The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Parikh, N.
Right arrow Articles by Sarin, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Parikh, N.
Right arrow Articles by Sarin, A.
The Journal of Immunology, 2004, 173: 6220-6227.
Copyright © 2004 by The American Association of Immunologists

The Bax N Terminus Is Required for Negative Regulation by the Mitogen-Activated Protein Kinase Kinase and Akt Signaling Pathways in T Cells1

Neha Parikh, Hadassah Sade2, Leo Kurian2 and Apurva Sarin3

National Center for Biological Sciences, Bangalore, Karnataka, India


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The Bcl-2 family proapoptotic protein, Bax, redistributes to the mitochondrion in response to varied stimuli, triggering loss of mitochondrial integrity and apoptosis. Suppression of MAPK kinase (MEK1) by the reagent UO126 in activated T cells maintained in the cytokine IL-2 disrupts cytoplasmic localization of Bax and cell survival. UO126 triggers mitochondrial translocation of ectopically expressed Bax-GFP, and both UO126 and dominant negative MEK-1 (DN-MEK1) trigger increased apoptosis in Bax-GFP-expressing T cell lines. Because inhibition of PI3K or its target Akt also triggers mitochondrial translocation of Bax in T cells and apoptosis in Bax-transfected cell lines, we generated Bax deletion mutants to identify the region(s) that confers sensitivity to regulation by MEK1 and Akt. A deletion mutant (Bax1–171) without the C terminus mitochondrial targeting sequence or an Akt target site (Ser184) localizes to the cytoplasm and triggers low level apoptosis that is enhanced by DN-Akt or DN-MEK1. A construct that lacks the first 29 aa (Bax-{delta}29) largely localizes to mitochondria, is highly apoptogenic, and is not inhibited by Akt or MEK1. Furthermore, Bax-{delta}29 overcomes IL-2-dependent survival in a T cell line, whereas Bax triggers comparatively low levels of apoptosis in these cells. Cytoplasmic localization and regulation by MEK1 and Akt are restored in a mutant deleted of the first 13 aa (Bax-{delta}13). Taken together, our results identify a region in the Bax N terminus that determines cellular localization regulated by MEK- and Akt-dependent signaling in T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
In mammalian cells apoptosis can be initiated by members of the TNF receptor family (1, 2) or by pathways integrated via the mitochondrion (3, 4). Members of the Bcl-2 family, which comprises both pro- and antiapoptotic proteins, are the principal regulators of mitochondrial integrity (5). Many proapoptotic members of this family are localized in the cytoplasm and translocate to the mitochondrion to initiate apoptotic cascades. The redistribution of proapoptotic proteins usually culminates in the loss of integrity of the mitochondrial outer membrane, culminating in the release of apoptogenic intermediates that propagate apoptotic pathways.

The Bcl-2 family proapoptotic protein Bax is critical for peripheral T cell homeostasis (6), and its overexpression results in decreased T cell number and survival (7). A combined deficiency of Bax and Bak compromises mitochondrially regulated death pathways in T cells (6). Lymphocyte survival is regulated by cytokines, which activate multiple signal transduction pathways via interactions through the cytoplasmic domains of their receptor subunits (8, 9, 10). Thus, IL-2 receptor engagement results in the activation of MAPK kinase (MEK1)4 (11, 12) and PI3K signaling (12, 13) in T cells. MEK1 has been previously implicated in protection from various apoptotic signals. The mechanisms range from transcriptional or post-translational regulation of proteins (14, 15, 16, 17) to the inhibition of apoptotic events after cytochrome c release (18, 19). The antiapoptotic function of PI3K has been described in multiple systems, and the serine-threonine kinase PKB/Akt is a key downstream target of PI3K in many models of suppression of apoptosis (20).

MEK1 or Akt regulation of Bax has been reported in diverse cellular systems (21, 22, 23), and the direct Akt-mediated phosphorylation of Bax as a mechanism of regulating its function has been recently demonstrated (23). However, most reports favor the model that Bax localization is regulated via its interactions with molecules such as 14-3-3 and Ku70 (24, 25) and other unidentified factors. A stretch of hydrophobic amino acids that may function as a mitochondrial localization sequence or transmembrane domain is present in the C-terminal region of Bax (26, 27). Additionally, various regions in the N terminus (25, 28) are implicated in the cytoplasmic sequestration of Bax. Thus, the multiple regulatory domains present in the molecule most likely facilitate tissue-specific regulation of Bax.

In one approach to understanding the regulation of Bax function, we have attempted to identify domains in Bax that confer sensitivity to regulatory signals that promote T cell survival. In this context we identify a region (spanned by aa 13–29) in the N terminus that is required for MEK1- and Akt-dependent regulation of Bax. Based on the analysis of various N and C terminus deletion mutants, we propose that MEK1 and/or Akt signaling requires the Bax N terminus to over-ride mitochondrial localization cues to sequester Bax in the cytosol of T cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and reagents

The murine T cell lines 2B4 and d11S and the human lymphoblastoid cell line Jurkat were used in these experiments. CTEV-2, a murine T cell line dependent on exogenous IL-2 for survival, was also used in some exper-iments. The generation of activated T cells has been described previously (17). Abs to phosphorylated Akt (pAkt), pERK, Bax, and Bcl-xL were obtained from Cell Signaling Technology (Beverly, MA). Abs to Bax, inhibitor of apoptosis protein-1 (IAP-1), IAP-2, p38 MAPK, ERK, GFP, p65-NF-{kappa}B, and Akt were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and Abs to GFP, cytochrome oxidase (Cox-4), cytochrome c, and Bcl 2 interacting mediator of cell death were obtained from BD Biosciences (Franklin Lakes, NJ). Digitonin was obtained from Sigma-Aldrich (St. Louis, MO), and all other chemicals were purchased from Calbiochem (San Diego, CA).

