Advertisement
JBC

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


     

Originally published In Press as doi:10.1074/jbc.M503041200 on July 1, 2005

J. Biol. Chem., Vol. 280, Issue 35, 31091-31100, September 2, 2005
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
280/35/31091    most recent
M503041200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
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 arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ray, R. M.
Right arrow Articles by Johnson, L. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ray, R. M.
Right arrow Articles by Johnson, L. R.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Protein Phosphatase 2A Regulates Apoptosis in Intestinal Epithelial Cells*

Ramesh M. Ray{ddagger}, Sujoy Bhattacharya, and Leonard R. Johnson

From the Department of Physiology, University of Tennessee Health Science Center, Memphis, Tennessee 38163

Received for publication, March 18, 2005 , and in revised form, June 28, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Polyamine depletion prevents apoptosis by increasing serine/threonine phosphorylation leading to either inactivation or activation of pro- and anti-apoptotic proteins, respectively. Despite evidence that protein kinases are regulators of apoptosis, a specific role for protein phosphatases in regulating cell survival has not been established. In this study, we show that polyamine depletion inhibits serine/threonine phosphatase 2A (PP2A). Inhibition of PP2A in cells depleted of polyamines correlated well with increased phosphorylation of Bad at Ser112. Bad Ser112 phosphorylation in response to tumor necrosis factor (TNF)-{alpha} treatment decreased with time in cells grown in control as well as those grown in the presence of {alpha}-difluoromethylornithine plus putrescine. However, a sustained increase in the levels of Bad Ser112 phosphorylation was maintained in response to TNF-{alpha} treatment in cells grown in the presence of {alpha}-difluoromethylornithine. Inhibition of PP2A by okadaic acid and fostriecin or PP2A small interfering RNA transfection significantly decreased TNF-{alpha}-induced apoptosis in control and polyamine-depleted cells. Inhibition of PP2A by okadaic acid: 1) increased Bad and Bcl-2 phosphorylation at Ser112 and Ser70, respectively; 2) increased ERK activity; 3) prevented JNK activation; 4) prevented cytochrome c release, and activation of caspases-9 and -3 in response to TNF-{alpha}. Inhibition of MEK1 by U0126 prevented phosphorylation of Bad at Ser112. These results indicate that polyamines regulate PP2A activity, and inhibition of PP2A in response to polyamine depletion increases steady state levels of Bad and Bcl-2 proteins and their phosphorylation and thereby prevents cytochrome c release, caspase-9, and caspase-3 activation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In eukaryotic cells, reversible protein phosphorylation catalyzed by protein kinases and protein phosphatases regulates numerous cellular processes including apoptosis (1). The kinases themselves are dephosphorylated and deactivated by a type-2A protein phosphatase. Kinases and phosphatases may even bind the same protein at different locations (2). Every third protein undergoes reversible phosphorylation in an average eukaryotic cell. Previous studies have implicated serine/threonine protein phosphatase-2A (PP2A)1 in a wide array of cellular functions including metabolism, transcription, translation, ion transport, development, proliferation, differentiation, and apoptosis (1, 3, 4). This includes increasing or decreasing enzyme activities (phospholipase, glycogen synthase kinase 3{beta}), marking a protein for degradation (I{kappa}B), allowing a protein to move from one cellular location to another, or enabling a protein to interact with or dissociate from other proteins (Bcl-2/Bcl-xL-associated death promoter (Bad)). The Bcl-2 family proteins are key regulators of apoptosis. Three subfamilies have been identified: 1) prosurvival Bcl-2 (e.g. Bcl-2, Bcl-xL, and Mcl-1) proteins; 2) the proapoptotic Bax (e.g. Bax and Bak) proteins; and 3) the BH3 domain-only (e.g. Bad, Bim, and Bid) proteins (5-7). Normal cellular homeostasis is maintained by suppression of proapoptotic proteins by various mechanisms, which include phosphorylation, intracellular localization, and heterodimerization with prosurvival Bcl-2 family proteins. The BH3-only proapoptotic protein Bad exerts its death promoting effects by heterodimerizing with Bcl-xL and Bcl-2 death antagonists at the mitochondrial membrane (8, 9).

The polyamines putrescine, spermidine, and spermine are present in virtually all eukaryotic cells. Polyamines are low molecular weight aliphatic amines and are highly charged cations at physiological pH. Putrescine, spermidine, and spermine are synthesized from a common precursor, ornithine, which is decarboxylated by the rate-limiting enzyme ornithine decarboxylase (ODC) to form putrescine. S-Adenosylmethionine decarboxylase, another rate-limiting enzyme in polyamine synthesis, converts S-adenosylmethionine into decarboxylated S-adenosylmethionine. Decarboxylated S-adenosylmethionine in turn acts as an aminopropyl donor in the synthesis of spermidine and spermine (10, 11). The role of polyamines in cell proliferation and differentiation has been extensively studied using enzyme inhibitors and polyamine analogs (12, 13). Interference of polyamine homeostasis not only alters cell proliferation but also may even cause cell death depending on the cell type or pharmacological agent used. Involvement of ODC in cell death processes first emerged from studies on neuronal cell death (14). Both up- and down-regulation of polyamine levels were reported during apoptosis.

Various studies using different cell systems have shown a rapid and marked increase of ODC activity during apoptotic cell death suggesting an active role for ODC in apoptosis (15). If polyamines play an active role in apoptosis, inhibition of polyamine synthesis should inhibit apoptosis. Tewari et al. (16) reported that degradation of ODC by forced expression of ODC antizyme, prevented cell death in human fibroblasts. Furthermore, inhibition of ODC activity with the highly specific ODC inhibitor {alpha}-difluoromethylornithine (DFMO) and subsequent depletion of polyamines inhibit apoptosis in almost all systems studied (15). Monti et al. (17) showed that polyamine depletion protected human promyelogenous leukemia cells (HL60) from 2-deoxy-D-ribose-induced apoptosis. Recent studies imply that a change in ODC activity per se is not obligatory for apoptosis. Noda et al. (18) showed that ischemia-reperfusion damage in rat intestinal mucosa resulted in elevated ODC activity accompanied by DNA fragmentation. Inhibition of ODC activity by DFMO did not affect DNA fragmentation, indicating that apoptosis was independent of ODC activity (19). Penning et al. (20) also showed that apoptosis stimulated by TNF-{alpha} was independent of ODC activity per se but was dependent on intracellular levels of spermine.

