generated, and Western blotting was per- Cs source at a dose rate of 2.35 Gy/min. Colony forming assays were formed using the antibody directed against either phosphoryl- performed as described previously (16). Briefly, cells were plated at 500 ated MEK1 or phosphorylated MEK2. Under these conditions, cells/dish and irradiated at a given dose. After irradiation, cells were we were unable to distinguish between the similarly sized returned to the 37 °C incubator. They were refed fresh media every 2 MEK1 and MEK2. However, the Western blot shown in the days. After 14 days, the colonies were fixed in ethanol and stained with crystal violet. Colonies were counted manually. Relative survival was upper panel of Fig. 1 shows that in HeLa cells, the 45-kDa determined by comparing colony number of cells irradiated at a given MEK1 and/or the 46-kDa MEK2 are phosphorylated in re- dose to the colony number of unirradiated cells plated at the same sponse to physiological doses of ionizing radiation (upper blot, density and cultured for the same amount of time. Fig. 1). To determine whether this activation of MEK1 and For cellular survival following treatment with vanadate, approxi- MEK2 is generalizable over cell lines, the same experiment was mately 2000 cells of each cell line (vector-only cells, K97A MEK1 cells, performed in a human non-transformed breast cell line (HBL- and K101A MEK2 cells) were plated 12 h prior to the commencement of the experiment. These cells were then treated with 50 mM vanadate 100), in a human pancreatic adenocarcinoma cell line (BxPC-3), (Sigma) for 8 h prior to irradiation. 16 h after irradiation with 5 Gy, and in a mouse pre-adipocyte cell line (NIH 3T3-L1, the phos- cells were washed extensively and refed with normal media. All cells phorylated sites recognized by the antibody are conserved in lines showed a strong G arrest by flow cytometry analysis. Three mouse MEK1 and MEK2 and human MEK1 and MEK2; see weeks later, cell survival was quantitated as described above (survival Refs. 56 and 57). In all of these cell lines either MEK1 or MEK2 was compared with cells treated with vanadate in the same manner, were phosphorylated in response to ionizing radiation and but not irradiated). For cellular survival following treatment with caf- feine, approximately 2000 cells of each cell line studied were plated as showed various dose responses (Fig. 1, bottom three blots). The above. The cells were irradiated with 5 Gy and then treated with 2 mM * in Fig. 1 refers to a 32-kDa cross-reacting band that is seen in caffeine (Calbiochem) 24 h after irradiation. 32 h after irradiation, flow all cell lines and that is unresponsive to serum starvation or cytometry showed recovery from G arrest in K101A MEK2 cells (rela- ionizing radiation. This cross-reactive band can be used to tive to irradiated/no caffeine cells) and no difference between irradiated standardize for equal protein loading (Fig. 1). In addition, vector-only cells and K97A MEK1 cells either treated or untreated with Western blots using antibody against total MEK1 or total caffeine (32 h after irradiation, vector-only cells and K97A MEK1 cells show normal cell cycle distribution; therefore, treatment with caffeine MEK2 indicate that ionizing radiation does not lead to in- at 24 h has no effect on the cell cycle profile). Survival was measured as creased total MEK protein in the time course of this experi- described above. ment (data not shown). Thus, these experiments show that DNA Repair Assays—Equal cell numbers were embedded into aga- phosphorylation of the MEKs in response to ionizing radiation rose plugs as described previously (8). The plugs were then exposed to is a general effect of ionizing radiation exposure. the indicated doses of ionizing radiation at 4 °C. To allow the cells to To show that MEK1 and MEK2 are not only phosphorylated repair damaged DNA, the cell plugs were then covered with 10% fetal bovine serum/Dulbecco’s modified Eagle’s media in a 37 °C incubator in response to ionizing radiation, but are activated as well, IP for the indicated period. The plugs were then digested overnight in a kinase assays were performed. Antibodies directed against the solution containing 10 mM Tris (pH 7.4), 20 mM NaCl, 50 mM EDTA, and non-conserved regions of MEK1 and MEK2 (N terminus) were 1 mg/ml proteinase K (Life Technologies, Inc.) at 50 °C. The plugs were used to limit cross-reactivity between the two proteins, and then embedded in a 0.7% agarose gel and subjected to pulsed field gel catalytically inactive ERK1 (K71A ERK1) was used as a sub- electrophoresis at 3 V/cm, 45-s pulse time at 14 °C for 48 h (CHEF II strate. Because MEK is downstream of Ras (30), as a positive system, Bio-Rad). Southern blotting was then performed using random prime labeled HeLa cell genomic DNA as a probe. Quantitation of repair control, activity of the MEKs from HeLa cells transformed with was performed using the Packard Electronic Autoradiography Instant v-ras were compared with activity of the MEKs from HeLa cells Imager. transfected with empty vector. Fig. 2A shows that MEK1 and Flow Cytometry—Cells were plated to 40% confluency on 100-mm MEK2 have much higher activity in cells transformed with plates. 18 h later, the cells were exposed to 5 Gy ionizing radiation v-ras, so these antibodies perform appropriately in IP kinase (except for the unexposed plate). At the indicated time point, the cells assays. HeLa cells were then serum-starved and exposed to were trypsinized, centrifuged, and washed 23 with PBS. 1 3 10 cells were then suspended in 1 ml of PBS supplemented with 5 mg/ml pro- various doses of ionizing radiation. As a positive control for pidium iodide. 