Plasmids

The mammalian expression vector encoding murine Bax was obtained from P. Vandenabeele (University of Ghent, Zvignaarde, Belgium). The Bax-GFP construct and the deletion mutants were made by PCR amplification and subcloning into the enhanced pGFP-N1 vector (BD Clontech, Palo Alto, CA) with GFP in-frame at the C terminus. The following primers were used to generate the different constructs used in this study: Bax-GFP forward, 5'-CCGCTCGAGATGGACGGGTCCGGG-3'; Bax-{delta}13 GFP forward, 5'-CCGCTCGAGATGACCAGCTCTGAA-3'; Bax {delta}29 GFP forward, 5'-CCGCTCGAGATGTTCATCCAGGAT-3'; Bax {delta}37 GFP forward, 5'-CCGCTCGAGATGGCTGGGGAGACA-3'; Bax-GFP reverse, 5'-CCCAAGCTTGCCCATCTTCTTCCA-3'; and Bax1–171-GFP reverse, 5'-CCCAAGCTTCTGCCATGTGGG-3'.

The sequences of all constructs were verified by automated sequencing (Microsynth, Balgach, Switzerland). The MEK1 constitutively active (CA-MEK1 or SA-MEK1) and dominant-negative (DN-MEK1) plasmids were originally produced in the laboratories of M. J. Weber (University of Virginia Health Sciences Center, Charlottesville, VA) and C. J. Marshall (Institute of Cancer Research, London, U.K.) and were obtained from S. Jameel (International Center for Genetic Engineering and Biotechnology, New Delhi, India). CA-Akt, DN-Akt1 (K179M), and CA-MEK1 (Upstate, Lake Placid, NY) were also used. The DN caspase-9 construct was a gift from C. Vincenz (University of Michigan Medical School, Ann Arbor, MI). Bcl-2 was a gift from S. Perwez Hussain (National Cancer Institute, National Institutes of Health, Bethesda, MD).

Transfections and induction of apoptosis

Electroporation and analysis of apoptotic nuclear damage in GFP-transfected cells using Hoechst 33342 have been described previously (29). Routinely, transfection efficiencies ranged between 35 and 40% in the 2B4 and d11S cell lines, as estimated by the number of GFP-positive cells. In all transfection-based experiments, the analysis was designed to score only GFP-positive cells, thereby minimizing variations that may arise from differences in transfection efficiencies across multiple experiments. For transfections, 1 µg of GFP; 3 µg of Bax-GFP or the deletion mutants; 6 µg of CA-Akt, CA-MEK1, DN-caspase-9, or Bcl-2; and 8 µg of DN-Akt or DN-MEK1 were used. UO126 (10 µM) or LY294002 (10 µM) was added 12 h post-transfection. For UV treatment, cells were exposed to UV for 10 s, washed once by centrifugation, and continued in culture for 18 h. For analysis of T cell blasts, day 5 activated T cells were washed twice with PBS to remove bound IL-2 and pretreated with 40 µM UO126 for 30–45 min, followed by the addition of IL-2. Apoptotic nuclear damage was assessed 18 h after initiation of culture unless specified otherwise. All experiments were performed a minimum of three times unless specified otherwise.

Subcellular fractionation

A digitonin-based buffer was used to separate the membrane (mitochondrial) fraction from the soluble (cytoplasmic) fraction of cell lysates. Briefly, 10 x 106 cells were pelleted by centrifugation, the pellet was resuspended in a digitonin-based buffer (40 µg/ml digitonin in 120 mM KCl, 10 mM NaCl, 1 mM KH2PO4, and 20 mM HEPES-Tris, pH 7.2, supplemented with protease inhibitors), and samples were kept on ice for 15 min. Samples were clarified by centrifugation at 7,500 x g to remove cell debris, followed by a centrifugation at 10,000 x g for 15 min. All manipulations were performed at 4°C. The resulting pellet was enriched in mitochondria, as confirmed by the mitochondrial marker protein Cox-4, and the supernatant was representative of the cytoplasmic fraction, confirmed by staining for Akt or NF-{kappa}B.

Cytochrome c release from isolated mitochondria

2B4 cells (20 x 106) were washed once with chilled PBS and resuspended in 200 µl of intracellular medium (ICM; 1 mM KH2PO4, 120 mM KCl, 10 mM NaCl, and 20 mM HEPES, pH 7.2) containing 40 µg/ml digitonin (a 5% stock of digitonin in DMSO was made fresh for each assay). Samples were incubated on ice for 30 min, with gentle tapping of the tube at 5-min intervals to aid lysis. The samples were centrifuged at 700 x g for 5 min at 4°C to remove cell debris. The pellet was discarded, and the supernatant was subjected to another centrifugation at 10,000 x g for 15 min at 4°C. The pellet, which represents the mitochondrial fraction, was washed once with ICM and finally resuspended in 70 µl of ICM and used for subsequent analysis. Thirty microliters of each fraction was incubated with 200 ng of either maltose binding protein (MBP) or MBP-Bax protein at 30°C for 20 min in a water bath. At the end of the incubation, the samples were centrifuged at 10,000 x g for 15 min at 4°C. Equivalent volumes of pellet (P) and the supernatant (Supn) fractions were boiled in 5x SDS lysis buffer, resolved on 12% SDS gels, and probed for cytochrome c and Cox-4 by Western blot analysis.

Western blot analysis

Whole cell lysates (0.5 x 106 cells) or samples obtained from fractionation procedures were resolved by SDS-PAGE and analyzed by Western blot analysis using standard protocols (17). Densitometric analysis was performed using Image gauge software version 3.0 (National Institutes of Health). The fold change in expression was calculated relative to the loading control, which was usually p38MAPK in most experiments. In densitometric analysis of the subcellular distribution of the Bax deletion mutants, values represent the band intensity relative to the total intensities of the mitochondrial and cytosolic fractions. Where calculated, these values are entered below the blots in the figures.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
MEK1 regulates the cytoplasmic localization of Bax in activated murine T cells

In activated T cells that are sustained in culture by cytokines such as IL-2, Bax is normally localized in the cytoplasm (Fig. 1A). Cytokine deprivation triggers the mitochondrial translocation of Bax (Fig. 1A) and culminates in apoptosis (data not shown). Disruption of IL-2-dependent activation of MEK1 signaling (assessed by the loss of phosphorylation of its downstream target, ERK-1/2) using the reagent UO126 (Fig. 1B) triggered a redistribution of Bax to mitochondria (Fig. 1C). The distribution of the inner mitochondrial membrane protein Cox-4 was used to establish the purity of the mitochondrial fraction. As can be seen from the estimation of band intensities by densitometry, the amount of Bax in the mitochondrial fraction was 3-fold higher in cells treated with UO126 than in cells cultured with IL-2 alone. Additionally, UO126 attenuated, in part, IL-2-mediated survival of T cells (Fig. 1D). To further explore the molecular mechanism of MEK1 regulation of the subcellular distribution of Bax, we screened for T cell lines in which ectopically expressed Bax was regulated by cytoplasmic sequestration.



View larger version (56K):
[in this window]
[in a new window]
  		FIGURE 1. MEK1 regulates Bax translocation in activated T cells. A, Day 5 T cell blasts were washed and continued in culture in the presence (+IL-2) or the absence (No IL-2) of IL-2. After 6 h, cells from both conditions were harvested, separated into cytosolic (Cyt) or mitochondrial (Mito) fractions, and analyzed by Western blot analysis for the distribution of Bax and Cox-4, as described in Materials and Methods. Values below the blots represent the densitometric estimation of protein relative to the total in the Cyt and Mito fractions. B, T cell blasts cultured in IL-2 were treated with 40 µM UO126 for 4 h. Total cell lysates were analyzed by Western blot analysis for the levels of phosphorylated ERK-1/2, and the expression of total ERK was used to establish parity of loading. C, T cell blasts were cultured as described in B. After 4 h, subcellular fractions were assessed for Bax expression by Western blot analysis. The purity of the fractions was confirmed by the distribution of Cox-4, and p38MAPK was used as the loading control (LC). D, Activated T cells were cultured with IL-2 in the presence or the absence of 40 µM UO126 for 24 h and scored for apoptotic nuclear damage.

 
Ectopically expressed Bax is poorly apoptogenic in 2B4 and d11S cell lines

Ectopic expression of Bax induced low levels of apoptosis in the 2B4 T cell hybridoma (Fig. 2A, {blacksquare}) compared with the human lymphoblastoid cell line Jurkat (Fig. 2A, {square}). The fusion protein, GFP-tagged Bax, was detected at the appropriate Mr (48 kDa) indicative of equivalent expression (Fig. 2A, inset, lanes 2 and 3, respectively). High endogenous levels of antiapoptotic proteins of the Bcl-2 family would render cells resistant to Bax-induced apoptosis. However, the levels of the antiapoptotic proteins, Bcl-xL and Bcl-2 (Fig. 2B), and the caspase antagonists, the IAPs, were comparable in the two cell lines (Fig. 2B). Bax-induced apoptosis in another T cell line, d11S, was marginally higher than that observed in 2B4 cells, but was substantially lower than that triggered in Jurkat cells (Fig. 2C). From a comparison of the expression of proapoptotic proteins in the Jurkat, d11S, and 2B4 cell lines, it was apparent that the expression of Bax or molecules such as Bcl 2 interacting mediator of cell death, which associate with Bax at the mitochondrial membrane, was higher in 2B4 and d11S than in the Jurkat cell line (Fig. 2D).



View larger version (44K):
[in this window]
[in a new window]
  		FIGURE 2. Comparison of sensitivity to Bax-induced apoptosis and the expression of pro- and antiapoptotic proteins in the 2B4, Jurkat, and d11S cell lines. A, 2B4 ({blacksquare}) or Jurkat ({square}) cells were transfected with GFP or Bax-GFP, and after 15 h, the GFP-positive cells were scored for apoptotic nuclear damage, as described in Materials and Methods. The data are representative of four independent experiments. Inset, Expression of GFP or Bax-GFP in 2B4 (lanes 1 and 2, respectively) and Bax-GFP in Jurkat (lane 3) using an Ab to GFP. B, 2B4 and Jurkat cell lysates were assessed for the expression of antiapoptotic proteins by Western blot analysis. p38MAPK was used to establish parity of loading. C, d11S cells were transfected with GFP or Bax-GFP, and after 18 h, the GFP-positive cells were scored for apoptotic nuclear damage, as described in Materials and Methods. The data are representative of five independent experiments. D, Lysates of 2B4, Jurkat, and d11S cells were assessed for the expression of the indicated proapoptotic proteins as indicated by Western blot analysis. p38MAPK was used to establish parity of loading.

 
Resistance to Bax-induced apoptosis could also arise from defects in apoptotic signaling pathways that are regulated by the mitochondrion. 2B4 cells, however, were susceptible to UV- or etoposide-triggered apoptosis (Fig. 3A), and dexamethasone-induced apoptosis was inhibited by Bcl-2 overexpression in these cells (Fig. 3B), indicating that the intermediates required for mitochondria-mediated apoptotic signaling were functional in the cell line. As another more direct readout of mitochondrial function, we determined that purified Bax protein induced the release of cytochrome c from mitochondria isolated from the 2B4 cell line (Fig. 3C). Cytochrome c released into the supernatant by MBP-tagged Bax (Fig. 3C, lane 4) was clearly higher than that released by MBP alone (Fig. 3C, lane 2). Thus, it appeared that reduced levels of Bax-induced apoptosis in 2B4 and d11S cells could not be attributed to the increased expression of proapoptotic proteins in these cells or defects in mitochondrial signaling. Therefore, in subsequent experiments we tested the effect of modulating MEK-dependent signaling on Bax localization and Bax-induced apoptosis in 2B4 and d11S cells.



View larger version (40K):
[in this window]
[in a new window]
  		FIGURE 3. Mitochondrial signaling is not defective in the 2B4 cell line. A, 2B4 cells were cultured alone or with etoposide or after UV exposure and were assessed for apoptotic nuclear damage after 18 h. B, GFP- or GFP- and Bcl-2-transfected 2B4 cells were assessed for dexamethasone-induced apoptosis after 8–14 h. C, MBP-Bax induced cytochrome c release from mitochondria isolated from 2B4 cells. The assay was performed as described in Materials and Methods. P, the pellet, which is the mitochondrial fraction. Values below the blot indicate the band intensity (assessed by densitometry) relative to the total (pellet and supernatant) in each condition.

 
MEK1 regulates the cytoplasmic sequestration of Bax in 2B4 cells

Bax-GFP was largely present in the cytoplasm of 2B4 cells, as determined by the detergent-based subcellular fractionation of Bax-transduced 2B4 cells (Fig. 4A, lane 1). After the addition of UO126, there was a shift in the distribution of Bax-GFP to the mitochondrial fraction (Fig. 4A, lane 4). Thus, cell fractions enriched for Cox-4 expressed a higher proportion of total Bax-GFP after UO126 treatment (Fig. 4A).



View larger version (23K):
[in this window]
[in a new window]
  		FIGURE 4. Disruption of MEK1 signaling in 2B4 cells induces Bax-GFP translocation to the mitochondria and apoptosis in T cell lines. A, 2B4 cells transfected with Bax-GFP were cultured overnight, and culture was then continued either alone or with 10 µM UO126 for 8 h. Cytosolic (Cyt) and mitochondrial (Mito) fractions obtained by subcellular fractionation from these two conditions were analyzed by Western blot analysis for Bax-GFP or Cox4. p38MAPK was used as the loading control (LC). The values below the blots indicate densitometric analysis for distribution of Bax-GFP. B, 2B4 cells transfected with GFP or Bax-GFP were cultured with or without UO126 (10 µM) for 15 h and assessed for apoptotic nuclear damage. The data are normalized with respect to nuclear damage in cells transfected with GFP alone. The inset shows the levels of pERK-1/2 and pAkt in 2B4 cells cultured with or without UO126 for 8 h. Total ERK-1/2 and p38MAPK served as loading controls. C, 2B4 cells transfected with GFP, GFP plus DN-MEK1, or Bax-GFP with or without DN-MEK1 were assessed for apoptotic nuclear damage after overnight culture. D, 2B4 cells were transfected with GFP, Bax-GFP, or Bax-GFP and Bcl-2. After 18 h, each sample was continued in culture for an additional 8 h in the presence or the absence of 10 µM UO126. Apoptotic nuclear damage in GFP-positive cells was normalized to nuclear damage in GFP-transfected cells cultured in the same condition. E, d11S cells transfected with GFP or Bax-GFP with or without DN-MEK1 were cultured for 24 h, and GFP-positive cells were assessed for apoptotic damage.

 
Disruption of MEK1 signaling by UO126 or the expression of DN-MEK1 also culminated in Bax-mediated apoptosis in 2B4 cells (Fig. 4, B and C). The induction of apoptosis in cells cotransfected with Bax and treated with UO126 was inhibited by coexpression of Bcl-2 (Fig. 4D), consistent with the activation of mitochondria-dependent apoptotic signaling triggered by Bax. Coexpression of DN-MEK1 also enhanced sensitivity to Bax-mediated apoptosis in the d11S cell line (Fig. 4E).

Inhibition of PI3K triggers Bax translocation and apoptosis

Recent studies have shown that the kinase Akt/PKB sequesters Bax in the cytosol, thereby preventing Bax-induced apoptosis (22, 23). Because kinase activation and functions are often cell type and context specific, we examined the role of PI3K in the regulation of Bax-induced apoptosis in 2B4 and d11S cells. Treating activated T cells with the PI3K inhibitor LY294002 triggered a redistribution of Bax to the mitochondrial fraction (Fig. 5A). Both LY294002 and the expression of dominant-negative Akt (DN-Akt) also revealed sensitivity to Bax-induced apoptosis in Bax-transfected 2B4 cells (Fig. 5, B and C). Apoptosis triggered by Bax and LY294002 was blocked by the coexpression of Bcl-2 (Fig. 5D). Similarly, cotransfection of DN-Akt enhanced Bax-induced apoptosis in the d11S cell line (Fig. 5E). Ectopic expression of both DN-Akt and DN-MEK1 did not result in a substantial increase in Bax-mediated apoptosis in 2B4 cells (Fig. 5F) compared with DN-MEK or DN-Akt alone, suggesting that both kinases may target one molecule or molecular complex to regulate Bax function.



View larger version (39K):
[in this window]
[in a new window]
  		FIGURE 5. Suppressing PI3K-dependent Akt signaling triggers mitochondrial translocation of Bax and apoptosis. A, T cell blasts were cultured with IL-2 in the absence (lanes 1 and 2) or the presence (lanes 3 and 4) of LY294002 (40 µM). After 4 h, subcellular fractions of cytosol (Cyt)- or mitochondria (Mito)-enriched fractions were assessed for Bax and Cox-4 expression by Western blot analysis. B, 2B4 cells transfected with GFP or Bax-GFP were cultured for 18 h, continued in culture with or without LY294002 (10 µM) for an additional 8 h, and assessed for apoptotic nuclear damage. The inset shows the levels of pAkt in 2B4 cells cultured with or without LY294002 for 8 h. Akt served as the loading control (LC). C, 2B4 cells were transfected with GFP or Bax-GFP with and without DN-Akt. Apoptotic nuclear damage was assessed in GFP-positive cells after 18–24 h. Apoptotic damage in cells transfected with DN-Akt alone was not higher than that triggered by GFP; hence, the values are not presented. D, 2B4 cells were transfected with either GFP or Bax-GFP, and each condition was also cotransfected with Bcl-2. After 18 h, each set was continued in culture for an additional 8 h in the presence or the absence of 10 µM LY294002. Apoptotic nuclear damage in GFP-positive cells was normalized as described in Fig. 3D. Results from two experiments are shown. E, d11S cells were transfected with GFP or Bax-GFP with and without DN-Akt. Apoptotic nuclear damage was assessed in the GFP-positive cells after 18–24 h. F, 2B4 cells were transfected with GFP or Bax-GFP, and both constructs were also cotransfected with DN-Akt and DN-MEK1. Apoptotic nuclear damage was assessed after 18–24 h.

 
MEK1 and Akt regulation of Bax deletion mutants

The findings to date indicate that Bax function can be regulated via Akt- and MEK1-dependent signaling. Post-transcriptionally this regulation could be effected by the phosphorylation of Bax, the phosphorylation of molecular interactors of Bax, or a combination of the two events. A recent study has described an Akt phosphorylation site (Ser184) in the Bax C terminus that sequesters Bax in the cytosol in neutrophils (23). However, aa 20–37 of the Bax N terminus function as a mitochondrial targeting sequence in a glioblastoma cell line (30). Therefore, we attempted to identify the regions in Bax that are required for regulation by MEK1 and Akt in T cells.

A Bax deletion mutant that is truncated at amino acid 171 (Bax1–171) and does not contain a stretch of hydrophobic amino acids at the C terminus was observed to largely localize to the cytoplasm (Fig. 6A) and triggered low levels of apoptosis (Fig. 6, D and E). This deletion also removes the recently described Akt phosphorylation site at Ser184. However, the addition of UO126 or LY294002 to Bax1–171-transfected cells triggered translocation of the deletion mutant to the mitochondrion (Fig. 6, B and C) and apoptosis in 2B4 cells (Fig. 6D). Additionally, both DN-MEK1 and DN-Akt increased the sensitivity of cells to Bax1–171-induced apoptosis (Fig. 6E). Apoptotic damage triggered by the coexpression of DN-Akt was lower than that triggered by the addition of LY294002 or by coexpressing DN-MEK1. However, it should be noted that despite deletion of the Akt phosphorylation site (Ser184), Akt-dependent regulation of Bax1–171 was not abrogated.



View larger version (29K):
[in this window]
[in a new window]
  		FIGURE 6. Bax1–171-GFP distribution and apoptotic activity in the 2B4 cell line. A, 2B4 cells transfected with Bax1–171-GFP were subjected to subcellular fractionation after overnight culture. The cytosolic (Cyt) and mitochondria (Mit)-rich fractions were analyzed for GFP and Cox-4 expression by Western blot analysis. B and C, 2B4 cells transfected with Bax1–171-GFP were cultured for 18 h and then continued in culture with 20 µM LY294002 (B) or 25 µM UO126 (C) for an additional 6–8 h. Cells were subjected to subcellular fractionation and analyzed as described in A. The fractions were also probed for NF-{kappa}B (not shown), which was present only in the cytosol and whole cell lysate (WCL). D, 2B4 cells transfected with GFP or Bax1–171-GFP were cultured for 18 h and then continued in culture with or without UO126 (10 µM) or LY294002 (10 µM) for an additional 8 h before being assessed for apoptotic nuclear damage. Nuclear damage was normalized to GFP-transfected cells. E, 2B4 cells were transfected with GFP or Bax1–171-GFP with and without DN-MEK1 or DN-Akt. Apoptotic nuclear damage was assessed in the GFP-positive cells after 18–24 h.

 
Deletion of the N-terminal aa 1–29 (Bax-{delta}29) resulted in a form of Bax that was primarily localized to the mitochondrion (Fig. 7, A and B) and triggered high levels of apoptosis in the 2B4 cell line (Fig. 7C). Apoptosis induced by Bax-{delta}29 was not inhibited by CA-Akt or CA-MEK (Fig. 7D), but was substantially reduced by DN-caspase-9 (Fig. 7E). Because the deletion of 29 aa may not completely disrupt the mitochondrial targeting sequence described in the N terminus (30), we generated a construct that was deleted for the first 37 aa. Bax-{delta}37 was not detected in the cytoplasmic fraction in 2B4 cells (Fig. 7F) and triggered high levels of apoptosis in the 2B4 and d11S cell lines (data not shown).



View larger version (43K):
[in this window]
[in a new window]
  		FIGURE 7. Characterization of N-terminal Bax deletion mutants in the 2B4 cell line. A, Cells transfected with Bax {delta}29-GFP were subjected to subcellular fractionation after overnight culture. The cytosolic (Cyt) and mitochondria (Mito)-rich fractions were analyzed for GFP, p38MAPK, and Cox-4 expression by Western blot analysis. B, Amounts of Bax {delta}29-GFP and Bax-GFP present in the mitochondrial fraction relative to the total protein in the cytosolic and mitochondrial fractions. C, Cells were transfected with GFP, Bax, or Bax {delta}29-GFP, and GFP-positive cells were assessed for apoptotic nuclear damage 15 h post-transfection. Nuclear damage plotted in the figure is normalized to GFP-transfected cells. The inset shows a Western blot analysis of 2B4 cells transfected with Bax {delta}29-GFP or GFP. D, 2B4 cells were transfected with GFP, Bax {delta}29-GFP, Bax {delta}29-GFP plus CA-Akt, or Bax {delta}29-GFP plus CA-MEK1. After 15 h, GFP-positive cells were scored for apoptotic nuclear damage. E, 2B4 cells transfected with GFP, Bax {delta}29-GFP, or Bax {delta}29-GFP and DN caspase-9 were assessed for the induction of apoptotic nuclear damage in GFP-positive cells after 15 h. F, Cells transfected with Bax-GFP or Bax {delta}37-GFP were subjected to subcellular fractionation as described in A. The fractions were assessed for GFP, Cox-4, and p38MAPK. Lanes 5 and 6 are whole cell lysates.

 
However, a mutant that lacked the first 13 aa (Bax-{delta}13) was localized to the cytoplasm (Fig. 8A) and triggered low levels of apoptosis comparable to the full-length construct (Fig. 8D). Treatment with LY294002 or UO126 triggered mitochondrial translocation of the ectopically expressed protein (Fig. 8, B and C). Furthermore, cotransfection of Bax-{delta}13 with DN-MEK1 or DN-Akt triggered apoptosis in 2B4 cells (Fig. 8D). If the N terminus of Bax confers sensitivity to inhibition by MEK1 and Akt, we reasoned that the cytokine IL-2-dependent survival should be abrogated by ectopic expression of Bax-{delta}29. To this end we show that in the IL-2-dependent CTEV-2 cell line, cytokine-dependent survival is disrupted by Bax-{delta}29 compared with the modest increase in apoptosis triggered by the enforced expression of Bax (Fig. 8E).



View larger version (45K):
[in this window]
[in a new window]
  		FIGURE 8. Regulation of Bax {delta}13-GFP in the 2B4 cell line. A, 2B4 cells transfected with Bax {delta}13-GFP were subjected to subcellular fractionation as described in Fig. 7A. The fractions were probed for GFP, Cox-4, NF-{kappa}B, or Akt. B and C, 2B4 cells transfected with Bax {delta}13-GFP were cultured for 18 h and then continued in culture with 20 µM LY294002 (B) or 25 µM UO126 (C) for an additional 6–8 h. Cells were subjected to subcellular fractionation and analyzed as described in A. D, Cells transfected with GFP and Bax {delta}13-GFP in the presence or the absence of DN-MEK1 or DN-Akt were analyzed for induction of apoptotic nuclear damage 18 h after transfection. E, Apoptotic nuclear damage triggered by Bax-GFP and Bax-{delta}29-GFP in the CTEV-2 cell line was assessed 6 h after transfection.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Bax is a proapoptotic member of the Bcl-2 family that is sequestered in the cytoplasm in healthy cells (5, 31) and translocates to the mitochondrion to induce apoptosis. Thus, Bax falls into a class of apoptotic proteins that are not only regulated at their sites of action, i.e., the mitochondrion and endoplasmic reticulum, but are also the target of cellular survival strategies that sequester proapoptotic proteins away from the mitochondrion. In this study we report that the kinases MEK and Akt regulate the subcellular distribution of Bax via a region in the N terminus in T cells.

What is the physiological relevance of the regulation of Bax in T cells? Gene knockout approaches have demonstrated a requirement for Bax or Bak in activated T cell death (6). Activated T cells generated in the course of the immune response receive extracellular survival cues from costimulatory molecules and cytokines (32), and memory T cells that survive the deletion of Ag-reactive T cells reportedly express high levels of ERK-1/2 (33), which is a target of MEK1. Furthermore, sustained expression of Akt has been shown to promote the long term survival of activated T cells (34). Thus, the regulation of Bax by Akt or MEK1 is significant in the context of understanding activated T cell homeostasis.

The analysis reported in this study has been facilitated by the availability of T cell lines that sequester ectopically expressed Bax in the cytoplasm and are susceptible to Bax cytotoxicity when manipulated by signals that are not themselves apoptogenic to these cells. Thus, inhibition of MEK1 or Akt signaling by either pharmacological approaches or DN constructs triggered apoptosis only in Bax-transfected 2B4 and d11S cells. Although regulation of Bax function via Akt-dependent phosphorylation has been recently reported in neutrophils (23), we believe that the kinases may not regulate Bax by direct phosphorylation alone. Firstly, the C-terminal deleted construct Bax1–171 does not contain the phosphorylation site targeted by Akt (23); nonetheless, LY294002 and the coexpression of DN-Akt regulated Bax1–171 function and localization (Fig. 6). Secondly, the N-terminal deletion mutant Bax-{delta}29 was not inhibited by CA-Akt (Fig. 7D), arguing against direct phosphorylation in the C-terminal region as the dominant mode of regulation of Bax in T cells. Finally, CA-MEK1 or CA-Akt did not inhibit Bax-induced apoptosis in the Jurkat cell line (data not shown). Given the potency of Bax, it is likely that multiple cellular mechanisms are in place to regulate Bax function. In this context it will undoubtedly be interesting to determine whether Ser184-mutated Bax is regulated by Akt and MEK1 in T cells.

The observation that the N-terminal truncation of 29 aa residues results in mitochondrial localization of Bax (Fig. 7, A and B) is consistent with the study by Wood and Newcombe (35). This is not in complete agreement with another study (30), which reported that deletion of aa 1–20 inhibited constitutive mitochondrial localization of a Bax isoform (Bax-{psi}) and its apoptotic activity in a Bax-deficient glioblastoma cell line. Both the N- and C-terminal regions in Bax have been reported to interact with 14-3-3, a ligand that sequesters other proapoptotic proteins of the Bcl-2 family in the cytosol (24), and a stretch of 53 N terminus aa in Bax is implicated in the interaction with Ku70 (25). These differences suggest that the mechanism of Bax regulation may be cell and stimulus specific. It should be noted that despite the demonstrated association of Ku70 with Bax, the deletion of Ku70 did not result in spontaneous translocation of Bax to the mitochondrion (25), indicating that multiple cytosolic factors (cell type specific) probably regulate Bax sequestration.

Our experiments with the deletion mutants implicate a region spanned by aa 13–29 at the Bax N terminus in the regulation of cytoplasmic localization by MEK and Akt. A recent study has shown that N-terminally deleted Bax functions like a BH3-only protein, requiring endogenous Bax for induction of apoptosis (36). However, we detect little/no endogenous Bax in the Jurkat cell line and report higher and comparable levels of expression of the endogenous protein in 2B4 and d11S cells. Thus, it is unlikely that Bax-{delta}29-induced apoptosis is mediated via the recruitment of endogenous Bax, because Jurkat cells are very susceptible to apoptosis induced by the deletion mutant (data not shown). Our experiments suggest a redundancy in the requirement for Akt- and MEK1-dependent signaling, and we hypothesize that Akt and MEK1 probably phosphorylate a cellular complex associated with Bax in T cells. The former may explain why the accumulation of activated T cells is not reported in mice genetically deficient for MEK1 or Akt.

In conclusion, our experiments in activated T cells show that one outcome of prosurvival signaling by cytokines is related to the regulation of Bax localization and thereby its apoptotic function. MEK-dependent regulation of Bax expression and translocation in primary activated T cells identifies a mechanism by which cytokines may block apoptosis by targeting premitochondrial events in T cells. Using an IL-2-dependent T cell line, we show that IL-2 could protect cells to some extent from Bax-induced apoptosis, but was unable to inhibit apoptosis triggered by the Bax-{delta}29 deletion mutant (Fig. 8D). Ongoing experiments are directed toward the identification of molecules associated with the N terminal of Bax under the influence of Akt and MEK1 signaling.


   Acknowledgments
 
We are grateful to Veronica Rodrigues (Division of Biological Sciences, Tata Institute of Fundamental Research, Mumbai, India) and M. K. Mathew and Gaiti Hasan (National Center for Biological Sciences, Bangalore, India) for their comments. The generous gift of reagents from Peter Vandenabeele and Claudius Vincenz is gratefully acknowledged.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by a Wellcome Trust International Senior Research Fellowship in Biomedical Science in India, the Wellcome Trust, U.K., and the Department of Science and Technology-Swarnajayanti Fellowship in Biology, Government of India (to A.S.). N.P. is supported by a fellowship from the Council for Scientific and Industrial Research, India. Back

2 H.S. and L.K. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Apurva Sarin, National Center for Biological Sciences, New Bellary Road, Bangalore 560065, Karnataka, India. E-mail address: sarina{at}ncbs.res.in Back

4 Abbreviations used in this paper: MEK, MAPK kinase; CA, constitutively active; Cox-4, cytochrome oxidase; DN, dominant-negative Akt; IAP, inhibitor of apoptosis protein; ICM, intracellular medium; MBP, maltose binding basic protein; p, phosphorylated. Back

Received for publication May 8, 2004. Accepted for publication August 29, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Ashkenazi, A., V. M. Dixit. 1998. Death receptors: signaling and modulation. Science 281:1305.[Abstract/Free Full Text]
  2. Wallach, D., E. E. Varfolomeev, N. L. Malinin, Y. V. Goltsev, A. V. Kovalenko, M. P. Boldin. 1999. Tumor necrosis factor receptor and Fas signaling mechanisms. Annu. Rev. Immunol. 17:331.[Medline]
  3. Desagher, S., J. C. Martinou. 2000. Mitochondria as the central control point of apoptosis. Trends Cell Biol. 10:369.[Medline]
  4. Zamzami, N., G. Kroemer. 2001. The mitochondrion in apoptosis: how Pandora’s box opens. Nat. Rev. Mol. Cell. Biol. 2:67.[Medline]
  5. Vander Heiden, M. G., and C. B. 1999. Thompson. Bcl-2 proteins: regulators of apoptosis or mitochondrial homeostasis? Nat. Cell. Biol. 1:E209.
  6. Rathmell, J. C., T. Lindsten, Z. Wei-Xing, R. M. Cinalli, C. T. Thompson. 2002. Deficiency in bak and bax perturbs thymic selection and lymphoid homeostasis. Nat. Immunol. 3:932.[Medline]
  7. Brady, H. J. M., G. S. Salomons, R. C. Bobeldijk, A. J. M. Berns. 1996. T cells from bax transgenic mice show accelerated apoptosis in response to stimuli but do not show restored DNA damage-induced cell death in the absence of p53. EMBO J. 15:1221.[Medline]
  8. Lin, J. X., T. S. Migone, M. Tsang, M. Friedmann, J. A. Weatherbee, L. Zhou, A. Yamauchi, E. T. Bloom, J. Mietz, S. John, et al 1995. The role of shared receptor motifs and common stat proteins in the generation of cytokine pleiotropy and redundancy by IL-2, IL-4, IL-7, IL-13 and IL-15. Immunity 2:331.[Medline]
  9. Lodolce, J. P., D. L. Boone, S. Chai, R. E. Swain, T. Dassopoulos, S. Trettin, A. Ma. 1998. IL-15 receptor maintains lymphoid homeostasis by supporting lymphocyte homing and proliferation. Immunity 9:669.[Medline]
  10. Maraskovsky, E., M. Teepe, P. J. Morrissey, S. Braddy, R. E. Miller, D. H. Lynch, J. J. Peschon. 1996. Impaired survival and proliferation in IL-7 receptor-deficient peripheral T cells. J. Immunol. 157:5315.[Abstract]
  11. Perkins, G. R., J Marvel, M. K. Collins. 1993. Interleukin 2 activates extracellular signal-regulated protein kinase 2. J. Exp. Med. 178:1429.[Abstract/Free Full Text]
  12. Moon, J. J., B. H. Nelson. 2001. Phosphatidylinositol 3-kinase potentiates, but does not trigger, T cell proliferation mediated by the IL-2 receptor. J. Immunol. 167:2714.[Abstract/Free Full Text]
  13. Gu, H., H. Maeda, J. J. Moon, J. D. Lord, M. Yoakim, B. H. Nelson, B. G. Neel. 2000. New role for Shc in activation of the phosphatidylinositol 3-kinase/Akt pathway. Mol. Cell. Biol. 20:7109.[Abstract/Free Full Text]
  14. Boucher, M. J., J. Morisset, P. H. Vachon, J. C. Reed, J. Laine, N. Rivard. 2000. MEK/ERK signaling pathway regulates the expression of Bcl-2, Bcl-XL, and Mcl-1 and promotes survival of human pancreatic cancer cells. J. Cell. Biochem. 79:355.[Medline]
  15. Pardo, O. E., A. Arcaro, G. Salerno, S. Raguz, J. Downward, M. J. Seckl. 2002. Fibroblast growth factor-2 induces translational regulation of Bcl-xL and Bcl-2 via a MEK-dependent pathway. J. Biol. Chem. 277:12040.[Abstract/Free Full Text]
  16. Scheid, M. P., V. Duronio. 1998. Dissociation of cytokine-induced phosphorylation of Bad and activation of PKB/Akt: involvement of MEK upstream of Bad phosphorylation. Proc. Natnl. Acad. Sci. USA 95:7439.[Abstract/Free Full Text]
  17. Varghese, J., H. Sade, P. Vandenabeele, A. Sarin. 2002. Head involution (Hid) triggered apoptosis requires caspase-8 but not FADD (Fas associated death domain) and is regulated by the extracellular signal-related kianse-I/2 (Erk) in mammalian cells. J. Biol. Chem. 277:35097.[Abstract/Free Full Text]
  18. Tashker, J. S., M. Olsen, S. Kornbluth. 2002. Post-cytochrome c protection from apoptosis conferred by a MAPK pathway in Xenopus egg extracts. Mol. Cell. Biol. 13:393.
  19. Erhardt, P., E. J. Schremser, G. M. Cooper. 1999. B-Raf inhibits programmed cell death downstream of cytochrome c release from mitochondria by activating the MEK/Erk pathway. Mol. Cell. Biol. 19:5308.[Abstract/Free Full Text]
  20. Datta, S. R., A. Brunet, M. E. Greenberg. 1999. Cellular survival: a play in three Akts. Genes Dev. 13:2905.[Free Full Text]
  21. Zhang, X. D., J. M. Borrow, X. Y. Zhang, T. Nguyen, P. Hersey. 2003. Activation of ERK1/2 protects melanoma cells from TRAIL-induced apoptosis by inhibiting Smac/DIABLO release from mitochondria. Oncogene 22:2869.[Medline]
  22. Tsuruta, F., N. Masuyama, Y. Gotoh. 2002. The Phosphatidylinositol 3-Kinase (PI3K)-Akt Pathway Suppresses Bax Translocation to Mitochondria. J. Biol. Chem. 277:14040.[Abstract/Free Full Text]
  23. Gardai, S. J., D. A. Hildeman, S. K. Frankel, B. B. Whitlock, S. C. Frasch, N. Borregaard, P. Marrack, D. L. Bratton, P. M. Henson. 2004. Phosphorylation of Bax serine 184 by Akt regulates its activity and apoptosis in neutrophils. J. Biol. Chem. 279:21085.[Abstract/Free Full Text]
  24. Nomura, M., S. Shimizu, T. Sugiyama, M. Narita, T. Ito, H. Matsuda, Y. Tsujimoto. 2003. 14–3-3 interacts directly with and negatively regulates pro-apoptotic Bax. J. Biol. Chem. 278:2058.[Abstract/Free Full Text]
  25. Sawada, M., W. Sun, P. Hayes, K. Leskov, D. A. Boothman, S. Matsuyama. 2003. Ku70 suppresses the apoptotic translocation of Bax to mitochondria. Nat. Cell. Biol. 5:320.[Medline]
  26. Wolter, K. G., Y. T. Hsu, C. L. Smith, A. Nechustan, X. G. Xi, R. J. Youle. 1997. Movement of Bax from the cytosol to mitochondria during apoptotsis. J. Cell Biol. 139:1281.[Abstract/Free Full Text]
  27. Nechustan, A., C. L. Smith, Y. T. Hsu, R. J. Youle. 1999. Conformation of the Bax C-terminus regulates subcellular location and cell death. EMBO J. 18:2330.[Medline]
  28. Goping, I. S., A. Gross, J. N. Lavoie, M. Nguyen, R. Jemmerson, K. Roth, S. J. Korsemeyer, G. C. Shore. 1998. Regulated targeting of BAX to mitochondria. J. Cell Biol. 143:207.[Abstract/Free Full Text]
  29. Sade, H., A. Sarin. 2003. IL-7 inhibits dexamethasone-induced apoptosis via Akt/PKB in mature, peripheral T cells. Eur. J. Immunol. 33:913.[Medline]
  30. Cartron, P. F., M. Priault, L. Oliver, K. Meflah, S. Manon, F. M. Vallette. 2003. The N-terminal end of Bax contains a mitochondrial-targeting signal. J. Biol. Chem. 278:11633.[Abstract/Free Full Text]
  31. Puthalakath, H., A. Strasser. 2002. Keeping killers on a tight leash: transcriptional and post-translational control of the pro-apoptotic activity of BH3-only proteins. Cell Death Differ. 9:505.[Medline]
  32. Schluns, K. S., L. Lefrancois. 2003. Cytokine control of memory T-cell development and survival. Nat. Rev. Immunol. 3:269.[Medline]
  33. Kaech, S. M., S. Hemby, E. Kersh, R. Ahmed. 2002. Molecular and functional profiling of memory CD8 T cell differentiation. Cell 111:837.[Medline]
  34. Song, J., S. Salek-Ardakani, P. R. Rogers, M. Cheng, L. Van Parijs, M. Croft. 2004. The costimulation-regulated duration of PKB activation controls T cell longevity. Nat. Immunol. 5:150.[Medline]
  35. Wood, D. E., E. W. Newcomb. 2000. Cleavage of Bax enhances its cell death function. Exp. Cell Res. 256:375.[Medline]
  36. Cartron, P. F., L. Oliver, P. Juin, K. Meflah, F. M. Vallette. 2004. The p18 truncated form of Bax behaves like a Bcl-2 homology domain 3-only protein. J. Biol. Chem. 279:11503.[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Parikh, N.
Right arrow Articles by Sarin, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Parikh, N.
Right arrow Articles by Sarin, A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS