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Copyright © 2003, The American Society for Cell Biology p38 Mitogen-Activated Protein Kinase Mediates Cell Death and p21-Activated Kinase Mediates Cell Survival during Chemotherapeutic Drug-induced Mitotic Arrest Karl Deacon,*† Pratibha Mistry,‡ Jonathan Chernoff,§ Jonathan L. Blank,* and Rajnikant Patel‡  Departments of *Cell Physiology and Pharmacology, and ‡ Biochemistry, University of Leicester, Leicester LE1 7RH, United Kingdom; and §The Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111 Marc Mumby, Monitoring Editor Corresponding author. E-mail address: rp31/at/le.ac.uk. †Present address: AstraZeneca R&D Charnwood, Bakewell Road,Loughborough, Leicester, LE11 5RH, United Kingdom. Received October 14, 2002; Revised November 22, 2002; Accepted December 27, 2002.  This article has been cited by other articles in PMC. | |||||||||||||||||||
Abstract Activation of the mitotic checkpoint by chemotherapeutic drugs such as taxol causes mammalian cells to arrest in mitosis and then undergo apoptosis. However, the biochemical basis of chemotherapeutic drug-induced cell death is unclear. Herein, we provide new evidence that both cell survival and cell death-signaling pathways are concomitantly activated during mitotic arrest by microtubule-interfering drugs. Treatment of HeLa cells with chemotherapeutic drugs activated both p38 mitogen-activated protein kinase (MAPK) and p21-activated kinase (PAK). p38 MAPK was necessary for chemotherapeutic drug-induced cell death because the p38 MAPK inhibitors SB203580 or SB202190 suppressed cell death. Dominant-active MKK6, a direct activator of p38 MAPK, also induced cell death by stimulating translocation of Bax from the cytosol to the mitochondria in a p38 MAPK-dependent manner. Dominant active PAK suppressed this MKK6-induced cell death. PAK seems to mediate cell survival by phosphorylating Bad, and inhibition of PAK in mitotically arrested cells reduced Bad phosphorylation and increased apoptosis. Our results suggest that therapeutic strategies that suppress PAK-mediated survival signals may improve the efficacy of current cancer chemotherapies by enhancing p38 MAPK-mediated cell death. | |||||||||||||||||||
INTRODUCTION During mitosis, a feedback mechanism called the spindle or mitotic checkpoint ensures that replicated chromosomes are segregated equally between the two daughter cells before division (reviewed in Shah and Cleveland, 2000 Studies in mammalian cells suggest that members of the mitogen-activated protein kinase (MAPK) family and the p21-activated kinase (PAK) family are involved in the regulation of cell survival and cell death (Xia et al., 1995 In the current study, we have examined the function of p38 MAPK in mitotically arrested HeLa cells. We show that antimicrotubule drugs cause concomitant activation of a p38 MAPK-mediated proapoptotic signaling pathway and a PAK-mediated prosurvival signaling pathway in the mitotically arrested cells. p38 MAPK stimulates apoptosis in mitotically arrested cells by inducing translocation of the proapoptotic protein Bax from the cytoplasm to the mitochondria, whereas PAK opposes p38 MAPK-induced cell death by phosphorylating the proapoptotic protein Bad. | |||||||||||||||||||
MATERIALS AND METHODS Cell Culture and Preparation of Cell Extracts Human cervical carcinoma cells (HeLa) were cultured as described previously (Patel et al., 1999  ). To obtain mitotically arrested cells, an asynchronous population of HeLa cells was treated with either nocodazole (3 μM), taxol (1 μM), vincristine (1 μM), or vinblastine (1 μM) for various times (0–30 h). Mitotic cells were collected by mechanical shake-off, washed in Dulbecco's phosphate-buffered saline (DPBS), and lysed in the appropriate buffer, depending on the protein kinase being assayed as described previously (Patel et al., 1998 ). Radioimmunoprecipitation (RIPA) assay buffer was used to prepare cell lysates for immunoblotting with the poly ADP-ribose polymerase (PARP) antibody. Cells remaining attached to the plates after shake-off were washed three times with DPBS (containing either nocodazole [3 μM], taxol [1 μM], vincristine [1 μM], or vinblastine [1 μM]) and lysed by scraping into the appropriate buffer. Cell lysates were centrifuged at 14,000 × g for 10 min at 4°C, and the protein content of the supernatant was determined using the Coomassie protein assay reagent (Pierce Chemical, Rockford, IL) before normalization and solubilization in two times SDS-PAGE sample buffer. Cell lysates for Western blotting with the Bad antibody or the phospho-specific Bad antibodies were solubilized in SDS-PAGE sample buffer before sonication for 10–15 s.Antibodies p38α, Erk1, Erk2, cdk1, anti-hemagglutinin epitope tag (HA), and PAKα were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-FLAG (M2), Cy3-conjugated anti-FLAG (M2), anti-γ-tubulin, anti-β-actin, horseradish peroxidase (HRP)-conjugated goat anti-mouse, HRP-conjugated goat anti-rabbit, and HRP-conjugated mouse anti-goat were from Sigma-Aldrich (St. Louis, MO). Anti-JNK1 (BD Biosciences PharMingen, San Diego, CA) anti-M30, which detects caspase cleavage, and cytokeratin 18 (Leers et al., 1999 ) was from Roche Diagnostics (Indianapolis, IN), along with anti-PARP (Roche Diagnostics). Alexa Fluor 488 goat anti-mouse IgG, Alexa Fluor 488 goat anti-rabbit IgG, and Alexa Fluor 594 rabbit anti-mouse IgG were from Molecular Probes (Eugene, OR). Phosphospecific anti-Bad and total anti-Bad were from New England Biolabs (Beverly, MA).Plasmids and Mutagenesis The expression vectors (FLAG epitope-tagged in pCDNA3) for p38α (murine), p38β (human), p38γ (human), and p38δ (human) were provided by J. Han (Scripps Research Institute, La Jolla, CA). The following expression vectors (all FLAG epitope-tagged in pCDNA3) were provided by R. Davis (University of Massachusetts Medical Center, Amherst, MA): dominant active (DA) MKK6 (S207D, T211D), kinase dead (KD) MKK6, glutathione S-transferase (GST)- p38α wild-type (WT), and GST-MKK6 (WT). The following expression constructs (all HA-epitope-tagged in the pXJ vector) were provided by E. Manser (Glaxo-Singapore, Singapore): PAKα (WT), DAPAKα (L106F), and KDPAKα (K298A). The expression vector for green fluorescent protein (GFP)-human bax was provided by R. Youle (National Institutes of Health, Bethesda, MD). The constructs for generating recombinant GST-fusion proteins of murine wild-type Bad (mBad) were provided by G. Bokoch (Scripps Research Institute). The mammalian expression vector for GST-mBad (pEBGmBad) was purchased from New England Biolabs. The pEBG empty vector was provided by D. Alessi (University of Dundee, Dundee, United Kingdom). In vitro mutagenesis of mBAD was carried out using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's protocol. Human MAD2 was cloned from a HeLa cell cDNA library (Stratagene) by polymerase chain reaction and subcloned into a mammalian expression vector (pCMV5) containing an N-terminal HA tag.Immunoprecipitation and Western Blot Analysis Immunoprecipitations were performed as described previously (Patel et al., 1998 ). The immunoprecipitated proteins and the cell extracts were resolved by SDS-PAGE and electroblotted onto Hybond-C nitrocellulose membrane by using a Hoeffer semidry blotting apparatus (Amersham Biosciences, Piscataway, NJ). Immunoreactive proteins were visualized using enhanced chemiluminescence (Amersham Biosciences). For immunoblotting with the phosphospecific Bad antibodies, the procedure recommended by the manufacturer was strictly followed.Protein Kinase Assays Immunecomplex kinase assays for JNK1, p38 MAPK, Erk1, Erk2, and cdk1 were performed as described previously (Patel et al., 1998 ) using GST-c-Jun, GST-activating transcription factor-2 (ATF-2), myelin basic protein (MBP), and histone H1 as substrates, respectively. The activity of the immunoprecipiated PAKα was assayed as described above for JNK1 (Patel et al., 1998 ) by using a GST-fusion containing the N-terminal 290 amino acids of mitogen-activated protein kinase kinase kinase (MEKK)2 as substrate (Deacon and Blank, 1999 ; Hagemann and Blank, 2001 ). Incorporated radioactivity was quantified by liquid scintillation counting of bands excised from SDS-PAGE gels.Flow Cytometry Analysis of the DNA content was performed by flow cytometry (FACScan; BD Biosciences, San Jose, CA) as described previously (Patel et al., 1998 ).Transfection of HeLa Cells HeLa cells were plated at a density of ~5 × 105 cells/well in a six-well plate or at 1 × 105 cells/well for coverslips in an eight-well plate. Eighteen to 24 h later, the cells were transfected with 1–4 μg of DNA by using FuGENE 6 according to the manufacturer's instructions (Roche Diagnostics). At 18–24 h after transfection, the cells on the coverslips were either fixed in cold (-20°C) methanol or incubated with MitoTracker red CMXRos (500 nM; Molecular Probes) for 15 min at 37°C, washed two times with DPBS, and fixed for 10 min in 4% (vol/vol) formaldehyde in DPBS. The cells were then processed for immunofluorescence microscopy as described below. The cells in the six-well plates were either washed two times with DPBS and lysed in the appropriate buffer to make cell extracts or collected (floating and attached cells) either for flow cytometry or for the determination of apoptosis as described above.Immunofluorescence and Confocal Microscopy HeLa cells were grown either on coverslips or in eight-well chambered slides (Nalge Nunc, Naperville, IL) and where indicated the cells were transfected as described above. The cells were washed three times in DPBS and fixed either in cold (-20°C) methanol for 30 min at -20°C or in 4% (vol/vol) formaldehyde in DPBS for 30 min at room temperature (for MitoTracker). Cells were rinsed five times with phosphate-buffered saline and the aldehyde-fixed cells permeabilized in 0.1% (vol/vol) Triton X-100 in phosphate-buffered saline for 10 min at room temperature. Cells were then blocked with 1% (wt/vol) bovine serum albumin (BSA) (in DPBS) for 45–60 min at room temperature. The following primary antibodies, diluted in 1% (wt/vol) BSA in DPBS, were used for immunocytochemistry: α-FLAG (M2), α-FLAG (M2) Cy3-conjugate, or α-HA. After incubation at room temperature for 60 min, the cells were washed five times with DPBS before incubation with the secondary antibody for a further 60 min at room temperature. The following secondary antibodies, diluted in 1% (wt/vol) BSA in DPBS, were used: sheep anti-mouse IgG, Texas Red conjugated sheep anti-mouse IgG, fluorescein isothiocyanate-conjugated and donkey anti-rabbit IgG, fluorescein isothiocyanate-conjugated. The cells were washed five times with DPBS and the nuclei labeled by staining with Hoechst 33258 (1 μg/ml in DPBS) for 5 min at room temperature. Cells were viewed on an inverted fluorescence microscope (TE3000; Nikon, Tokyo, Japan), and images were captured through a 100× objective (numerical aperture 1.4) by using an Orca ER charge-coupled device camera (Hamamatsu Photonics, Bridgewater, NJ). For deconvolution the images were captured in the z-plane at 1-μm intervals by using a 100× piezo-driven objective (numerical aperture 1.4). The images were volume deconvolved and merged using Openlab software (Improvision, Coventry, United Kingdom). The Openlab images were subsequently compiled in Photoshop (Adobe Systems, Mountain View, CA). Confocal scanning microscopy was performed on a Leitz DM IRB/E TCS4D (Leica, Wezlar, Germany). A krypton/argon laser was used for fluorescence excitation of GFP (488 nm), MitoTracker red (568 nm), Hoechst 33342 (364 nm), and Cy5 (647 nm). Images were processed and maximum projections made using Scanware (Leica). Tiff files for all four fluorochromes were merged using OpenLab software.Preparation of Recombinant GST Fusion Proteins Recombinant GST-c-Jun, GST-ATF-2, GST-MEKK2 (N terminus), GST-MKK6 (wt), and GST-p38α were prepared as described previously (Deacon and Blank, 1999 ).Reagents SB203580 and SB202190 (Calbiochem, San Diego, CA). Nocodazole, taxol, vincristine, vinblastine, cytochalasin D, phorbol 12-myristate 13-acetate (PMA), anisomycin, and histone H1 (type IIIs; Sigma-Aldrich). MBP (Invitrogen, Carlsbad, CA). All other reagents were of analytical grade and obtained from Sigma-Aldrich. | |||||||||||||||||||
RESULTS p38 MAPK Is Activated Preferentially in Mitotically Blocked HeLa Cells Treatment of HeLa cells with nocodazole (3 μM) resulted in MAD2 localization to condensed chromatin in the mitotically arrested cells, confirming that the checkpoint was activated (Figure 1A). Analysis of the nocodazole-treated cells by flow cytometry (Figure 1B) indicated the presence of two populations: a mitotic population with high cdk1 activity (Figure 2, A and B) and an attached, nonmitotic population with low cdk1 activity (Figure 2, B and C).
We evaluated the activity of the MAPKs in the nocodazole-treated cells by using immunocomplex kinase assays. p38 MAPK was activated primarily in the mitotically arrested population (Figure 2A). Immunoblot analysis of p38α immunoprecipitates indicated that the activation of p38 MAPK was not due to variations in the level of immunoprecipitated p38 protein. In contrast to p38 MAPK, both JNK and Erk MAP kinases were activated primarily in the attached cells (Figure 2, A and B). The activation of MAP kinase family members was evaluated further using alternative drugs that perturb microtubule dynamics and cause mitotic arrest. As observed with nocodazole, treatment of HeLa cells with either taxol (1 μM), vincristine (1 μM), or vinblastine (1 μM) caused mitotic arrest (as shown by high cdk1 activity; Figure 2C) and activated p38 MAPK selectively in the mitotically arrested population. With the exception of taxol, which activated JNK equally in both the mitotic and the attached cells, vincristine, vinblastine, and nocodazole activated JNK preferentially in the attached cells (Figure 2C). All four drugs also activated the Erk MAP kinases preferentially in the attached cell population (Figure 2C). Importantly, treatment of HeLa cells with cytochalasin D (5 μM) or EDTA (0.5 mM) to induce cell rounding or detachment did not activate p38 but did stimulate JNK activity (our unpublished data). Furthermore, p38 MAPK was selectively activated in mitotic mouse NIH3T3 cells arrested with either nocodazole, taxol, vincristine, and vinblastine (our unpublished data). These results indicate that p38 MAPK is specifically activated in mammalian cells mitotically arrested by microtubule-interfering drugs. We also evaluated whether p38 MAPK was activated in a synchronized population of mitotic HeLa cells. In normal mitotic cells (Mit), containing high cdk1 activity, p38 MAPK activity was not detected compared with cells arrested in mitosis with nocodazole (Noc; Figure 2D). To further confirm that p38 MAPK activation was not necessary for normal mitosis, we used the p38 MAPK inhibitors SB203580 and SB202190 (Lee et al., 1994 Because p38 MAPK is activated only in the mitotically arrested cells, we performed a time-course analysis of p38 activation at intervals after the addition of nocodazole (3 μM) to asynchronous HeLa cells. JNK activity was also assayed in parallel. At 6 h after nocodazole addition, when the majority of the cells was still attached (>90%), p38 MAPK activity was low, whereas JNK activity was maximal (Figure 3, A and B). From 12 h onward, we were able to collect purely mitotic cells by shake-off as assessed by high cdk1 activity. The activation of p38 MAPK correlated closely with that of cdk1, whereas JNK activity had returned to near basal levels 12 h after nocodazole addition (Figure 3, A and B). Furthermore, removal of nocodazole from the mitotically arrested cells resulted in exit from mitosis, as shown by the inactivation of cdk1. This inactivation of cdk1 correlated with the inactivation of p38 (Figure 3, C and D), providing further evidence that p38 MAPK activation is specifically associated with mitotic arrest.
PAK Is Also Activated Preferentially in Mitotically Arrested Cells We examined the activation of the PAKα in mitotically arrested HeLa cells. PAKα and p38 MAPK activity was analyzed at intervals after the addition of nocodazole (3 μM) to exponentially growing cells. PAKα activation paralleled p38 MAPK activation, both occurring selectively in the mitotic population at 12 h and remaining elevated for up to 30 h. Neither PAKα nor p38 MAPK activation was observed in the attached population over the same time period (Figure 4A). Similar results were obtained after treatment of HeLa cells with taxol (our unpublished data). HeLa cells treated with taxol (1 μM), vincristine (1 μM), or vinblastine (1 μM) also selectively activated both p38 MAPK and PAK in the mitotically arrested cells (Figure 4B). Treatment of HeLa cells with either cytochalasin D (5 μM) or EDTA (0.5 mM) did not activate PAK (our unpublished data). Furthermore, removal of the mitotic block in nocodazole-arrested cells resulted in inactivation of PAK as cells exited mitosis, as assessed by inactivation of cdk1 (Figure 4C). However, PAK inactivation seemed to be delayed relative to that of p38 MAPK. Collectively, these results suggest that activation of both PAK and p38 MAPK is specifically associated with mitotic arrest.
To determine whether PAK is an upstream activator of p38 MAPK in HeLa cells we cotransfected either wild-type (WT) or dominant active (DA) PAK (L106F) with WT p38α. DAPAK, in comparison with WT PAK, was highly active as assessed by its ability to phosphorylate GST-Mekk2 but was unable to activate cotransfected p38α, as measured GST-ATF2 phosphorylation (Figure 4D). The transfected p38α was, however, activated by anisomycin as assessed by phosphorylation of GST-ATF2. These data suggest that DAPAK is not coupled to p38 MAPK activation in HeLa cells. p38 MAPK Activation Stimulates Apoptotic Cell Death, whereas PAK Activation Promotes Cell Survival The effect of nocodazole on cell death was assessed in both the attached and the mitotically arrested populations by either immunoblotting with an anti-PARP antibody or immunostaining with the M30 antibody. Nocodazole-induced poly (ADP-ribose) polymerase (PARP) cleavage was apparent in the mitotic cells at 12 h after drug addition and continued to increase over the time course examined (Figure 5A). Treatment of HeLa cells with either taxol (1 μM), vincristine (1 μM), or vinblastine (1 μM) also caused PARP cleavage, preferentially in the mitotic population (Figure 5B). Nocodazole also induced cleavage of cytokeratin 18 and caused a time-dependent increase in the number of apoptotic cells specifically in the mitotic cell population (Figure 5C, right).
To determine whether p38 MAPK was involved in this apoptotic response, p38 MAPK inhibitors were used. SB203580 (20 μM) or SB202190 (10 μM) reduced the number of apoptotic cells in the mitotic population by 46 and 42%, respectively, as assessed by cytokeratin 18 cleavage (Figure 5D). The relationship between p38 MAPK activity and apoptosis was examined further by transfecting HeLa cells with dominant active (DA) MAP kinase kinase, MKK6 (S207D, T211D). Consistent with previous reports (Derijard et al., 1995
To address the function of PAK in mitotically arrested cells, DAPAKα (L106F) was transfected into HeLa cells, either separately or together with DAMKK6 (Figure 6D), and their effect on survival assayed using the M30 antibody. In the absence of any treatment, 2% of the cells were found to be apoptotic (Figure 6E). Transfection with the empty vector alone increased the number of apoptotic cells to 12%. In contrast, 54% of DAMKK6-transfected cells were found to be apoptotic as assessed by the presence of both cleaved cytokeratin 18 and fragmented chromatin, whereas a signaling inactive mutant of MKK6 (S207A, T211A) did not induce apoptosis (14%). Coexpression of DAPAK with DAMKK6 suppressed DAMKK6-induced apoptosis to near basal levels (18%), whereas KDPAK did not affect the level of MKK6-induced apoptosis. We also determined whether PAK aids cell survival of nocodazole-arrested mitotic cells. Treatment of DAPAK-transfected HeLa cells with nocodazole (3 μM for 24 h) reduced the number of apoptotic cells in the mitotic population by 56% (nocodazole: percentage of M30 positive cells ± SEM; 34.2 ± 3.4, n = 3; nocodazole + DAPAK: 15 ± 2.2, n = 3). These results suggest that nocodazole activates two signaling pathways in mitotically arrested cells that have opposing effects on cell survival. Activation of p38 MAPK either by nocodazole or MKK6 stimulates cell death, whereas activation of PAK stimulates cell survival. DAMKK6 Induces Bax Translocation from the Cytoplasm to the Mitochondria and PAK Phosphorylates Bad To examine the mechanism by which p38 MAPK and PAK exert their effect on cell survival, we examined whether these enzymes regulate the activity of the bcl-2 family members Bad and Bax. The effect of p38 MAPK on the intracellular distribution of Bax was investigated by cotransfecting HeLa cells with DAMKK6 and a GFP-Bax fusion construct. Both GFP-Bax (Figure 7A) and MKK6 (Figure 7B) were distributed homogeneously throughout the cytoplasm of exponentially growing cells when expressed alone. Cotransfection of GFP-Bax with DAMKK6 resulted in a dramatic change in the distribution of Bax from a homogeneous distribution to a more punctate, perinuclear distribution (Figure 7C). Early morphological changes (16 h after transfection) included cell rounding and membrane blebbing (Figure 7C). At later times (36 h after transfection), chromatin condensation and fragmentation was accompanied by cell shrinkage (Figure 7D). In control experiments where KDMKK6 was cotransfected with GFP-Bax, Bax remained homogeneously distributed throughout the cytoplasm (Figure 7E). In addition, cotransfection of DAMKK6 with a plasmid expressing GFP alone caused no redistribution of GFP (Figure 7F). To determine whether the DAMKK6-induced redistribution of Bax was mediated through activation of p38 MAPK, cells cotransfected with GFP-Bax and DAMKK6 were treated with either SB203580 or SB202190. In cells transfected with GFP-Bax alone, Bax showed a punctate, perinuclear distribution in 14% of the cells. However, in cells coexpressing both GFP-Bax and DAMKK6, 55% of the cells displayed a punctate, perinuclear distribution of Bax. In the presence of either SB203580 (20 μM) or SB202190 (10 μM) the translocation of Bax to the perinuclear region was reduced to near basal levels (16%) (Figure 7G).
We determined whether DAMKK6 was inducing redistribution of GFP-Bax to the mitochondria. In cells cotransfected with GFP-Bax and DAMKK6 (Figure 8, A–E), the punctate distribution of GFP-Bax (Figure 8A) coincided with mitochondrial staining by MitoTracker red (Figure 8B). These results demonstrate that the DAMKK6-induced activation of p38 MAPK is sufficient to stimulate Bax translocation from the cytosol to the mitochondria. Next, we determined whether Bax also translocates to the mitochondria in nocodazole-arrested mitotic cells. HeLa cells transfected with GFP-Bax were treated with nocodazole (3 μM) for 24 h and subsequently stained with an antibody to cytochrome c to detect the mitochondria (Figure 8, F–I). After 24 h of exposure to nocodazole between 20 and 30% of the transfected, mitotic cells displayed a punctate, perinuclear distribution of Bax (Figure 8F), which overlapped with a mitochondrial-rich region of the cell (Figure 8G). These cells were undergoing apoptosis as assessed by the presence of fragmented chromatin (Figure 8H). In nocodazole-arrested, nonapoptotic cells GFP-Bax was distributed homogeneously throughout the cytoplasm and did not colocalize with the mitochondria, which were distributed concentrically around the condensed chromatin (our unpublished data). Control nocodazole-arrested mitotic cells expressing GFP alone (Figure 8, J–M) also did not show a punctate, perinuclear pattern of Bax fluorescence despite being clearly apoptotic, based on membrane blebbing (Figure 8J) and DNA fragmentation (Figure 8L). These experiments indicated that Bax translocates from the cytoplasm to the mitochondria in nocodazole-arrested mitotic cells as they undergo apoptosis.
Consistent with previous data regarding Bad phosphorylation by PAK (Schurmann et al., 2000
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DISCUSSION We have identified the signaling events associated with activation of the spindle checkpoint by drugs that perturb microtubule dynamics. The results of this study demonstrate that 1) chemotherapeutic drugs cause activation of both p38 MAPK and PAK preferentially in the mitotically arrested cells; 2) chemotherapeutic drug-induced cell death is mediated through p38 MAPK, whereas chemotherapeutic drug-induced cell survival is mediated through PAK; 3) p38 MAPK activity induces the translocation of the cell death activator Bax, from the cytosol to the mitochondria; and 4) PAK phosphorylates Bad, thereby providing a mechanism to inhibit its proapoptotic function. Our data indicate that the mitotic checkpoint is activated in nocodazole-arrested cells as assessed by the presence of the checkpoint protein MAD2 on the condensed chromatin. In these mitotic checkpoint-arrested cells, we demonstrate for the first time the concomitant activation of both p38 MAPK and PAK. Activation of either p38 MAPK or PAK was not observed in the attached cell population where both JNK and the Erk MAP kinases were found to be activated. The function of either JNK or Erk in the attached population is currently unknown. However, our studies indicate that Erk may suppress JNK-mediated cell death in this cell population (our unpublished data). Previous studies that have examined chemotherapeutic drug-activated signaling pathways have reported activation of JNK, p38 MAPK and Erk MAP kinases either alone or in combination (Wang et al., 1998 The upstream signaling events that lead to the activation of p38 MAPK and PAK during mitotic arrest remain to be identified. Although it has been reported that PAKs can activate p38 MAPK (Bagrodia et al., 1995 The downstream target(s) of p38 MAPK that cause cell death in the mitotically arrested cells are largely unknown. A recent observation (Ghatan et al., 2000 The activation of PAKα by chemotherapeutic drugs in a mitotically arrested cell population has not been described previously. We have shown in this study that DAPAKα suppresses MKK6-induced apoptosis, suggesting that one function of native PAKα may be to suppress cell death in the mitotically blocked cells. Indeed, our data have shown that expression of DAPAKα confers a survival advantage during nocodazole-induced mitotic arrest, whereas inhibition of PAK activation enhances nocodazole-induced cell death. In mammalian cells PAK isoforms have been shown to play distinct roles in apoptosis. Both PAK1 (PAKα) and PAK4 have been shown to protect cells from apoptosis induced by serum withdrawal, UV irradiation, or tumor necrosis factor-α in fibroblasts, or by growth factor withdrawal in lymphoid cells (Schurmann et al., 2000 The target(s) of PAKα that regulate cell survival in mitotically blocked cells are also unknown. One mechanism through which PAK may protect lymphoid progenitor cells from apoptosis, after growth factor withdrawal, is by phosphorylation of the BH3-only proapoptotic protein Bad (Schurmann et al., 2000 In summary, the present study provides evidence that both cell survival and cell death pathways are activated in mitotically arrested cells. A p38 MAPK-activated and Bax-dependent pathway contributes to cell death, whereas a PAK-activated and Bad-dependent pathway contributes to cell survival. The results of our study have implications for the design of future chemotherapeutic drug therapies that target the mitotic checkpoint. In particular, the identification of other survival pathways that are activated in response to the chemotherapeutic agents, and a search for agents that suppress them may considerably increase the effectiveness of the current anticancer therapies. | |||||||||||||||||||
Acknowledgments We thank Ed Manser, Roger Davis, Jiahuai Han, Gary Bokoch, and Richard Youle for generously providing various expression plasmids. Roger Snowden for assistance with flow cytometry and Kulvinder Sikand for assistance with confocal microscopy. This work was supported by grants from the Wellcome Trust and the Biotechnology and Biological Sciences Research Council (to R.P. and J.L.B.). | |||||||||||||||||||
Notes Article published online ahead of print. Mol. Biol. Cell 10.1091/mbc.E02-10-0653. Article and publication date are at www.molbiolcell.org/cgi/doi/10.1091/mbc.E02-10-0653. | |||||||||||||||||||
References
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