1 ml of Vindelov’s solution (10 mM Tris (pH 8.0), 10 mM increased kinase activity, cells were restimulated with 20% NaCl, 0.7 units/ml RNase, 50 mg/ml propidium iodide, 0.1% Nonidet serum (Fig. 2B, far left lane). MEK1 shows a slight increase in P-40) was then added, and the cells were allowed to incubate in the dark activity at both 2.5 and 10 Gy, whereas MEK2 is strongly at 4 °C overnight. Flow cytometry was performed using a Becton Dick- activated by 10 Gy of ionizing radiation (Fig. 2B). The same inson FACS Caliber using the Vanderbilt University Flow Cytometry experiment was then performed using an IP-depletion strategy Core. Data acquisition software is CellQuest Version 3.1. Excitation was with 488 nm air-cooled argon-ion laser at 15 milliwatts. Emission aimed at eliminating the remaining MEK1 and MEK2. Cells Role of MEK2 in G /M Checkpoint Recovery 2735 exposure. Overexpression of Dominant Negative MEK2, but Not Domi- nant Negative MEK1, Increases Sensitivity of the Cell to Ioniz- ing Radiation—To determine whether the activation of MEK1 and MEK2 is significant for the response of the cell to ionizing radiation, dominant negative forms of these two proteins were used. Two dominant negative forms of MEK1 were obtained (60). The first, K97A MEK1, replaces a lysine in the ATP- binding domain, with an alanine, such that the kinase cannot bind ATP to transfer the phosphate. The second, S222A MEK1, replaces a serine, which is essential for activation, with an alanine, such that full activation cannot be achieved (generous gifts of Edwin Krebs, University of Washington, Seattle). These two dominant negative constructs have been previously shown to slow cell growth and inhibit activation by EGF and serum stimulation (60). In addition, as a control for overexpression of MEK1, a constitutively active form of MEK1 (S222E MEK1) was also obtained (gift of Edwin Krebs, University of Washing- ton, Seattle). We subcloned these three constructs into pREP4 (Invitrogen). This vector replicates episomally in HeLa cells and contains the hygromycin resistance gene driven by the cytomegalovirus promoter. The constructs were transfected into HeLa cells, selected for 3 weeks in hygromycin, and ap- proximately 1000 clones were pooled. Because activity of dom- inant negative proteins is dependent on their expression rela- tive to wild-type protein, we wanted to show expression of the dominant negative constructs in these cells relative to wild- type MEK1 expression. Lysates were generated and Western blots were performed (with equivalent lysate protein concen- FIG.1. MEK1 and MEK2 are phosphorylated in response to tration) using an antibody against MEK1. This antibody will ionizing radiation. Cells were serum-starved for 2 days and then recognize both endogenous wild-type protein and overex- exposed to the different doses of ionizing radiation. 10 min after expo- pressed dominant negative protein. Fig. 3A shows that relative sure, the cells were lysed, and equal amounts of lysates were electro- to cells stably transfected with vector only, both K97A MEK1 phoresed. Western blotting was performed using an antibody that recognizes active, phosphorylated MEK1 (Ser-218 and Ser-222 cells and S222A MEK1 cells have greatly overexpressed dom- phosphorylation) or MEK2 (Ser-222 and Ser-226 phosphorylation). The inant negative MEK1 (Fig. 3A). In addition, the constitutively cell lines used to show MEK phosphorylation in response to ionizing active S222E MEK1 is also expressed to a high degree (Fig. 3A). radiation were (top to bottom) as follows: HeLa cells, a human cervical To show that these constructs affect the activity of MEK1 in cancer cell line; HBL-100 cells, a human non-transformed breast epi- thelial cell line; BxPC-3 cells, a human pancreatic adenocarcinoma cell response to ionizing radiation, IP kinase assays were per- line and NIH 3T3-L1 cells, a mouse pre-adipocyte cell line. MEK1 runs formed using exposed and unexposed cells. The cells containing as a 45-kDa band and MEK2 runs as a 46-kDa band and are indistin- the stably transfected empty vector show the 1.6 –1.8-fold acti- guishable in this assay. The * refers to a 32-kDa band that cross-reacts vation of MEK1 upon exposure to ionizing radiation, whereas with the antibody in all cell lines and is unresponsive to both serum starvation and to ionizing radiation. This band can be used to normalize the cells containing the dominant negative constructs showed for equal protein loading. In addition, Western blots against total MEK no activation upon exposure to ionizing radiation (Fig. 3B). In protein indicated that in the time course of this experiment, MEK addition, the S222E MEK1 cell line shows elevated basal ac- protein levels were not up-regulated by ionizing radiation exposure (data not shown). tivity which can be increased approximately 1.4-fold upon ion- izing radiation exposure (Fig. 3B). To test the K97A MEK1 and the S222A MEK1 dominant were metabolically labeled with [ S]methionine, and immu- negative effects of constructs on cell survival in response to nodepletion was performed using antibodies directed against ionizing radiation, colony forming assays were performed. This either c-fos, MEK1, or MEK2. Lysates were cleared by adding assay measures the ability of a cell to survive ionizing radiation protein A/G-agarose and, after 1 h incubation, retaining the exposure and to proliferate following ionizing radiation expo- supernatant. These depleted lysates were then immunoprecipi- sure (16). Approximately 500 cells were plated per ionizing tated with either MEK1 and MEK2. The antibody to MEK1 radiation dose. The plates were exposed to the indicated dose of immunoprecipitates a 45-kDa protein when depleted either of ionizing radiation, and colony formation was scored 2 weeks c-fos or MEK2, whereas this antibody precipitates decreased later. The S222A MEK1 cell line had slightly decreased sur- 45-kDa protein when depleted of MEK1 (Fig. 2C, left three vival at both 4 and 6 Gy of ionizing radiation, whereas the lanes). The antibody directed against MEK2 immunoprecipi- K97A MEK1 cell line only showed slightly decreased survival tates a 46-kDa protein when depleted of either c-fos or MEK1 at 6 Gy of ionizing radiation (Fig. 3C). Neither of these cell lines but not when depleted of MEK2 (Fig. 2C, right three lanes). Thus, the antibodies employed in Fig. 2 do not show a high showed significant difference from the S222E MEK1 cell line (Fig. 3C), so the dominant negative MEK1 constructs have degree of cross-reactivity. Immunodepletion was then coupled with the IP kinase assay to show activity of MEK1 and MEK2. little effect on HeLa cell survival in response to ionizing After serum starvation and depletion of either MEK1 or MEK2, radiation. IP kinase assays were performed. Fig. 2D shows that both Because MEK2 is activated in IP kinase assays to a greater MEK1 and MEK2 show increased kinase activity upon expo- extent then MEK1, it is possible that a dominant negative form sure to 10 Gy of ionizing radiation and that MEK2 shows of MEK2 would have a larger effect on the ability of HeLa cells increased activity relative to MEK1 upon ionizing radiation to survive ionizing radiation exposure. To test the effect of a 2736 Role of MEK2 in G /M Checkpoint Recovery FIG.2. MEK1 and MEK2 are activated by ionizing radiation. A, as a positive control to show that the antibodies obtained could be used to perform MEK1 and MEK2 IP kinase assays, IP kinase assays were performed on cells stably transfected with v-ras or cells stably transfected with empty vector. Cells were serum-starved, and equal amounts of lysate were immunoprecipitated with an antibody that recognizes only MEK1 or only MEK2. The substrate K71A ERK1 (kinase-dead) was then added to the immunoprecipitate in the presence of [g- P]ATP. The kinase reaction was halted by the addition of SDS-PAGE buffer followed by boiling. The reaction was then electrophoresed and autoradiographed. B, IP kinase assays were performed on serum-starved HeLa cells after exposure to serum, 2.5 Gy ionizing radiation, and 10 Gy ionizing radiation. Equal amounts of protein were immunoprecipitated with antibody directed against MEK1 or MEK2. Phosphorylation of the K71A ERK1 substrate is shown in the autoradiograph. C, to test specificity of the antibodies and their suitability for IP depletion/IP kinase assays, cells were metabolically labeled with [ S]methionine. Lysates were pooled and divided into three tubes. For control immunodepletions, to one tube antibody to c-fos was added. To another, antibody to MEK1 was added, and to the last, antibody to MEK2 was added. After overnight incubation, protein A/G-agarose was added for an hour. After centrifugation, each supernatant was divided in two, and immunoprecipitations were performed using antibody to either MEK1 or MEK2. The S-labeled MEK1 or MEK2 is indicated in the autoradiograph of the electrophoresed immunoprecipitation product. D, immunodepletion was followed by IP kinase assays. HeLa cells were serum-starved. Equal amounts of lysates were immunodepleted of either MEK1 or MEK2, and then IP kinase assays were performed using K71A ERK1 as a substrate. Again, although both MEK1 and MEK2 are activated by ionizing radiation, MEK2 shows a greater increase in activation relative to unstimulated cells. Each IP kinase assay presented in Fig. 2 was performed a minimum of three times with similar results each time. dominant negative MEK2, we mutated wild-type MEK2 (57, survival, but at higher doses of radiation (.3 Gy), these cells 61) (generous gift of Natalie Ahn, University of Colorado, Boul- are significantly more radiosensitive (Fig. 4C). Since cells con- der) to K101A MEK2. This mutation was designed to be anal- taining wild-type MEK2 show no differences in survival rela- ogous to the K97A MEK1 mutation, as this region of MEK2 is tive to the vector-only cells, the decreased survival of cells highly conserved with MEK1 (56, 57). By analogy, the K101A expressing dominant negative MEK2 is not simply an overex- MEK2 mutation should render MEK2 unable to bind ATP to pression phenomenon. Thus, whereas both MEK1 and MEK2 transfer the phosphate to its substrate. Both the dominant are activated in response to ionizing radiation, only expression negative K101A MEK2 and wild-type MEK2 were subcloned of dominant negative MEK2 leads to radiosensitivity. into the pREP4 vector and transfected into HeLa cells. After 3 Dominant Negative MEK2 Has No Effect on Double-strand weeks of hygromycin selection, approximately 1000 colonies DNA Repair but Leads to Defective G /M Checkpoint Control— were pooled. Western blots were performed (equivalent protein Two explanations are possible for the effect of MEK2 on radi- concentrations of lysates) using an antibody that recognizes ation survival. First, MEK2 may influence DNA repair. Ioniz- both endogenous, wild-type MEK2 and transfected, dominant ing radiation causes double-strand DNA breaks, and a number negative MEK2. Fig. 4A shows that K101A MEK2 and wt of proteins that cause radiation hypersensitivity do so by not MEK2 are highly expressed relative to cells stably transfected allowing efficient double-strand break repair (62). In addition, with vector alone (Fig. 4A). To show that expression of the the MEK activator, c-mos, has been shown to influence cellular dominant negative MEK2 leads to decreased MEK2 activation genomic stability (63). For these reasons, the effect of MEK2 on in response to ionizing radiation, IP kinase assays were per- double-strand break repair was tested using the K101A MEK2, formed on these cells using kinase-inactive ERK1 as substrate. K97A MEK2, and vector-only cell lines. Equal numbers of cells The cells containing only the empty vector show a 4 – 6-fold were embedded in agarose plugs and exposed to 10 Gy of up-regulation of MEK2 activity in response to ionizing radia- ionizing radiation. These cells were then allowed to repair the tion, whereas the K101A MEK2 cells show no activation in damaged DNA for 30, 60, or 120 min or not allowed to repair (0 response to ionizing radiation (Fig. 4B). In addition, expression min) the damage. After the plugs were digested overnight with of wild-type MEK2 shows higher basal MEK2 activity that proteinase K, they were embedded in a 0.7% agarose gel and can be increased only slightly in response to ionizing radiation subjected to pulse-field electrophoresis. Southern blotting was (Fig. 4B). then performed, and percent repair was quantitated. A repre- Colony forming assays were then performed to determine the sentative experiment is shown in Fig. 5A. Under these electro- influence of MEK2 on the cell’s survival response to ionizing phoretic conditions, the damaged, unrepaired DNA migrates as radiation. 500 cells were plated 12 h prior to radiation expo- a discrete band, whereas the undamaged or repaired DNA sure. Cells were exposed to the given dose, and colony forma- barely migrates out of the agarose plug. The results of three tion was scored 2 weeks later. At low doses of ionizing radia- independent experiments are quantitated in Fig. 5. There are tion, the K101A MEK2 cells do not show decreased cell no significant differences in the abilities of these three cell lines Role of MEK2 in G /M Checkpoint Recovery 2737 FIG.3. Cells that are stably transfected with dominant negative MEK1 show only modestly increased sensitivity to ionizing radiation. A, cell lines that were stably transfected with K97A MEK1, S222A MEK1, or S222E MEK1 were generated from pools of approximately 1000 clones. As a control, stably transfected cells containing only empty vector were also made. To show expression, lysates from the four cell lines (vector, K97A MEK1, S222A MEK1, and S222E MEK1) were generated, and Western blots were performed using an antibody that recognizes the C terminus of MEK1 (Santa Cruz Biotechnology) to probe the blot. The K97A MEK1, the S222A MEK1, and the S222E MEK1 cell lines all overexpress transfected MEK1 as shown by this blot. B, to test whether endogenous MEK1 becomes activated in response to ionizing radiation in the K97A MEK1 and S222A MEK1, two separate plates of each cell line (including vector-only cells) were serum-starved. IP kinase assays were then performed after one plate of cells was exposed to 15 Gy radiation, whereas the other plate of cells was not exposed. Phosphorylation of kinase-dead (K71A ERK2) was then quantitated. Each data point was performed in triplicate. Counts/min of phosphate transferred per mg of substrate is shown in a graph. C, survival of the cells stably transfected with the given MEK1 construct was measured using the colony forming assay. Approximately 500 cells were plated for an indicated time point. Each plate was then exposed to the given dose of ionizing radiation. Cells were refed every 2 days for 2 weeks. Colonies were then stained by crystal violet and counted manually. Relative survival refers to the survival relative to an unirradiated plate of cells from the same cell line plated on the same day at the same density. Each experiment was performed in triplicate. Relative survivals and S.E. are graphed. All cell lines were used within 3 weeks of generation or within 2 weeks of thawing. to repair double-strand DNA breaks (Fig. 5). This result is survival between the K101A MEK2 cell line and the vector-only expected because the K101A MEK2 survival curve (Fig. 4C) cell line is maximal at this dose. At the indicated time points shows a shoulder at low doses of ionizing radiation, implying after exposure, cells were harvested and stained with pro- the ability to repair sublethal DNA damage (3). pidium iodine, and flow cytometry was performed. The vector- Since the MAP kinase pathway has been shown to be neces- only cells and the K97A MEK1 cells show G arrest 12 h after sary for progression through G /M (50, 52), a second explana- exposure with recovery 24 h after exposure (Fig. 6, vector-only tion for the effect of dominant negative MEK2 on ionizing cells are shown in upper panels and K97A MEK1 cells are radiation sensitivity might be a dysregulated G /M cell cycle shown in middle panels). In contrast, the K101A MEK2 cells checkpoint. In HeLa cells, the G /M checkpoint shows the show slower G /M arrest. Full arrest eventually occurs after 2 2 greatest arrest due to ionizing radiation damage (48). To de- approximately a 5–7-h delay (Fig. 6). However, the major cell termine the effect of MEK2 on the G /M checkpoint, the dom- cycle dysfunction in the K101A MEK2 cell line is an inability to inant negative K101A MEK2 cell line was used. As controls, recover from G /M arrest. At both the 24- and 36-h time points, the vector-only cell line, the K97A MEK1 cell line and the significant numbers of K101A MEK2 cells are still arrested in S222A MEK1 cell line were also used. Asynchronously growing G (Fig. 6). At these time points, both the vector-only cells and cells were exposed to 5 Gy ionizing radiation; the difference in the K97A MEK2 cells have recovered and show relatively nor- 2738 Role of MEK2 in G /M Checkpoint Recovery FIG.4. Cells that express dominant negative MEK2 are hypersensitive to ionizing radiation. A, a site-directed MEK2 mutant (K101A MEK2) designed to mimic the K97A MEK1 dominant negative was made. Both the wild-type MEK2 gene and the K101A MEK2 gene were transfected into HeLa cells independently. After 3 weeks of hygromycin selection (pREP4 vector), approximately 1000 colonies from each transfection were pooled. Again, a separate control cell line containing only the vector was also made. Lysates from the three cell lines (K101A MEK2 cells, vector-only cells, and wild-type MEK2 cells) were generated, and Western blotting was performed using an antibody that recognizes the N terminus of MEK2. Both the wild-type MEK2 cells and the K101A MEK2 cells overexpress MEK2 as shown by this blot. B, to test whether the stably transfected K101A MEK2 blocked MEK2 activation in response to ionizing radiation, IP kinase assays were performed. Two serum-starved plates of cells were used. One was exposed to 10 Gy ionizing radiation and the other was not exposed. Phosphorylation of kinase-dead (K71A ERK2) was then quantitated. Each data point was performed in triplicate. Counts/min phosphate transferred per mg of substrate is shown in a graph. C, survival of the cells stably expressing the given MEK2 construct was measured using the colony forming assay. Again, relative survival refers to the survival relative to an unirradiated plate of cells from the same cell line plated on the same day at the same density. Each experiment was performed in triplicate. Relative survivals and S.E.s are graphed. All cell lines were used within 3 weeks of generation or within 2 weeks of thawing. mal cell cycle profiles (Fig. 6). The S222A MEK1 cell line before ionizing radiation exposure. 16 h after radiation expo- showed similar profiles to the K97A MEK1 cell line (data not sure, cells were washed extensively, refed with normal growth shown). Since effective G /M arrest and recovery from that media, and colony forming assays were performed. Fig. 7A arrest are essential for the ability of the cell to respond effec- shows that treatment of K101A MEK2 cells with ionizing ra- tively to ionizing radiation, and since inhibiting MEK2 activity diation and vanadate leads to an increased G arrest when with a dominant negative construct leads to a slightly delayed compared with K101A MEK2 cells treated with ionizing radi- G /M arrest and to a grossly delayed recovery from that arrest, ation alone (Fig. 7A, middle panels). This forced G arrest does 2 2 it is likely that activation of MEK2 in response to ionizing recover radioresistance in the K101A MEK2 cell line, as van- radiation influences the G /M checkpoint control of the cell. adate-treated K101A MEK2 cells show similar radiosensitivity To determine whether an inability to arrest in G or an to untreated K101A MEK2 cells (Fig. 7B). inability to recover from G arrest is responsible for the radio- To study recovery from the G /M arrest, caffeine was used. 2 2 sensitivity of the K101A MEK2 cell line, pharmacological ma- Caffeine is typically regarded as an agent that sensitizes cells nipulation was performed. Vanadate has been shown to inhibit to ionizing radiation (65, 66). Treatment of cells with caffeine dephosphorylation of CDC2, thereby leading to an arrest in G prior to irradiation abolishes the G arrest and leads to radia- 2 2 (64). K101A MEK2 cells were exposed to vanadate for 8 h tion sensitivity (65, 66). We used caffeine in a slightly different Role of MEK2 in G /M Checkpoint Recovery 2739 FIG.5. Cells that express dominant negative MEK1 or dominant negative MEK2 do not show a defect in double- strand DNA break repair. A, the ability of each cell line to repair double-strand DNA breaks was measured by embedding the cells from the three cell lines (K101A MEK2, vector- only, or K97A MEK1) in agarose plugs and exposing the plugs to 10 Gy ionizing radiation. The cells were allowed to repair the damage for the given amount of time before the plugs were di- gested with proteinase K and subjected to pulse-field electrophoresis. Southern blot- ting was then performed using random- prime labeled human DNA as a probe, and the data were then quantitated. A representative experiment is shown. B, the graph shows the quantitation of three DNA repair experiments. Percent repair was determined by comparing the amount of damaged DNA at a certain time point to the amount of damaged DNA at 0 min of repair (after standardi- zation to equal amounts of DNA). Percent repair and S.E.s are graphed. DISCUSSION manner. By exposing K101A MEK2 cells to caffeine 24 h after ionizing radiation exposure, we were able to use caffeine to The RAF/ERK signaling pathway is generally regarded to be force the K101A MEK2 cells to recover from the G /M arrest in 2 strongly responsive to mitogenic signals and only weakly re- a timely manner. Fig. 7A shows that irradiated, non-caffeine- sponsive to stressful signals (11, 32, 36). The only stressful treated K101A MEK2 cells have significant numbers of cells stimulus that strongly activates the MAP kinase pathway is arrested in G 32 h after ionizing radiation exposure. When ionizing radiation (37– 41). Whereas a number of cell stressors these cells are treated with caffeine 24 h after irradiation, the and DNA-damaging agents cause a G /S arrest, physiologic cells show a normal cell cycle distribution 8 h later (Fig. 7A, doses of ionizing radiation cause a G /M arrest (42). In this right panels). Thus, caffeine can be used to force recovery from work, we have studied the possibility that ionizing radiation the G /M arrest in otherwise terminally arrested K101A MEK2 activates the RAF/ERK signaling pathway to influence G /M cells. This forced G /M arrest recovery reverses the radiosen- checkpoint kinetics. We have shown that both MEK1 and sitivity of K101A MEK2 cells upon ionizing radiation exposure MEK2 are activated in response to ionizing radiation (Figs. 1 (Fig. 7B), whereas treatment of vector-only cells or K97A and 2). This activation has functional significance as dominant MEK1 cells at these time points with either caffeine or vana- negative MEK2 is essential for cell survival in response to date has no appreciable effect on cell survival (Fig. 7B). There- ionizing radiation (Fig. 4). We have also shown that expression fore, the radiosensitivity of cells that express dominant nega- of dominant negative MEK2 leads to a delayed induction of the tive MEK2 is most likely due to an inability to recover from G G /M cell cycle checkpoint and a vastly decreased ability to arrest and not due to an inability to arrest in G in a timely recover from that checkpoint once it is established (Fig. 6). manner. 2740 Role of MEK2 in G /M Checkpoint Recovery FIG.6. Cells expressing dominant negative MEK2 show delayed induction of the G arrest and decreased ability to recover from that G arrest. Asynchronously growing cells from the respective cell lines (vector only, top; K97A MEK1, middle; K101A MEK2, bottom) were exposed to 5 Gy ionizing radiation. Propidium iodine staining was performed at the indicated time points. Flow cytometry was then performed. This experiment was performed five times with similar results all five times. A representative experiment is shown. Similar results were obtained using the S222A MEK1 cells line (not shown). Finally, we show using pharmacological means that it is the The importance of the MAP kinase pathway in all phases of faulty recovery from the G /M arrest and not the delayed G /M cell cycle regulation is being increasingly recognized. The MEK 2 2 arrest that is responsible for the radiosensitivity in cells that inhibitor, PD98059, has been shown to reverse the nerve express dominant negative MEK2 (Fig. 7). growth factor-induced G arrest in fibroblasts (68). In addition, Previous work on activation of the MAP kinase pathway by expression of a constitutively active form of MEK1 induces ionizing radiation focused on the activation of the components differentiation in PC12 cells (69) and in megakaryocytes (70). of the pathway and not on the cellular effects of that activation. These findings indicate that in some cell lines, MEK activity We have carried that work a step further by showing activation leads to G /G arrest. In other instances, however, such as upon 0 1 of MEK1 and MEK2 at doses (2.5 Gy) that are likely to be growth factor stimulation and upon cellular transformation by physiologically relevant. In addition, we have focused on the v-ras and v-raf, MEK activity is necessary for progression effect that activation of these kinases has on cell survival and through G (67, 71). Inhibition of MEK activity in pre-adipo- cell cycle checkpoint control. Although there is a pharmacolog- cytes also enhances adipocyte differentiation (72). Constitu- ical agent (PD98059) that inhibits activation of MEK1 (IC 5 tively active MEK1 will also transform some cell lines (71). 5 mM) and MEK2 (IC 5 50 mM) (54, 67), the doses required to These findings imply that MEK activity helps the cell progress completely inhibit MEK2 activity (100 mM) were toxic to HeLa through G . Therefore, the data surrounding the activity of cells. For this reason, dominant negative MEK1 and MEK2 MEK in G cell cycle regulation is largely contradictory. The constructs were employed. The dominant negative constructs effect of MEK on G cell cycle regulation is largely dependent allowed us to selectively block MEK1 and MEK2 activity, re- on the circumstance and cell line tested. spectively. Our results show that the inhibition of MEK2 acti- Recently, the MAP kinase pathway has also been implicated vation causes cells to become both radiosensitive and check- in G cell cycle regulation. In Xenopus oocytes, MAP kinase point-defective. These defects in cells expressing dominant activity has been shown to be necessary for progression negative MEK2 suggest that MEK activation by ionizing radi- through G , possibly by inhibiting an inhibitor of cyclin ation is a specific component of the stress response of the cell B/CDC2 kinase activity (49 –51). c-mos (an activator of MEK1 and not a nonspecific effect of cellular stress as previously and MEK2) has been shown to be essential for progression suggested (41). through G (52). In addition, there is mounting circumstantial 2 Role of MEK2 in G /M Checkpoint Recovery 2741 FIG.7. Delayed recovery from the G arrest leads to radiosensitivity in the K101A MEK2 cell line. A, flow cy- tometry was performed to monitor G ar- rest after irradiation and treatment of the cells with the various pharmacological agents. Far left profile, cell cycle profile of asynchronously growing K101A MEK2 cells. Middle profile, the timing of the ex- periment is shown in the upper panel. Below this is cell cycle profile of K101A MEK2 cells irradiated with 5 Gy and har- vested 16 h later. Full arrest has not oc- curred at this time point. The bottom panel shows the cell cycle profile of K101A MEK2 cells pretreated with vanadate fol- lowed by irradiation with 5 Gy. Cells treated with vanadate plus irradiation in this manner show a significantly in- creased G arrest relative to K101A MEK2 cells treated with only irradiation. Far right panels, the upper panel shows the time course of the experiment. Below this panel is a cell cycle profile of K101A MEK2 cells irradiated with 5 Gy and har- vested 32 h later. These cells show a sig- nificant number still arrested in G . The bottom panel shows the cell cycle profile of K101A MEK2 cells irradiated with 5 Gy and treated with caffeine 24 h following irradiation. Eight hours following caffeine treatment (32 h following irradiation), cells were harvested. Caffeine treatment leads to a recovery from the G arrest and allows a cell cycle profile which is now indistinguishable from asynchronously growing cells. B, survival of cells treated with 5 Gy ionizing radiation and either caffeine or vanadate in the time course described above. Neither caffeine nor van- adate lead to a change in cell survival in the vector-only cells or the K97A MEK1 cells. Vanadate treatment (forced G /M arrest) had no effect on K101A MEK2 cell survival, whereas caffeine treatment (forced recovery from G /M arrest) leads to significantly increased survival. evidence that the MAP kinase pathway plays an essential role nous cells) to fully enter G arrest. In addition, these cells take in meiosis. In Caenorhabditis elegans, activation of the MAP remarkably longer to recover from G arrest (Fig. 6). Because kinase pathway is necessary for meiotic cell cycle progression our cell survival assays require the cells to recover from the (73), and in mouse oocytes, MAP kinase becomes activated at insulting agent and to proliferate after that recovery, we are metaphase and becomes localized to the microtubule-organiz- not measuring only cell death but instead measuring both cell ing centers in meiotic maturation (53). Because the repair of death and proliferation capacity following recovery. Thus, a double-strand DNA breaks seen during physiological crossing- dysfunctional recovery from G arrest would manifest in our over during meiosis is similar to the repair of ionizing radia- assay by showing increased cell death and decreased prolifer- tion-induced double-strand DNA breaks (74), activation of the ation capacity, leading to decreased colony formation. We show MAP kinase cascade may lead to similar effects in meiosis and through the use of caffeine to force recovery from G arrest that DNA repair. To this point, however, the MEKs have not been the delay in recovery from radiation-induced G arrest medi- implicated in the maintenance of stress-induced cell cycle ates the radiosensitivity of the K101A MEK2 cells. Activation checkpoints or in progression through those checkpoints. In of MEK2 by ionizing radiation likely influences the progression this article, we have shown that cells that express dominant through the G checkpoint. Because ionizing radiation causes a negative MEK2 have a defective G /M arrest. Although these G /M arrest, this could provide an explanation for the finding 2 2 cells eventually arrest at levels seen in controls, these cells that the RAF/ERK pathway is activated by ionizing radiation take slightly longer (approximately 5–7 h longer in asynchro- but not activated by other stress stimuli. 2742 Role of MEK2 in G /M Checkpoint Recovery Acknowledgments—We thank Edwin Krebs (University of Washing- Riches, D. W. H. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 1614 –1618 32. Minden, A., Lin, A., McMahon, M., Lange-Carter, C., Derijard, B., Davis, R. J., ton, Seattle), Natalie Ahn (University of Colorado, Boulder), and Kun- Johnson, G. L., and Karin, M. (1994) Science 266, 1719 –1723 Liang Guan (University of Michigan, Ann Arbor) for their generous 33. Derijard, B., Raingeaud, J., Barrett, T., Wu, I.-H., Han, J., Ulevitch, R. J., and gifts of plasmids. We thank Marylin Thompson and Michael Freeman Davis, R. J. (1995) Science 267, 682– 685 for critical comments on the manuscript and Philip Browning, Steve 34. Whitmarch, A. J., Shore, P., Sharrocks, A. D., and Davis, R. J. (1995) Science Hann, David Miller, and P. Anthony Weil for helpful discussions con- 269, 403– 407 cerning the data. 35. Zanke, B. W., Rubie, E. A., Winnett, E., Chan, J., Randall, S., Parsons, M., Boudreau, K., McInnis, M., Yan, M., Templeton, D. J., and Woodgett, J. R. REFERENCES (1996) J. Biol. Chem. 271, 29876 –29881 36. Su, B., and Karin, M. (1996) Curr. Opin. Immunol. 8, 402– 411 1. Barlow, C., Hirotsune, S., Paylor, R., Liyanage, M., Eckhaus, M., Collins, F., 37. Sklar, M. D. (1988) Science 239, 645– 647 Shiloh, Y., Crawley, J. N., Ried, T., Tagle, D., and Wynshaw-Boris, A. (1996) 38. Kasid, U., Suy, S., Dent, P., Ray, S., Whiteside, T. L., and Sturgill, T. W. (1996) Cell 86, 159 –171 Nature 382, 813– 816 2. Hartley, K. O., Gell, D., Smith, G. C. M., Zhang, H., Divecha, N., Connelly, 39. Kharbanda, S., Saleem, A., Shafman, T., Emoto, Y., Weichselbaum, R., and M. A., Admon, A., Lees-Miller, S. P., Anderson, C. W., and Jackson, S. P. Kufe, D. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 5416 –5420 (1995) Cell 82, 849 – 856 40. Stevenson, M. A., Pollack, S. S., Coleman, C. N., and Calderwood, S. K. (1994) 3. Savitsky, K., Bar-Shira, A., Gilad, S., Rotman, G., Ziv, Y., Vanagaite, L., Tagle, Cancer Res. 54, 12–15 D. A., Smith, S., Uziel, T., Sfez, S., Ashkenazi, A., Pecker, I., Frydman, M., 41. Suy, S., Anderson, W. B., Dent, P., Chang, E., and Kasid, U. (1997) Oncogene Harnik, R., Patanjali, S. R., Simmons, A., Clines, G. A., Sartiel, A., Gatti, R. 15, 53– 61 A., Chessa, L., Sanal, O., Lavin, M. F., Jaspers, N. G. J., Taylor, A. M. R., 42. Bernhard, E. J., Maity, A., Muschel, R. J., and McKenna, W. G. (1995) Radiat. Arlett, C. F., Miki, T., Weissman, S. M., Lovett, M., Collins, F. C., and Environ. Biophys. 34, 79–83 Shiloh, Y. (1995) Science 268, 1749 –1753 43. Lohrer, H. D. (1996) Experientia (Basel) 52, 316 –328 4. Jackson, S. P. (1996) Cancer Surv. 28, 261–279 44. Bezzubova, O., Silbergleit, A., Yamaguchi-Iwai, Y., Takeda, S., and 5. Jhappan, C., Morse, H. C., III, Fleischmann, R. D., Gottesman, M. M., and Buerstedde, J. M. (1997) Cell 89, 185–193 Merlino, G. (1997) Nat. Genet. 17, 483– 486 45. Sargent, R. G., Brenneman, M. A., and Wilson, J. H. (1997) Mol. Cell. Biol. 17, 6. Sharan, S. K., Morimatsu, M., Albrecht, U., Line, D.-S., Regel, E., Dinh, C., 267–277 Sands, A., Eichele, G., Hasty, P., and Bradley, A. (1997) Nature 386, 46. Tsukamoto, Y., Kato, J., and Ikeda, H. (1997) Nature 388, 900 –903 804 – 809 47. Yaneva, M., Kowalewski, T., and Lieber, M. R. (1997) EMBO J. 16, 5098 –5112 7. Patel, K. J., Yu, V. P. C. C, Lee, H., Cororan, A., Thistlethwaite, F. C., Evans, 48. Maity, A., Kao, G. D., Muschel, R. J., and McKenna, W. G. (1997) Int. J. M. J., Colledge, W. H., Friedman, L. S., Ponder, B. A. J., and Venkitaraman, Radiat. Oncol. Biol. Phys. 37, 639 – 653 A. R. (1998) Mol. Cell 1, 347–357 49. Jin, P., Gu, Y., and Morgan, D. O. (1996) J. Cell Biol. 134, 963–970 8. Abbott, D. W., Freeman, M. L., and Holt, J. T. (1998) J. Natl. Cancer Inst. 90, 50. Abrieu, A., Doree, M., and Picard, A. (1997) Mol. Biol. Cell 8, 249 –261 978 –985 51. Abrieu, A., Fisher, D., Simon, M.-N., Doree, M., and Picard, A. (1997) EMBO 9. Conner, R., Berwistle, D., Mee, P. J., Ross, G. M., Swift, S., Grigorieva, E., J. 16, 6407– 6413 Tybulewicz, V. L. J., and Ashworth, A. (1997) Nat. Genet. 17, 423– 430 52. Gotoh, Y., and Nishida, E. (1995) Mol. Reprod. Dev. 42, 486 – 492 10. Friedman, L. S., Thistlethwaite, F. C., Patel, K. J., Yu, V. P, Lee, H., 53. Verlhac, M.-H., de Pennart, H., Maro, B., Cobb, M. H., and Clarke, H. J. (1993) Venkitaraman, A. R., Abel, K. J., Carlton, M. B, Hunter, S. M., Colledge, W. Dev. Biol. 158, 330 –340 H, Evans, M. J., and Ponder, B. A. (1998) Cancer Res. 58, 1338 –1343 54. Alessi, D. R., Cuenda, A., Cohen, P., Dudley, D. T., and Saltiel, A. R. (1995) 11. Yan, M., Dal, T., Deak, J. C., Kyriakis, J. M., Zon, L. I., Woodgett, J. R., and J. Biol. Chem. 270, 27489 –27494 Templeton, D. J. (1994) Nature 372, 798 – 800 55. Allessi, D. R., Saito, Y., Campbell, D. G., Cohen, P., Sithanandam, G., Rapp, 12. Yuan, Z.-M., Huang, Y., Whang, Y., Sawyers, C., Weichselbaum, R., U., Ashworth, A., Marshall, C. J., and Cowley, S. (1994) EMBO J. 13, Kharbanda, S., and Kufe, D. (1996) Nature 382, 272–274 1610 –1619 13. Baskaran, R., Wood, L. D., Whitaker, L. L., Canman, C. E., Morgan, S. E., Xu, 56. Wu, J., Harrison, J. K., Dent, P., Lynch, K. R., Weber, M. J., and Sturgill, T. W. Y., Barlow, C., Baltimore, D., Wynshaw-Boris, A., Kastan, M. B., and Wang, (1993) Mol. Cell. Biol. 13, 4539 – 4548 J. Y. (1997) Nature 387, 516 –519 57. Zheng, C.-F., and Guan, K.-L. (1993) J. Biol. Chem. 268, 11435–11439 14. Kharbanda, S., Pandey, P., Jin, S., Inoue, S., Bharti, J., Yaun, Z.-M., 58. Alessi, D. R., Cohen, P., Ashworth, A., Cowley, S., Leevers, S. J., and Marshall, Weichselbaum, R., Weaver, D., and Kufe, D. (1997) Nature 386, 732–735 C. J. (1995) Methods Enzymol. 255, 279 –290 15. Shafman, T., Khanna, K. K., Kedar, P., Spring, K., Kozlov, S., Yen, T., Hobson, 59. Abbott, D. W., and Holt, J. T. (1997) J. Biol. Chem. 272, 32454 –32462 K., Gatei, M., Zhang, N., Watters, D., Egerton, M., Shiloh, Y., Kharbanda, 60. Seger, R., Seger, D., Reszka, A. A., Munar, E. S., Eldar-Finkelman, H., S., Kufe, D., and Lavin, M. F. (1997) Nature 387, 520 –523 Dobrowolka, G., Jensen, A. M., Campbell, J. S., Fischer, E. H., and Krebs, 16. Abbott, D. W., and Holt, J. T. (1997) J. Biol. Chem. 272, 14005–14008 E. G. (1994) J. Biol. Chem. 269, 25699 –25709 17. Halliwell, B., and Gutteridge, J. M. C. (1984) Biochem. J. 219, 1–14 61. Mansour, S. J., Candia, J. M., Gloor, K. K., and Ahn, N. G. (1996) Cell Growth 18. Halliwell, B., and Gutteridge, J. M. C. (1986) Arch. Biochem. Biophys. 246, Differ. 7, 243–250 501–514 62. Jackson, S. P. (1996) Cancer Surv. 28, 261–279 19. Kyriakis, J. M., and Avruch, J. (1996) J. Biol. Chem. 271, 24313–24316 63. Fukasawa, K., and Vande Woude, G. F. (1997) Mol. Cell. Biol. 17, 506 –518 20. Chen, Y. R., Wang, X., Templeton, D., Davis R. J., and Tan, T. H. (1996) J. Biol. 64. Morla, A. O., Draetta, G., Beach, D., and Wang, J. Y. (1989) Cell 58, 193–203 Chem. 271, 31929 –31936 65. Bernhard, E. J., Maity, A., Muschel, R. J., and McKenna, W. G. (1994) Radiat. 21. Shafman, T. D., Saleem, A., Kyriakis, J., Weichselbaum, R., Kharbanda, S., and Kufe, D. W. (1995) Cancer Res. 55, 3242–3245 Res. 140, 193–340 66. Hain, J., Jaussi, R., and Wurgler, F. E. (1994) Cell. Signalling 6, 539 –550 22. Wang, C. Y., Mayo, M. W., and Baldwin, A. S., Jr. (1996) Science 274, 784 –787 23. Sherman, M. L., Datta, R., Hallahan, D. E., Weichselbaum, R. R., and Kufe, 67. Dudley, D. T., Pang, L., Decker, S. J., Bridges, A. J., and Saltiel, A. R. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7686 –7689 D. W. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 5663–5666 24. Hallahan, D. E., Sukhatme, V. P., Sherman, M. L., Virudachalam, S., Kufe, D., 68. Pumiglia, K. M., and Decker, S. J. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 448 – 452 and Weichselbaum, R. R. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 2156 –2160 69. Cowley, S., Paterson, H., Kemp, P., and Marshall, C. J. (1994) Cell 77, 841– 852 70. Whalen, A. M., Galasinski, S. C., Shapiro, P. S., Nahreini, T. S., and Ahn, N. G. 25. Datta, R., Rubin, E., Sukhatme, V., Qureshi, S., Hallahan, D., Weichselbaum, R. R., and Kufe, D. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 10149 –10153 (1997) Mol. Cell. Biol. 17, 1947–1958 71. Mansour, S. J., Matten, W. T., Hermann, A. S., Candia, J. M., Rong, S., 26. Hallahan, D. E., Gius, D., Kuchibhotla, J., Sukhatme, V., Kufe, D. W., and Weichselbaum, R. R. (1993) J. Biol. Chem. 268, 4903– 4907 Fukasawa, K., Wande Woude, G. F., and Ahn, N. G. (1994) Science 265, 966 –970 27. Hallahan, D. E., Dunphy, E., Virudachalam, S., Sukhatme, V. P., Kufe, D. W., and Weichselbaum, R. R. (1995) J. Biol. Chem. 270, 30303–30309 72. Font de Mora, J., Porras, A., Ahn, N., and Santos, E. (1997) Mol. Cell. Biol. 17, 6068 – 6075 28. Crews, C. M., and Erikson, R. L. (1993) Cell 74, 215–217 29. Guan, K.-L. (1994) Cell. Signalling 6, 581–589 73. Church, D. L., Guan, K. L., and Lambie, E. J. (1995) Development 121, 2525–2535 30. Seger, R., and Krebs, E. G. (1995) FASEB J. 9, 726 –735 31. Winston, B. W., Lange-Carter, C. A., Gardner, A. M., Johnson, G. L., and 74. Roeder, G. S. (1997) Genes Dev. 11, 2600 –2621
Journal
Journal of Biological Chemistry – Unpaywall
Published: Jan 1, 1999