Our laboratory has studied the growth and repair of the gastrointestinal mucosa and has examined these processes in both rats and in cultured intestinal epithelial cells (IEC-6), a non-transformed line derived from rat crypt cells (21, 22). Previously we have shown that polyamine depletion inhibits apoptosis induced in response to camptothecin, TNF-{alpha}, and {gamma}-irradiation in IEC-6 cells (23, 24). In an attempt to delineate the mechanisms regulating apoptosis, we have shown that polyamine depletion causes sustained activation of ERKs, which in turn prevents the activation of pro-apoptotic N-terminal c-Jun kinase (25, 26). Furthermore, polyamine depletion with DFMO caused constitutive activation of Akt and NF-{kappa}B (27). In addition, each of these pathways controls the levels or activities of Bcl-2 family proteins that are responsible for determining whether cytochrome c is released from mitochondria. The activities of many of the components of these pathways and the Bcl-2 proteins are regulated by serine-threonine phosphorylation. We have also shown that Akt and ERKs are activated immediately following exposure of IEC-6 cells to TNF-{alpha}. These activities peak around 3-4 h and then decrease to basal levels by 9 h (26, 27). In polyamine-depleted cells, however, the activities of these enzymes increase to higher levels and then plateau. Thus, it appears that there is no inactivation of Akt and ERK in DFMO-treated cells. These findings suggest that increased serine/threonine phosphorylation might be because of inactivation of PP2A in polyamine-depleted cells, and that inactivation protected the cells from apoptosis. Earlier reports also indicated that polyamines activate PP2A specifically compared with PP1 (28, 29). Therefore, in the present study we investigated the role of PP2A in the regulation of apoptosis in polyamine-depleted cells.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Cell cultureware was purchased from Corning Glass works (Corning, NY). Media and other cell culture reagents were obtained from Invitrogen. Dialyzed fetal bovine serum (dFBS) was purchased from Sigma. FuGENE 6 transfection reagent was purchased from Roche Diagnostics. Geneticin (G418) was purchased from Invitrogen. TNF-{alpha} was obtained from Pharmingen. The enhanced chemiluminescence (ECL) Western blot detection system was purchased from PerkinElmer Life Sciences. DFMO was a gift from ILEX OncologyTM Inc. (San Antonio, TX). Caspase-3 substrate DEVD-pNA was purchased from Biomol%20Research%20Laboratories">Biomol Research Laboratories (Plymouth Meeting, PA). Anti-PP2A and caspase-3 antibodies, PP2A-specific and non-targeting control siRNA were purchased from Santa Cruz Biotechnology. Phospho-ERK, total ERK, and caspase-3 antibodies were purchased from Cell Signaling (Beverly, MA). Anti-phosphotyrosine antibody was purchased from Upstate (Charlottesville, VA). Okadaic acid and fostriecin were purchased from Calbiochem, EMD Biosciences (La Jolla, CA), and Sigma, respectively. FuGENE 6 transfection reagent and Cell Death Detection ELISA Plus kit were purchased from Roche. The IEC-6 cell line (ATCC CRL 1592) was obtained from American Type Culture Collection (Manassas, VA) at passage 13. The cell line was derived from normal rat intestine and was developed and characterized by Quaroni et al. (30). IEC-6 cells originate from intestinal crypt cells as judged by morphological and immunologic criteria. They are nontumorigenic and retain the undifferentiated character of epithelial stem cells. Tests for mycoplasma were always negative. All chemicals were of the highest purity commercially available.

Cell Culture—IEC-6 cell stock was maintained in T-150 flasks in a humidified, 37 °C incubator in an atmosphere of 90:10, air:CO2. The medium consisted of Dulbecco's modified Eagle's medium (DMEM) with 5% heat inactivated FBS and 10 µg of insulin and 50 µg of gentamicin sulfate per ml. The stock flask was passaged weekly, fed 3 times per week, and passages 15-22 were used. To set up experiments, the cells were trypsinized with 0.05% trypsin and 0.53 mM EDTA and counted by a Beckman Coulter counter (model Z1). General experimental protocols for using IEC-6 cells have previously been described (31). Briefly, IEC-6 cells were plated at 6.24 x 104 cells/cm2 in DMEM as described, fed every other day, and serum starved during the 24 h before harvesting. We have previously reported that maximal polyamine depletion in IEC-6 cells occurs after 4 days of treatment with DFMO, with putrescine undetectable after 6 h of DFMO, spermidine depleted after 24 h, and spermine reduced to a stable 40% after 4 days of DFMO (32).

Apoptosis—Cells were plated (day 0) at a density of 6.25 x 104 cells/cm2 in DMEM/dFBS with or without 5 mM DFMO and DFMO plus 10 µM putrescine in triplicate for each group. The DFMO plus putrescine group served as a control to show that the effects of DFMO were because of polyamine depletion and not DFMO itself. In all experiments the results with this group of cells were identical to those of the untreated controls. Cells were fed on day 2. On day 3, the culture medium was removed and replaced with serum-free medium. On day 4, TNF-{alpha} (20 ng/ml) plus cycloheximide (25 µg/ml) was added to the serum-free medium for 3 h, with the appropriate vehicle added. Me2SO was used as vehicle in most experiments involving inhibitors.

siRNA Transfection—PP2A-specific siRNA sequence (20-25 nucleotides) was derived from the Mus musculus protein phosphatase 2a, catalytic subunit {alpha} isoform (GenBankTM accession number NM_019411 [GenBank] ), which is 97.84% homologous to Rattus norvegicus PP2A (GenBank accession number NM_017039 [GenBank] ). FuGENE 6 transfection reagent was mixed with 10 µM PP2A or 10 µM control (non-targeting un-conjugated) siRNA (3:3.4 µl) in serum-free medium, to a total volume of 100 µl and incubated for 30 min at room temperature. IEC-6 cells at a relatively early passage were grown to 50% confluence in 12-well plates. For transfection, the cell monolayer was rinsed with serum-free medium and the siRNA/FuGENE mixture was added dropwise onto the cell monolayer and further incubated for 30 h at 37 °C. Cells were serum starved for 24 h, treated with TNF-{alpha}/CHX for 3 h, and subjected to quantitative DNA fragmentation analysis. Fluorescein isothiocyanate-conjugated control siRNA was used to monitor the efficiency of transfection.

Quantitative DNA Fragmentation Enzyme-linked Immunosorbent Assay—Cells were grown in 12-well culture plates for both DNA fragmentation and protein determination. After treatment, floating cells were discarded, and the attached cells were washed twice with Dulbecco's phosphate-buffered saline. Cells were lysed and centrifuged to remove the nuclei. An aliquot of the nuclei-free supernatant was placed in streptavidin-coated wells, respectively. A mixture of anti-histone-biotin and anti-DNA peroxidase-conjugated antibody for DNA fragmentation was added to the wells and incubated for 2 h at room temperature. After incubation, the sample was removed and the wells were washed 3 times with incubation buffer. After the final wash was removed, 100 µl of the peroxidase substrate, 2,2'-azino-di(3-ethylbenzthiazolin-sulfonate), was placed in the wells and incubated at room temperature. The absorbance was read at 405 nm using a microplate reader. Results were expressed as absorbance 405 nm/min/mg protein.

Immunoprecipitation—Cell extracts containing equivalent amounts of protein (100-200 µg) were incubated for 2 h at 4 °C in the presence of 2 µg of Bcl-2 or anti-phosphotyrosine or PP2A catalytic subunit-specific antibodies and adsorbed overnight at 4 °C to protein A-Sepharose beads. Samples were centrifuged at 2000 x g for 5 min and washed twice with 50 mM Tris-HCl, pH 7.0, containing 100 µM CaCl2. Protein A-Sepharose beads were resuspended in either SDS sample buffer or serine/threonine assay buffer and used for Western blot analysis or to assay PP2A activity, respectively.

Measurement of PP2A Activity—PP2A protein immunoprecipitated as described above was used to determine enzyme activity using a PP2A immunoprecipitation phosphatase assay kit from Upstate USA Inc. following the manufacturer's instructions. The specificity of the immunoprecipitation assay was tested by the addition of 2 nM okadaic acid, a concentration that specifically inhibits PP2A activity in vitro.

Western Blot Analysis—Cells were first washed with ice-cold phosphate-buffered saline and then lysed for 10 min in ice-cold extraction buffer containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM {beta}-glycerophosphate, 1 mM Na3VO4, and a protease inhibitor mixture. Lysates were centrifuged at 10,000 x g for 10 min at 4 °C. Supernatants from 25 to 50 µg of protein were trichloroacetic acid precipitated and eluted in 1x SDS sample buffer for 5 min and separated on 10-15% SDS-PAGE. Proteins were transferred overnight to Immobilon-P membranes (Millipore, Bedford, MA) and probed with the indicated antibodies overnight at 4 °C in TBS buffer containing 0.1% Tween 20 and 5% nonfat dry milk (blotting grade, Bio-Rad). Membranes were subsequently incubated with secondary antibody-conjugated to horseradish peroxidase at room temperature for 1 h, and the immunocomplexes were visualized by the ECL detection system. Blots were stripped and probed with the indicated antibodies to determine equal loading of the samples.

Cytochrome c Release—IEC-6 cells were grown in control and DFMO containing media as described earlier in the section on apoptosis. Cells were either treated with 10 nM okadaic acid or left untreated for 1 h. TNF-{alpha}/CHX was added to each well, and plates were further incubated for 30 min. 1 µl of mitosensor reagent, prepared in incubation buffer, was added to each well and further incubated for 30-45 min at 37 °C and washed 3 times with cold Dulbecco's phosphate-buffered saline. Fluorescence images were captured using a microscope with a bandpass filter to detect fluorescein and rhodamine. Aggregates of the mitosensor dye localized in the mitochondria exhibit red fluorescence, whereas its monomers in the cytoplasm, indicative of apoptotic cells, exhibit green fluorescence.

Densitometry and Statistics—All data are expressed as mean ± S.E. All experiments were repeated three times, with triplicate samples. Analysis of variance and appropriate post-hoc testing determined the significance of the differences between means. Values of p < 0.05 were regarded as significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Polyamine Depletion Decreases PP2A Activity—Earlier reports from our laboratory have shown that polyamine depletion as a consequence of ODC inhibition by DFMO increases basal levels as well as TNF-{alpha}-induced phosphorylation of ERKs, I{kappa}B{alpha}, STAT3, and AKT (25-27). These proteins were phosphorylated on either serine or threonine residues, and phosphorylation persisted longer in polyamine-depleted cells compared with controls. These data suggest that the enzyme responsible for dephosphorylation of these proteins is inactivated in polyamine-depleted cells. We examined the activity of PP2A, which is inactivated by tyrosine phosphorylation. Cell lysates were first immunoprecipitated with a phosphotyrosine-specific antibody and then subjected to Western blotting using an antibody specific for the PP2A catalytic subunit (PP2Ac). Results in Fig. 1A demonstrate that polyamine depletion did not alter the level of PP2Ac protein. However, tyrosine phosphorylation was significantly higher in cells grown in DFMO containing medium compared with those grown in control and DFMO plus putrescine containing media. Furthermore, polyamine depletion of cells significantly decreased PP2A enzyme activity (2-3-fold) compared with controls (Fig. 1B).

Effect of Inhibition of PP2A on TNF-{alpha}-induced Apoptosis—Okadaic acid is the most widely used inhibitor of the serine/threonine protein phosphatases. PP2A is inhibited most strongly, whereas PP2B is less sensitive, and PP2C is not inhibited (33). Okadaic acid at the dose and conditions of our experiments is considered a selective inhibitor of PP2A. Fostriecin is the most selective small molecule inhibitor of protein phosphatases described to date and has a greater potency against PP2A than PP1 (34, 35). We used okadaic acid and fostriecin to inhibit PP2A in the presence and absence of TNF-{alpha}, and measured DNA fragmentation as an index of apoptosis. Okadaic acid and fostriecin significantly decreased (65-70%) apoptosis in cells grown in control conditions (Fig. 2A). Cells grown in DFMO containing medium had significantly less apoptosis compared with cells grown in control conditions, and okadaic acid further decreased apoptosis almost 50% in these cells. We also determined the level of tyrosine phosphorylation of PP2A as described earlier (Fig. 1A) to ascertain the specificity of okadaic acid. Fig. 2B shows that okadaic acid increased the tyrosine phosphorylation and inactivation of PP2A in control cells and those treated with TNF-{alpha}. Interestingly, TNF-{alpha} itself increased tyrosine phosphorylation of PP2A, suggesting that the initial response to TNF-{alpha} results in both anti- and proapoptotic signals. Although, TNF-{alpha} treatment in the presence or absence of okadaic acid increases PP2A phosphorylation, total amounts of PP2A protein levels remained unchanged. These results indicate that PP2A inactivation protects cells from apoptosis, and that decreased PP2A activity in response to polyamine depletion might be responsible for protection in these cells.



View larger version (31K):
[in this window]
[in a new window]
  		FIG. 1.
Polyamine depletion inhibits PP2A activity. IEC-6 cells were grown to confluence for 3 days in DMEM, 5% dFBS with or without 5 mM DFMO and DFMO plus 10 µM putrescine and serum deprived for 24 h. A, tyrosine phosphorylation of PP2A. Equal amounts of protein (200 µg) were immunoprecipitated (IP) using an anti-phosphotyrosine specific antibody as described under "Experimental Procedures" and 50 µg of whole cell extract were separated by SDS-PAGE, transferred to membranes, and probed with an antibody specific for the PP2A catalytic subunit (PP2Ac). Representative blots from three independent observations are shown. B, PP2A activity. Immunoprecipitated PP2A beads were resuspended in 60 µl of Ser/Thr assay buffer containing 750 µM phosphopeptide for 10 min at 30 °C in a shaking incubator followed by brief centrifugation. An equal volume (25 µl) from each sample was transferred into each well of the 1/2 volume microtiter plate. 100 µl of malachite green phosphate detection solution was added to each well and read at 650 nm between 10 and 15 min. Values are mean ± S.E. *, p < 0.05 compared with control and DFMO + putrescine (PUT). WB, Western blot.

 
Although okadaic acid and fostriecin are specific inhibitors of PP2A at doses used in this study, we used a molecular approach to prevent expression of PP2A protein by PP2A siRNA transfection. Transfection of cells with PP2A siRNA resulted in a 50% decrease in PP2A enzyme activity compared with cells transfected with control siRNA as well as untransfected cells (Fig. 3A). Cells transfected with siRNA for PP2A also showed a significant decrease in the expression of PP2A protein compared with control siRNA-transfected cells (Fig. 3B). Transfection with PP2A siRNA significantly reduced DNA fragmentation in response to TNF-{alpha} compared with cells incubated with transfection reagent alone (Fig. 3C). One group of cells grown under the conditions of siRNA transfection was treated with 100 nM fostriecin, and showed almost 60% inhibition of DNA fragmentation (Fig. 3C), which is similar to that shown in Fig. 2A. Fluorescein isothiocyanate-conjugated control siRNA was used to monitor the transfection efficiency (data not shown).



View larger version (27K):
[in this window]
[in a new window]
  		FIG. 2.
Okadaic acid and fostriecin protect IEC-6 cells from apoptosis. A, DNA fragmentation. IEC-6 cells were grown to confluence for 3 days in DMEM, 5% dFBS with or without 5 mM DFMO and serum deprived for 24 h prior to treatment with TNF-{alpha}/CHX for 3 h in the presence and absence of 15 nM okadaic acid or 100 nM fostriecin. DNA fragmentation was measured using a colorimetric enzyme-linked immunosorbent assay kit as described under "Experimental Procedures." Values are mean ± S.E. *, p < 0.05 compared with respective TNF-{alpha}-treated cells. B, tyrosine phosphorylation of PP2A. Equal amounts of protein (200 µg) were immunoprecipitated using anti-phosphotyrosine (p-Tyr)-specific antibody as described under "Experimental Procedures" and were separated by SDS-PAGE, transferred to membranes, and probed with an antibody specific for the PP2A catalytic subunit. Membrane was also stripped and probed for PP2A. Representative blots from three independent observations are shown. WB, Western blot.

 
PP2A Mediates Bad Ser112 Phosphorylation via Mitogen-activated Protein Kinase—Inactivation of Bad involves the phosphorylation of serine residues at positions 112, 136, and 155, which decrease its affinity for Bcl-xL at the mitochondrial membrane and increases its cytoplasmic binding to protein 14-3-3 (1, 8, 9). Decreased PP2A activity in polyamine-depleted cells should increase the phosphorylation of Bad, and consequently inactivate it. Cells grown in the presence of DFMO had significantly higher levels of Bad protein phosphorylated at Ser112 compared with controls without any changes in the total amount of Bad protein (Fig. 4A). Because TNF-{alpha} also induced tyrosine phosphorylation and subsequent inactivation of PP2A (Fig. 2B), we determined the phosphorylation of Bad during 1.5-6.0 h exposure to TNF-{alpha}. Phosphorylation of Bad (Ser112) decreased progressively during the 1.5-6.0 h exposure to TNF-{alpha} in cells grown in control groups (Fig. 4B). However, a significantly higher level of Bad phosphorylation at Ser112 was evident in polyamine-depleted cells than in either control or DFMO plus putrescine groups. Unlike control cells, phosphorylation of Bad Ser112 was maintained during the entire time course in polyamine-depleted cells. Total Bad protein levels were almost identical in all three groups, changing only slightly during the entire time course. Levels of actin were the same in all three groups. Next, we determined whether PP2A inhibition by okadaic acid increased phosphorylation of Bad at Ser112. Okadaic acid increased Bad phosphorylation at Ser112 of control cells both in the presence and absence of TNF-{alpha}, indicating that inhibition of PP2A inactivates Bad by phosphorylation at Ser112. Bad was constitutively phosphorylated at Ser112 in polyamine-depleted cells.



View larger version (21K):
[in this window]
[in a new window]
  		FIG. 3.
siRNA-mediated inhibition of PP2A expression and apoptosis. IEC-6 cells were transfected with either control or PP2A-specific siRNA as described under "Experimental Procedures." A, cell extracts were used to determine the activity of PP2A. Immunoprecipitated PP2A beads were resuspended in 60 µl of Ser/Thr assay buffer containing 750 µM phosphopeptide for 10 min at 30 °C in a shaking incubator followed by brief centrifugation. An equal volume (25 µl) from each sample was transferred into each well of the 1/2 volume microtiter plate. 100 µl of malachite green phosphate detection solution was added to each well and read at 650 nm between 10 and 15 min. Values are mean ± S.E. *, p < 0.05 compared with untransfected and control siRNA transfected. B, expression of PP2A. Equal amounts of protein from control and PP2A siRNA-transfected cells were separated by SDS-PAGE, transferred to membrane, and probed with an antibody specific for the PP2A catalytic subunit. Membrane was also stripped and probed for actin. A representative blot from three observations is shown. C, DNA fragmentation. Cells were grown in control medium at 50% confluence. Cells grown in control medium and transfected with PP2A-specific siRNA or treated with transfection reagent alone for 30 h were incubated with or without TNF-{alpha}/CHX for 3 h and then subjected to DNA fragmentation analysis. Cells grown in control media treated with transfection reagent alone and another group of control cells pretreated with fostriecin were also treated with TNF-{alpha}/CHX for 3 h and then subjected to DNA fragmentation analysis. Values are mean ± S.E. *, p < 0.05 compared with untransfected cells treated with TNF-{alpha}.

 



View larger version (35K):
[in this window]
[in a new window]
  		FIG. 4.
Bad Ser112 phosphorylation. IEC-6 cells were grown to confluence for 3 days in DMEM, 5% dFBS (C) with or without 5 mM DFMO (D) and DFMO plus 10 µM putrescine (DP) and serum deprived for 24 h prior to treatment. A, Bad Ser(P)112. Equal amounts of protein were separated by SDS-PAGE, transferred to membranes, and probed with either total or phospho-Bad (Ser112)-specific antibodies followed by a secondary antibody conjugated to horseradish peroxidase. A representative blot from three observations is shown. B, time course of Bad phosphorylation. Cells were exposed to TNF-{alpha}/CHX for 1.5, 3, and 6 h. Equal amounts of protein were separated by SDS-PAGE, transferred to membranes, and probed with antibodies recognizing either total or serine 112-phosphorylated Bad proteins. Membranes were stripped and probed with {beta}-actin-specific antibody. Representative blots from three independent observations are shown. C, inhibition of PP2A and Bad phosphorylation. Cells grown in control and DFMO containing media were pretreated with okadaic acid (PP2A inhibitor) for 1 h followed by TNF-{alpha}/CHX for 3 h. Equal amounts of protein were separated by SDS-PAGE, transferred to membranes, and probed with either total or phospho-Bad (Ser112)-specific antibodies followed by a secondary antibody conjugated to horseradish peroxidase. A representative blot from three observations is shown.

 
Activation of MEK-ERK has been shown to phosphorylate Bad on Ser112, leading to its sequestration in the inactive form in the cytoplasm (36, 37). PP2A has also been shown to regulate the mitogen-activated protein kinase pathway in response to growth factors and other stimuli (38). Our previous observations that constitutively active MEK1 expression protects cells from apoptosis in response to TNF-{alpha} (26), and the current findings that DFMO inhibits PP2A activity suggest that in addition to preventing the dephosphorylation of Bad, PP2A inhibition might also cause sustained activation of the MEK/ERK pathway. Therefore, we determined the effect of okadaic acid on ERK activation. Fig. 5A shows that okadaic acid increased basal as well as TNF-{alpha}-induced activation of ERKs in cells grown under control conditions. In agreement with our previous report, cells grown in the presence of DFMO had a significant increase in basal ERK activity. In cells grown in a DFMO containing medium supplemented with putrescine, ERK activation followed a pattern identical to that seen in those grown under control conditions. Fig. 5B shows the time course of ERK activation during the induction of apoptosis by TNF-{alpha}. In control cells, ERK specific activity increased significantly by 3.0 h followed by a gradual decrease to near basal levels. In contrast, ERK activation gradually and significantly increased with time during the entire 9 h in polyamine-depleted cells. Earlier we found that U0126, a specific inhibitor of MEK1, inhibited basal as well as TNF-{alpha}-induced ERK activation, significantly increased apoptosis, and abolished the protection conferred by the depletion of polyamines (26). Cells grown in control and DFMO containing media were treated with or without U0126 (10 µM) and TNF-{alpha} for 3 h, and cell extracts were analyzed by Western blotting for the detection of Bad Ser112 phosphorylation. Results depicted in Fig. 5C show that the inhibition of MEK1 significantly decreased Bad Ser112 phosphorylation in cells grown in both control and DFMO containing media, suggesting that MEK1/ERK mediates phosphorylation of Bad at Ser112.



View larger version (30K):
[in this window]
[in a new window]
  		FIG. 5.
PP2A mediates ERK activation and Bad Ser112 phosphorylation. A, ERK activity. Cells were grown for 3 days in DMEM, 5% dFBS in the presence or absence of 5 mM DFMO and 10 µM putrescine, serum starved, and treated with 10 ng/ml TNF-{alpha} and 25 µg/ml CHX with or without 15 nM okadaic acid for a period of 3 h. Cell lysates were assayed for ERK activity and resolved on SDS-PAGE, transferred to membranes, and probed with phospho-ELK antibody. The blot shown is representative of three independent observations. B, time-dependent ERK1/2 phosphorylation in response to TNF-{alpha}. IEC-6 cells grown to confluence for 3 days in DMEM, 5% dFBS with or without 5 mM DFMO, were serum deprived for 24 h before treatment with TNF-{alpha} (10 ng/ml) and cycloheximide (25 µg/ml) for 3, 6, and 9 h. 25 µg of protein was separated on SDS-PAGE for Western blot analysis using a phospho-ERK1/2 specific antibody. The membranes were stripped and subsequently probed with ERK1/2 antibody to determine the total protein level. Densitometric analysis is shown as specific activity from three blots. Values are mean ± S.E. *, p < 0.05) compared with 3.0 h TNF-{alpha} treated; {dagger}, significantly higher (p < 0.05) compared with respective TNF-{alpha}-treated control. C, inhibition of MEK. Cells were grown for 3 days in DMEM, 5% dFBS in the presence or absence of 5 mM DFMO, serum starved, and treated with 10 ng/ml TNF-{alpha} and 25 µg/ml CHX with or without 10 µM U0126 for a period of 3 h. Cell lysates were resolved on SDS-PAGE, transferred to membranes, and probed with total and phospho-Bad Ser112 antibodies. The blot shown is representative of three independent observations.

 
PP2A Mediates Bcl-2 Phosphorylation—Recent studies have shown that phosphorylation of Bcl-2 at Ser70 increases its activity and anti-apoptotic effect and that mitochondrial PP2A regulates Bcl-2 phosphorylation (39-41). Therefore, the inhibition of PP2A might increase Bcl-2 phosphorylation in polyamine-depleted cells. Cells were grown in control medium and media containing DFMO or DFMO plus putrescine as described in earlier experiments. Cells grown in control medium were also treated with okadaic acid for 1 h and other groups were left untreated. Cell extracts from the above groups were immunoprecipitated using a Bcl-2-specific antibody, resolved on SDS-PAGE, and analyzed by Western blot to detect the levels of Bcl-2 phosphorylation at Ser70 using an antibody specifically recognizing Ser70-phosphorylated Bcl-2. Polyamine depletion increased Bcl-2 and Bcl-2 Ser(P)70 compared with the group of controls (Fig. 6A). Okadaic acid significantly increased Bcl-2 phosphorylation at Ser70 (about 2.0-fold). Bcl-2 phosphorylation at Ser70 decreased following TNF-{alpha} treatment (3 h) in control cells, and this decrease was prevented by okadaic acid (Fig. 6B). Fig. 6, C and D, depicts the densitometric analysis of the results shown in Fig. 6, A and B, respectively. These data suggest that PP2A regulates the phosphorylation of Bad and Bcl-2 proteins and thereby regulates the integrity of mitochondria and the subsequent downstream signaling events leading to apoptosis.

Inhibition of PP2A Prevents Release of Cytochrome c and Activation of Caspases-9 and -3—Because Bcl-2 and Bad regulate mitochondrial permeability transition, we determined whether the inhibition of PP2A prevents TNF-{alpha}-induced cytochrome c release and caspase-9 and caspase-3 activation (Fig. 7). Cells grown in control medium were treated with TNF-{alpha} in the presence of okadaic acid or fostriecin for 1 h, and those grown in medium containing DFMO were treated with TNF-{alpha} alone. All were stained with mitosensor dye to detect cytochrome c release. Mitosensor dye bound to cytochrome c in the cytoplasm exhibits green fluorescence, whereas its aggregation in mitochondria exhibits red fluorescence. A significantly higher proportion of TNF-{alpha}-treated cells had green fluorescence indicating the release of cytochrome c (Fig. 7A). Cells grown in the presence of DFMO, okadaic acid, or fostriecin had few cells stained green, indicating a significantly smaller change in mitochondrial permeability. Cytochrome c release from mitochondria activates apoptosis activating protein factor-mediated caspase-9 activation. Activated caspase-9 subsequently activates caspase-3, an executioner caspase causing apoptosis. TNF-{alpha} increased the activation of caspase-9 (Fig. 7B, lane 2) and caspase-3 (Fig. 7C, lane 2) in control cells as judged by the cleavage of procaspases into lower molecular weight products indicative of the active forms of the caspases. Okadaic acid almost completely prevented the TNF-{alpha}-induced activation of caspase-9 (Fig. 7B, lane 4), and caspase-3 (Fig. 7C, lane 4), analogous to cells grown in the presence of DFMO and treated with TNF-{alpha} (Fig. 7, B and C, lane 6).

PP2A Inhibition Prevents JNK Activation—Activation of N-terminal c-Jun kinase (JNK) mediates apoptosis through cytochrome c release, and sustained activation of ERKs by expression of constitutively active MEK1 prevents activation of JNK and subsequent release of cytochrome c (25, 26). Because the inhibition of PP2A increased ERK activation, we determined the effect of okadaic acid on JNK activation. Fig. 8 shows that inhibition of PP2A significantly decreased JNK activation in response to TNF-{alpha} as indicated by the levels of phosphorylated forms of JNK protein (phospho-JNK). JNK activation was completely prevented in cells grown in the presence of DFMO and treated with TNF-{alpha} (Fig. 8, lane 6), which is in agreement with our previous report (25). Total JNK protein was the same in all samples.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Polyamines have been shown to regulate various cellular functions such as proliferation, migration, and apoptosis. DFMO, a suicide inhibitor of ODC causes rapid depletion of intracellular putrescine (within 3-6 h) in intestinal epithelial cells. Depletion of spermidine occurs after 24 h and the levels of spermine decrease to 40% following the 96-h DFMO treatment (32). Reports from our laboratory have shown that proliferation, migration, and apoptosis are inhibited in cells grown in the presence of DFMO for 4 days (31, 42, 43). We have demonstrated that depletion of intracellular polyamines decreases apoptosis induced by camptothecin, {gamma}-irradiation, or TNF-{alpha} (24, 31). Polyamine depletion increases basal as well as TNF-{alpha}-induced ERK, AKT, and NF-{kappa}B activation leading to protection from apoptosis (25-27). Constitutively active MEK1 expression inhibited JNK activation, cytochrome c release, and apoptosis, which is similar to the effects of polyamine depletion. It is unlikely that polyamine depletion affected each of these pathways individually. The activity of each of these proteins is influenced by serine/threonine phosphorylation. In view of the above we hypothesize that polyamine depletion either activated anti-apoptotic proteins or deactivated proapoptotic proteins by altering their phosphorylation at serine/threonine residues.

Because multiple kinases are required for serine/threonine phosphorylation of ERKs, AKT, and I{kappa}B{alpha} (44, 45), we predicted that the common regulator could be a serine/threonine phosphatase. Tung et al. (28) as well as others (29) have reported that polyamines increase PP2A activity and that spermine is a more potent activator than spermidine or putrescine. PP2A activity was significantly reduced in polyamine-depleted cells compared with controls or cells grown in DFMO plus putrescine (Fig. 1B). Because tyrosine phosphorylation inactivates PP2A, we determined whether decreased activity correlated with its tyrosine phosphorylation. Cells grown in DFMO containing medium had significantly more tyrosine-phosphorylated PP2A compared with controls, and the levels of PP2A protein were the same in all three groups (Fig. 1A). Inhibition of PP2A by okadaic acid and fostriecin significantly reduced apoptosis in both groups of cultures in response to TNF-{alpha} (Fig. 2A). Transfection of cells with PP2A siRNA significantly decreased the expression of PP2A protein (Fig. 3A), enzyme activity (Fig. 3B), and apoptosis, indicated by decreased DNA fragmentation (Fig. 3C). Cells grown under conditions used for siRNA transfection, treated with fostriecin to inhibit PP2A activity rather than PP2A protein translation, were also protected against TNF-{alpha}-induced apoptosis similar to those transfected with PP2A siRNA. Decreased apoptosis in okadaic acid-treated cells grown under control conditions correlated well with increased tyrosine phosphorylation of PP2A (Fig. 2B). These results clearly indicate that PP2A regulates apoptosis in IEC-6 cells and that inhibition of PP2A in response to polyamine depletion might be responsible for the protection of these cells.



View larger version (33K):
[in this window]
[in a new window]
  		FIG. 6.
PP2A inhibition increases Bcl-2 Ser70 phosphorylation. A, cells were grown for 3 days in DMEM, 5% dFBS in the presence or absence of 5 mM DFMO and 10 µM putrescine and serum starved for 24 h. Cells grown in control medium were also treated with 15 nM okadaic acid for a period of 3 h before harvesting and preparing the lysates. Equal amounts of protein were separated by SDS-PAGE, transferred to membranes, and probed with either total or phospho-Bcl-2 (Ser70)-specific antibodies followed by a secondary antibody conjugated to horseradish peroxidase. A representative blot from three observations is shown. B, cells grown in control medium as mentioned above were treated with or without 10 ng/ml TNF-{alpha} and 25 µg/ml CHX and with or without 15 nM okadaic acid for a period of 3 h. Cell lysates were resolved on SDS-PAGE, transferred to membranes, and probed with total and phospho-Bcl-2 Ser70 antibodies. The blot shown is representative of three independent observations. Densitometric analyses of the results shown in A and B are shown as C and D, respectively. *, p < 0.05) compared with untreated control; {dagger}, significantly higher (p < 0.05) compared with both TNF-{alpha} treated and untreated cells. PUT, putrescine. C, control; D, DFMO; DP, DFMO + putrescine.

 
Proapoptotic Bad modulates the function of Bcl-2 by direct interaction through its BH3 domain (9). However, formation of such heterodimers is determined by the phosphorylation state of the Bad. Phosphorylated Bad associates with 14-3-3 in the cytoplasm, which permits Bcl-2 localization to the mitochondrial membrane. In its unphosphorylated state, Bad is targeted to the mitochondrial surface where it binds Bcl-2, preventing its anti-apoptotic activity and causing cytochrome c release. Phosphorylation of Ser112 and Ser136 creates binding sites for the interaction of Bad with 14-3-3 proteins (9), whereas phosphorylation of Ser155 triggers dissociation of Bad from Bcl-2 (46-49). Our results in Fig. 4A indicate that cells grown in DFMO had significantly higher levels of Bad phosphorylation at Ser112 compared with cells grown in control groups, suggesting that polyamine depletion either increases Bad phosphorylation or decreases its dephosphorylation. Because Ser112 phosphorylation of Bad is mitogen-activated protein kinase-dependent (36), and ERK1/2 are constitutively activated in polyamine-depleted cells (26), we determined the effect of okadaic acid on ERK1/2 activity. As anticipated, inhibition of PP2A significantly increased ERK activity both in the presence and absence of TNF-{alpha} (Fig. 5A). In cells grown in the presence of DFMO, okadaic acid further enhanced activation of ERK compared with controls (Fig. 5A). ERK specific activity significantly increased within 3 h of TNF-{alpha} treatment followed by a gradual decline within 9 h in cells grown under control conditions (Fig. 5B). However, cells grown in the presence of DFMO showed a gradual increase in the specific activities of ERKs throughout the 9 h (Fig. 5B). We have shown that time dependent decreases and increases in activities of ERKs correlated well with increased and decreased DNA fragmentation in control cells and polyamine-depleted cells, respectively (26). These results imply that the inhibition of PP2A allows sustained activation of ERKs, which might be responsible for the increased phosphorylation of Bad at Ser112. Inhibition of MEK1 by U0126 completely blocked Bad phosphorylation at Ser112 in response to okadaic acid in the presence and absence of TNF-{alpha} in both control and DFMO groups (Fig. 5C). Recently Chatfield and Estman (49) have shown that the inhibition of PP2A by okadaic acid induced the phosphorylation of ERK and Bcl-2. The above results indicate that the MEK/ERK pathway mediates Ser112 phosphorylation of Bad in IEC-6 cells, and the inhibition of PP2A in response to polyamine depletion prevents its dephosphorylation.



View larger version (51K):
[in this window]
[in a new window]
  		FIG. 7.
PP2A inhibition on cytochrome c release, caspase-9, and caspase-3 activation. A, cytochrome c release. Cells were grown for 3 days in DMEM, 5% dFBS in the presence or absence of 5 mM DFMO and serum starved for 24 h. Cells were treated with 10 ng/ml TNF-{alpha} and 25 µg/ml CHX. Cells grown in control medium were also treated with okadaic acid or fostriecin 1 h before TNF-{alpha}. After washing with Hanks' balanced salt solution, cells were processed as described under "Experimental Procedures" using the ApoAlert mitochondrial membrane sensor kit. A representative of six observations is shown. B, caspase-9. Cells were grown for 3 days in DMEM, 5% dFBS in the presence or absence of 5 mM DFMO and 10 µM putrescine (PUT), serum starved, and treated with or without 10 ng/ml TNF-{alpha} and 25 µg/ml CHX, and with or without 15 nM okadaic acid for a period of 3 h. Cell lysates were resolved on SDS-PAGE, transferred to membranes, and probed with caspase-9 antibody. C, caspase-3. Membranes for caspase-9 were stripped and probed with caspase-3 antibody. The blots shown are representative of three independent observations.

 



View larger version (41K):
[in this window]
[in a new window]
  		FIG. 8.
PP2A inhibition and JNK. Cells were grown for 3 days in DMEM, 5% dFBS in the presence or absence of 5 mM DFMO, serum starved, and treated with or without 10 ng/ml TNF-{alpha} and 25 µg/ml CHX in the presence or absence of 15 nM okadaic acid for a period of 3 h. Cell lysates were resolved on SDS-PAGE, transferred to membranes, and probed with total and phospho-JNK specific antibodies. A representative of three observations is shown.

 
The activity of Bcl-2 is modulated by dimerization with Bax and phosphorylation. The phosphorylation status of Bcl-2 has been used as a survival sensor during stress (41, 50). Under favorable conditions, Bcl-2 is phosphorylated, whereas in response to apoptotic insults, rapid dephosphorylation of Bcl-2 correlates with apoptosis. The serine residue at position 70 has been identified as the functional phosphorylation site in Bcl-2 responsible for its anti-apoptotic effects. In addition to phosphorylation at this single site, multisite phosphorylation of Bcl-2 occurs at Thr69, and Ser87 via the kinases ERK1 and ERK2. In view of the above, we investigated whether PP2A is involved in the regulation of Bcl-2 phosphorylation. Polyamine-depleted cells contained significantly higher levels of phospho-Ser70-Bcl-2 than controls (Fig. 6A). Polyamine depletion also increased the levels of Bcl-2 protein (Fig. 6A, lane 2), confirming our earlier report (51). TNF-{alpha} treatment decreased Bcl-2 phosphorylation, whereas pretreatment of cells with okadaic acid prevented the TNF-{alpha}-mediated down-regulation of Bcl-2 phosphorylation (Fig. 6B). These data indicate that PP2A regulates phosphorylation of Bcl-2 at Ser70, and that decreased PP2A activity in polyamine-depleted cells might be responsible for the higher levels of phosphorylated Bcl-2 and its anti-apoptotic activity. Increased Ser112 and Ser70 phosphorylation of Bad and Bcl-2, respectively, in response to okadaic acid and polyamine depletion suggest that PP2A plays a central role in the regulation of the mitochondrial pathway in these cells. As this predicts, okadaic acid and fostriecin significantly decreased TNF-{alpha}-induced cytochrome c release (Fig. 7A, compare panel 1 with 3 and 4). Furthermore, cytochrome c release from polyamine-depleted cells was also inhibited, approximately to the levels seen in cells treated with inhibitors of PP2A (Fig. 7A, compare panels 2 with 3 and 4).

Caspases, a group of cysteine proteases, cleave a number of different cellular substrates. They are synthesized as inactive precursors and are the effectors of cell death. Sequential activation of caspases results in cleavage of key target proteins leading to the dismantling of the cell. Data presented in Fig. 7 reaffirm that the inactivation of PP2A prevented the release of cytochrome c from mitochondria, which is evident by the almost complete prevention of caspase-9 activation (Fig. 7, B and C, lane 4). Because caspase-9 cleaves procaspase-3 and activates it, samples from the above experiment (Fig. 7B) were probed for caspase-3. Okadaic acid prevented TNF-{alpha}-induced caspase-3 activation in control cells compared with untreated cells (Fig. 7C). It is also apparent from our previous reports and current observations that polyamine depletion, analogous to okadaic acid, prevented release of cytochrome c and caspase-9-mediated caspase-3 activation in these cells (Fig. 7, A and B). Addition of exogenous putrescine to DFMO completely restored caspase-9, and caspase-3 activation, further confirming that these effects are because of depletion of polyamines and not to DFMO (Fig. 7, A and B).

Prevention of JNK activation either by SP600125 or expression of constitutively active MEK inhibited cytochrome c release and the subsequent activation of caspase-9 and caspase-3 in IEC-6 cells (16). Because okadaic acid produced effects similar to those observed upon prevention of JNK activation, we determined whether inhibition of PP2A also prevented JNK activation. As shown in Fig. 8, okadaic acid significantly decreased TNF-{alpha}-induced JNK activation (Fig. 8, fourth lane) without any change in the levels of JNK protein. These data together with those in Fig. 5A showing that okadaic acid activates ERKs indicate that PP2A probably regulates activation of JNK via MEK1.

As shown in Fig. 9, our previous results, and current observations indicate that the inhibition of PP2A in response to polyamine depletion increased ERK phosphorylation, which in turn increases phosphorylation of Bad at Ser112 and inactivates it and prevents dephosphorylation of Bad and Bcl-2 increasing anti-apoptotic activity. Modulation of Bad and Bcl-2 activities by the inhibition of PP2A decreases cytochrome c release from mitochondria and prevents the activation of caspases-9 and -3. Moreover, increased ERK activity following the inhibition of PP2A prevented the activation of proapoptotic JNK. Thus, polyamine depletion prevents apoptosis in IEC-6 cells by regulating serine/threonine phosphorylation of pro- and anti-apoptotic proteins. PP2A accounts for the observed increases or decreases in the anti- or proapoptotic proteins, respectively, in response to polyamine depletion.



View larger version (14K):
[in this window]
[in a new window]
  		FIG. 9.
A schematic representation of the PP2A-influenced apoptotic signaling pathway in polyamine-depleted IEC-6 cells. Polyamine depletion inhibits PP2A. Decreased PP2A activity sustains the phosphorylation of Bcl-2 and ERK1/2, increasing their activities. It also sustains the phosphorylation of Bad, decreasing its activity. ERK inhibits JNK, and all these effects act to decrease cytochrome c release and apoptosis. (+ and - refer to changes in activities and phosphorylated Bcl-2 and Bad.)

 

   FOOTNOTES
 
* This work was supported by NIDDK National Institutes of Health Grant DK-16505 and a Thomas A. Gerwin Endowment. 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. Back

{ddagger} To whom correspondence should be addressed: Dept. of Physiology, University of Tennessee Health Science Center, 894 Union Ave., Memphis, TN 38163. Tel.: 901-448-7168; Fax: 901-448-7126; E-mail: rray{at}physio1.utmem.edu.

1 The abbreviations used are: PP2A, serine/threonine protein phosphatase-2A; ODC, ornithine decarboxylase; DFMO, {alpha}-difluoromethylornithine; ERK, extracellular signal-regulated kinase; IEC, intestinal epithelial cell; dFBS, dialyzed fetal bovine serum; siRNA, small interfering RNA; DMEM, Dulbecco's modified Eagle's medium; CHX, cyclohexamide; JNK, c-Jun N-terminal kinase. Back


   ACKNOWLEDGMENTS
 
We sincerely acknowledge Greg Short and Danny Morse for help preparing the figures.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Garcia, A., Cayla, X., Guergnon, J., Dessauge, F., Hospital, V., Rebollo, M. P., Fleischer, A., and Rebollo, A. (2003) Biochimie (Paris) 85, 721-726
  2. Sim, A. T., and Scott, J. D. (1999) Cell Calcium 26, 209-217[CrossRef][Medline] [Order article via Infotrieve]
  3. VanHoof, C., and Goris, J. (2003) Biochim. Biophys. Acta 1640, 97-104[Medline] [Order article via Infotrieve]
  4. Janssens, V., and Goris, J. (2001) Biochem. J. 353, 417-439[CrossRef][Medline] [Order article via Infotrieve]
  5. Adams, J. M., and Cory, S. (2001) Trends Biochem. Sci. 26, 61-66[CrossRef][Medline] [Order article via Infotrieve]
  6. Green, D. R., and Reed, J. C. (1998) Science 281, 1309-1312[Abstract/Free Full Text]
  7. Gross, A., McDonnell, J. M., and Korsemeyer, S. J. (1999) Genes Dev. 13, 1899-1911[Free Full Text]
  8. Yang, E., Zha, J., Jockel, J., Boise, L. H., Thompson, C. B., and Korsemeyer, S. J. (1995) Cell 80, 285-291[CrossRef][Medline] [Order article via Infotrieve]
  9. Zha, J., Harada, H., Yang, E., Jockel, J., and Korsemeyer, S. J. (1996) Cell 87, 619-628[CrossRef][Medline] [Order article via Infotrieve]
  10. Seiler, N., Delcros, J. G., and Moulinoux, J. P. (1996) Int. J. Biochem. Cell Biol. 28, 843-861[CrossRef][Medline] [Order article via Infotrieve]
  11. Persson, L., Svensson, F., and Lovkist-Wallstrom, E. (1996) in Polyamines in Cancer: Basic Mechanisms and Clinical Approaches (Nishioka, K., ed) pp. 19-43, Landis, Austin, TX
  12. Marton, L. J., and Pegg, A. (1995) Annu. Rev. Pharmacol. Toxicol. 35, 55-91[CrossRef][Medline] [Order article via Infotrieve]
  13. Kramer, D. L., Fogel, P. M., Diegelman, P., Cooley, J. M., Bernacki, R. J., McManis, J. S., Bergeron, R. J., and Porter, C. W. (1997) Cancer Res. 57, 5521-5527[Abstract/Free Full Text]
  14. Bernstein, H. G., and Muller, M. (1999) Prog. Neurobiol. 57, 485-505[CrossRef][Medline] [Order article via Infotrieve]
  15. Schipper R. G., Penning, L. C., and Verhofstad, A. A. (2000) Semin. Cancer Biol. 10, 55-68[CrossRef][Medline] [Order article via Infotrieve]
  16. Tewari, M., Hamid, Q. A., Tuncay, O. C., and Tewari, D. S. (1998) Oral Oncol. 34, 538-542[CrossRef][Medline] [Order article via Infotrieve]
  17. Monti, M. G., Ghiaroni, S., Pernecco, L., Barbieri, D., Marverti, G., and Franceschi, C. (1998) Life Sci. 62, 799-806[CrossRef][Medline] [Order article via Infotrieve]
  18. Noda, T., Iwakiri, R., Fujimoto, K., Matsuo, S., and Aw, T. Y. (1998) Am. J. Physiol. 274, G270-G276[Medline] [Order article via Infotrieve]
  19. Lin, C. K., Zou, H. Y., Kaptein, J. S., Yen, C. F., Kalunta, C. I., Nguyen, T. T., Park, E., and Lad, P. M. (1997) Exp. Cell Res. 237, 231-241[CrossRef][Medline] [Order article via Infotrieve]
  20. Penning, L. C., Schipper, R. G., Vercammen, D., Verhofstad, A. A., Denecker, T., Beyaert, R., and Vandenabeele, P. (1998) Cytokine 10, 423-431[CrossRef][Medline] [Order article via Infotrieve]
  21. Wang, J. Y., and Johnson, L. R. (1991) Gastroenterology 100, 333-343[Medline] [Order article via Infotrieve]
  22. McCormack, S. A., Viar, M. J., and Johnson, L. R. (1992) Am. J. Physiol. 263, G426-G435[Medline] [Order article via Infotrieve]
  23. Ray, R. M., Viar, M. J., Yuan, Q., and Johnson, L. R. (2000) Am. J. Physiol. 278, C480-C489
  24. Deng, W., and Johnson, L. R. (April 28, 2005) Am. J. Physiol. 10.1152/ajpgi.00564.2004[Abstract/Free Full Text]
  25. Bhattacharya, S., Ray, R. M., Viar, M. J., and Johnson, L. R. (2003) Am. J. Physiol. 285, G980-G991
  26. Bhattacharya, S., Ray, R. M., and Johnson, L. R. (2004) Am. J. Physiol. 286, G479-G490
  27. Bhattacharya, S., Ray, R. M., and Johnson, L. R. (2005) Apoptosis 10, 759-776[CrossRef][Medline] [Order article via Infotrieve]
  28. Tung, H. Y., Pelech, S., Fisher, M. J., Pogson, C. I., and Cohen, P. (1985) Eur. J. Biochem. 149, 305-313[Medline] [Order article via Infotrieve]
  29. Cornwell, T., Mehta, P., and Shenolikar, S. (1986) J. Cyclic Nucleotide Protein Phosphor. Res. 11, 373-382[Medline] [Order article via Infotrieve]
  30. Quaroni, A., Wands, J., Trelstad, R. L., and Isselbacher, K. J. (1979) J. Cell Biol. 80, 248-265[Abstract/Free Full Text]
  31. Ray, R. M., Viar, M. J., Yuan, Q., and Johnson, L. R. (2000) Am. J. Physiol. 278, C480-C489
  32. McCormack, S. A., Viar, M. J., and Johnson, L. R. (1993) Am. J. Physiol. 264, G367-G374[Medline] [Order article via Infotrieve]
  33. Favre, B., Turowski, P., and Hemmings, B. A. (1997) J. Biol. Chem. 272, 13856-13863[Abstract/Free Full Text]
  34. Walsh, A. H., Cheng, A., and Honkaneu, R. E. (1997) FEBS Lett. 416, 230-234[CrossRef][Medline] [Order article via Infotrieve]
  35. Lewy, D. S., Gauss, C. M., Soenen, D. R., and Boger, D. L. (2002) Curr. Med. Chem. 22, 2005-2032
  36. Scheid, M. P., Schubert, K. M., and Duronio, V. (1999) J. Biol. Chem. 274, 1108-1113[Abstract/Free Full Text]
  37. Fang, X., Yu, S., Eder, A., Mao, M., Bast, R. C., Jr., Boyd, D., and Millis, G. B. (1999) Oncogene 18, 6635-6640[CrossRef][Medline] [Order article via Infotrieve]
  38. Alessi, D. R., Gomez, N., Moorhead, G., Lewis, T., Keyse, S. M., and Cohen, P. (1995) Curr. Biol. 5, 283-295[CrossRef][Medline] [Order article via Infotrieve]
  39. May, W. S., Tyler, P. G., Ito, T., Armstrong, D. K., Qatsha, K. A., and Davidson, N. E. (1994) J. Biol. Chem. 269, 26865-26870[Abstract/Free Full Text]
  40. Ito, T., Deng, X., Carr, B., and May, W. S. (1997) J. Biol. Chem. 272, 11671-11673[Abstract/Free Full Text]
  41. Ruvolo, P. P., Deng, X., Ito, T., Carr, B. K., and May, W. S. (1999) J. Biol. Chem. 274, 20296-20300[Abstract/Free Full Text]
  42. Ray, R. M., Zimmerman, B. J., McCormack, S. A., Patel, T. B., and Johnson, L. R. (1999) Am. J. Physiol. 276, C684-C691[Medline] [Order article via Infotrieve]
  43. Ray, R. M., McCormack, S. A., Covington, C., Viar, M. J., Johnson, L. R., and Zheng, Y. (2003) J. Biol. Chem. 278, 13039-13046[Abstract/Free Full Text]
  44. Zolneirowicz, S. (2000) Biochem. Pharmacol. 60, 1225-1235[CrossRef][Medline] [Order article via Infotrieve]
  45. Tan, Y., Demeter, M. R., Ruan, H., and Comb, M. J. (2000) J. Biol. Chem. 275, 25864-25869
  46. Ayllon, V., Martinez, A., Garcia, A., Cayla, X., and Rebello, A. (2000) EMBO J. 19, 2237-2246[CrossRef][Medline] [Order article via Infotrieve]
  47. Chiang, C. W., Kanies, C., Kim, K. W., Fang, W. B., Parkhurst, C., Xie, M., Henry, T., and Yang, E. (2003) Mol. Cell. Biol. 23, 6350-6362[Abstract/Free Full Text]
  48. Wang, H. G., Pathan, N., Ethell, I. M., Krajewaski, S., Yamaguchi, Y., Shibasaki, F., Bobo, T., Franke, T. F., and Reed, J. C. (1999) Science 284, 339-343[Abstract/Free Full Text]
  49. Chatfield, K., and Estmal, A. (2004) Biochem. Biophys. Res. Commun. 323, 1313-1320[CrossRef][Medline] [Order article via Infotrieve]
  50. Ruvolo, P. P., Deng, X., and May, W. S. (2001) Leukemia 15, 515-522[CrossRef][Medline] [Order article via Infotrieve]
  51. Yuan, Q., Ray, R. M., and Johnson, L. R. (2002) Am. J. Physiol. 282, C1290-C1297

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Cardiovasc ResHome page
G. Kewalramani, P. Puthanveetil, F. Wang, M. S. Kim, S. Deppe, A. Abrahani, D. S. Luciani, J. D. Johnson, and B. Rodrigues
AMP-activated protein kinase confers protection against TNF-{alpha}-induced cardiac cell death
Cardiovasc Res, June 12, 2009; (2009) cvp166v2.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
R. K. Dagda, R. A. Merrill, J. T. Cribbs, Y. Chen, J. W. Hell, Y. M. Usachev, and S. Strack
The Spinocerebellar Ataxia 12 Gene Product and Protein Phosphatase 2A Regulatory Subunit B{beta}2 Antagonizes Neuronal Survival by Promoting Mitochondrial Fission
J. Biol. Chem., December 26, 2008; 283(52): 36241 - 36248.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
B. S. Marasa, L. Xiao, J. N. Rao, T. Zou, L. Liu, J. Wang, E. Bellavance, D. J. Turner, and J.-Y. Wang
Induced TRPC1 expression increases protein phosphatase 2A sensitizing intestinal epithelial cells to apoptosis through inhibition of NF-{kappa}B activation
Am J Physiol Cell Physiol, May 1, 2008; 294(5): C1277 - C1287.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
S. Jin, R. M. Ray, and L. R. Johnson
TNF-{alpha}/cycloheximide-induced apoptosis in intestinal epithelial cells requires Rac1-regulated reactive oxygen species
Am J Physiol Gastrointest Liver Physiol, April 1, 2008; 294(4): G928 - G937.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
J. A. Lee and D. C. Pallas
Leucine Carboxyl Methyltransferase-1 Is Necessary for Normal Progression through Mitosis in Mammalian Cells
J. Biol. Chem., October 19, 2007; 282(42): 30974 - 30984.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
F. J. Barreyro, S. Kobayashi, S. F. Bronk, N. W. Werneburg, H. Malhi, and G. J. Gores
Transcriptional Regulation of Bim by FoxO3A Mediates Hepatocyte Lipoapoptosis
J. Biol. Chem., September 14, 2007; 282(37): 27141 - 27154.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
V. Yanamadala, H. Negoro, L. Gunaratnam, T. Kong, and B. M. Denker
G{alpha}12 Stimulates Apoptosis in Epithelial Cells through JNK1-mediated Bcl-2 Degradation and Up-regulation of I{kappa}B{alpha}
J. Biol. Chem., August 17, 2007; 282(33): 24352 - 24363.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
C. Y. Chung, J. B. Koprich, S. Endo, and O. Isacson
An Endogenous Serine/Threonine Protein Phosphatase Inhibitor, G-Substrate, Reduces Vulnerability in Models of Parkinson's Disease
J. Neurosci., August 1, 2007; 27(31): 8314 - 8323.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
N. Bhasin, S. R. Cunha, M. Mudannayake, M. S. Gigena, T. B. Rogers, and P. J. Mohler
Molecular basis for PP2A regulatory subunit B56{alpha} targeting in cardiomyocytes
Am J Physiol Heart Circ Physiol, July 1, 2007; 293(1): H109 - H119.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
M.-J. Hsu, C. Y. Hsu, B.-C. Chen, M.-C. Chen, G. Ou, and C.-H. Lin
Apoptosis Signal-Regulating Kinase 1 in Amyloid {beta} Peptide-Induced Cerebral Endothelial Cell Apoptosis
J. Neurosci., May 23, 2007; 27(21): 5719 - 5729.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
R. M. Ray, H. Guo, M. Patel, S. Jin, S. Bhattacharya, and L. R. Johnson
Role of myosin regulatory light chain and Rac1 in the migration of polyamine-depleted intestinal epithelial cells
Am J Physiol Gastrointest Liver Physiol, April 1, 2007; 292(4): G983 - G995.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. ProteomicsHome page
W. Liu, A. M. Silverstein, H. Shu, B. Martinez, and M. C. Mumby
A Functional Genomics Analysis of the B56 Isoforms of Drosophila Protein Phosphatase 2A
Mol. Cell. Proteomics, February 1, 2007; 6(2): 319 - 332.
[Abstract] [Full Text] [PDF]

Home page
 GutHome page
L Frelin, E D Brenndorfer, G Ahlen, M Weiland, C Hultgren, M Alheim, H Glaumann, B Rozell, D R Milich, J G Bode, et al.
The hepatitis C virus and immune evasion: non-structural 3/4A transgenic mice are resistant to lethal tumour necrosis factor {alpha} mediated liver disease
Gut, October 1, 2006; 55(10): 1475 - 1483.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
X. Li, M. G. Schwacha, I. H. Chaudry, and M. A. Choudhry
A role of PP1/PP2A in mesenteric lymph node T cell suppression in a two-hit rodent model of alcohol intoxication and injury
J. Leukoc. Biol., March 1, 2006; 79(3): 453 - 462.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
280/35/31091    most recent
M503041200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
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 arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ray, R. M.
Right arrow Articles by Johnson, L. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ray, R. M.
Right arrow Articles by Johnson, L. R.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2005 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement