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First published online 30 November 2004
doi: 10.1242/jcs.01573

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p38, but not p38ß, inhibits the phosphorylation and presence of c-FLIP_S in DISC to potentiate Fas-mediated caspase-8 activation and type I apoptotic signaling

Fraser Laboratories, Department of Medicine, McGill University Health Centre and Royal Victoria Hospital, Montreal, Quebec, H3A 1A1, Canada

View larger version (24K): [in a new window]   Fig. 1. Phosphorylation and activation of p38 and JNK precedes caspase-8 induction in Fas-mediated apoptosis in Jurkat cells. Following incubation with Fas-mAb, cells were either stained with annexin-V to detect apoptosis or were lysed and subjected to immunoblot analysis. (A) Fas-induced apoptosis was detectable by 4 hours, increased with time and was maximal by 24 hours. (B) Time-dependent increase occurred in the phosphorylation of p38 (blot 1) and JNK (blot 3) in the absence of any change in the total levels of p38 (blot 2) and JNK (blot 4). (C) The in vitro activity of each kinase was assessed following immunoprecipitation with the respective phosphospecific antibodies. Increased phosphorylation of p38 correlated with its ability to phosphorylate ATF-2 in vitro (blot 1) and of JNK with its ability to phosphorylate JUN (blot 2). (D) The formation of significant amounts of cleaved fragments of caspase-8 (p44/43) and p16 was detectable only beyond 2 hours.

View larger version (23K): [in a new window]   Fig. 2. Specificity of the p38 and JNK inhibitors. (A) Cells were incubated with Fas-mAb for 1 hour in the absence (lane 2) or presence of PD169316 (lane 3), SP600125 (lane 4) or both (lane 5). Fas-induced increase in p38 activity was inhibited by PD169316 but not SP600125. (B) Phosphorylation of JUN by JNK was inhibited by SP600125 but was unaffected by PD169316. Inhibition of both kinases was observed only in cells treated with both inhibitors.

View larger version (20K): [in a new window]   Fig. 3. Effect of the inhibition of p38 and JNK on Fas-mediated apoptosis. Jurkat cells were incubated with Fas-mAb (150 ng/ml) in the absence and presence (15 µM) of SP600125 and/or p38 inhibitors for 4 hours. (A) Fas-signaled apoptosis (lane 2) was enhanced 2.8-fold by PD169316 (lane 3) and 4-fold by SP600125 (lane 4). (B) The reduction in mitochondrial membrane potential (m) in cells undergoing Fas-mediated apoptosis (lane 2) was enhanced by SP600125 (lane 4) but not PD169316 (lane 3). PD169316 suppressed the sensitizing effect of SP600125 (lane 5). These inhibitors by themselves (individually or together) did not trigger apoptosis or decrease of m (lanes 6-8). The sensitizing effect of SP600125 on Fas-induced apoptosis (panel C) and the reduction in m (panel D) was abrogated by two other p38 inhibitors SB203580 and SB202190 but not by the inactive derivative SB202474 (mean±s.e.m., n=12).

View larger version (17K): [in a new window]   Fig. 4. Effect of p38 and JNK inhibitors on Fas-mediated activation of caspases. (A) Fas-mediated activation of caspase-8 (lane 2) was increased to a greater extent in cells treated with SP600125 in the absence (lane 4) or presence of PD169316 (lane 5) than that seen with the latter alone (lane 5). (B) Caspase-9 activation was potentiated by SP600125 (lane 4). PD169316 inhibited caspase-9 activation in Fas-activated cells both in the absence (lane 2) and in presence of SP600126 (lane 5). (C) Caspase-3 activity was higher in Fas-activated cells in presence of SP600125 (lane 4) than PD169316 (lane 3). PD169316 blunted the potentiating effect of SP600125 (lane 5). (D) Immunoblot analysis demonstrating the effects of PD169316 and SP600125 on the formation of cleaved fragments of caspase-8 (blot 1), caspase-9 (blot 3) and caspase-3 (blot 5). Increased presence of cleaved caspase-9 and caspase-3 was detected in cells treated with SP600125 (lane 4), but not PD169316 alone (lane 3) or in the presence of the former (lane 5). The differential effects of these inhibitors occurring distal to caspase-8 were confirmed by the differences in cyt c release (blot 4) and PARP cleavage (blot 6) and the lack of a difference in the extent of cleavage of Bid into tBid (blot 2). (Data are representative of three experiments).

View larger version (11K): [in a new window]   Fig. 5. Fas-mediated apoptosis is associated with the activation of p38 and ß isoforms. (A) A pan anti-p38 antibody was used to immunoblot immunoprecipitated p38 and p38ß, by using their respective specific antibodies. Fas-activation did not affect the cellular level of these kinases. (B) Fas induced phosphorylation of p38 and p38ß was detected by immunoprecipitating phosphorylated p38 using phospho-specific anti-p-38 antibody and immunoblotting with antibodies specific for p38 (blot 1) or p38ß (blot 2). (C) The expression of p38 is selectively abrogated in si-p38 cells (top panel) whereas p38ß was suppressed only in si-p38ß cells (bottom panel) (D) Expression of JNK1 and JNK2 was decreased in si-JNK1/2 cells.

View larger version (40K): [in a new window]   Fig. 6. Effect of translational silencing of p38, p38ß and JNK1/2 on Fas-mediated apoptotic signaling. (A) Dot-plot analysis demonstrates the potentiation of Fas-mediated apoptosis in si-p38ß and si-JNK1/2 cells, and its suppression in si-p38 cells compared with that in si-C cells. Apoptotic cells are identified by an increase in annexin-V-FITC positive, PI negative cells (bottom right quadrants). (B) The percentage of cells with decreased m (region L) compared with that of cells with high or normal m (region H) was enhanced to a greater extent in si-JNK1/2 cells than in si-p38ß cells, but was decreased in si-p38 cells. (C and D) Fas-induced cleavage of procaspase-8, procaspase-9 and procaspase-3 was evident in si-C cells and was inhibited in si-p38 cell but enhanced in si-p38ß cells (compare lanes 3 and 4 with lane 2, blots 1,2,3, panel C) and in si-JNK cells (compare lanes 3 and 2, panel D). The changes in caspase-9 cleavage correlate with parallel differences in the cytosolic presence of cyt c.

View larger version (14K): [in a new window]   Fig. 7. Fas-induced apoptosis in Jurkat cells is potentiated by ectopically introduced Flag-tagged p38 and suppressed by the dominant negative variant p38-AGF. (A) Immunoblot analysis using anti-p38 antibody (lanes 1-3) and anti-Flag antibody (lanes 4-6) demonstrate the expression of transfected Flag-p38 (lanes 2,5) and Flag-p38-AGF (lanes 3,6). (B) Cells expressing Flag-p38 displayed threefold greater sensitivity to Fas-mediated apoptosis than the native Jurkat cells (30.6±1.1 vs 11.4±0.6% apoptotic cells, lanes 3 and 2). By contrast, Flag-p38-AGF expression rendered the cells resistant to Fas-activation (lane 4). (C) The decrease in mitochondrial membrane potential (m) in Jurkat cells (37±2.6%) was increased in cells expressing Flag-p38 (66±1.2%) but was suppressed by Flag-p38-AGF (10.7±0.7%). The transfected proteins did not affect the basal levels of apoptosis or m (lanes 1,5 and 6).

View larger version (24K): [in a new window]   Fig. 8. DISC composition is altered in p38 and JNK inhibited cells. (A) Lysates of native and Fas-activated cells in the absence and presence of PD169316 and/or SP600125 were immunoprecipitated with a non-agonistic anti-Fas antibody and analyzed for the presence of Fas-associated proteins. The increased presence of FADD in the DISC and the concomitant decrease in the presence of c-FLIPS, but not c-FLIPL, is seen in PD169316-treated native (lane 2) and Fas-activated cells (lane 6) (top panel). Similar changes were observed in presence of SP600125 in the native (lane 3) and Fas-activated (lane 7) cells. These inhibitors did not exert additive effects either in the native (lane 4) or in Fas-activated (lane 8) cells. (B) Presence of c-FLIPS, but not c-FLIPL, in DISC is increased and induced reciprocal changes in DISC-associated FADD and procaspase-8 in si-p38 cells but not in si-p38ß and si-JNK1/2 cells (top panel) in the absence any change in protein in the levels of the all the proteins tested (bottom panel). Data are representative of three separate experiments.

View larger version (30K): [in a new window]   Fig. 9. p38, but not p38ß is essential for maintaining c-FLIPS in non-phosphorylated state. Phosphorylation of c-FLIPS was determined by Pro Q diamond phosphoprotein gel stain following immunoprecipitation with anti-c-FLIP antibody. Phosphorylation of c-FLIPS is enhanced in si-p38, but inhibited in si-p38ß or si-JNK1/2 cells compared with that seen in native Jurkat cells (top panel). No change in the total c-FLIPS levels was observed in any of the cells (bottom panel). (B) Jurkat cells expressing control (si-C) or p38 siRNA (si-p38) and flag-p38 or flag-p38-AGF were metabolically labeled with sodium ortho[32P]phosphate. Phosphorylated c-FLIP was immunoprecipitated from the lysates with anti-c-FLIP antibody that recognizes both c-FLIPL and c-FLIPS, electrophoresed on SDS-polyacrylamide gels and subjected to autoradiographic analysis. Phosphorylation of c-FLIPS was higher in (si-p38, lane 4) than in cells expressing the dominant negative mutant flag-p38-AGF (lane 3) whereas minimal phosphorylation was seen in native Jurkat (lane 1) si-C (lane 2) and Flag-p38 (lane 5) cells. By contrast, c-FLIPL phosphorylation was unaffected by p38.

View larger version (26K): [in a new window]   Fig. 10. Effect of PD169316 on SP600125-induced sensitization of Fas-mediated apoptosis in Jurkat cells lacking p38, p38ß and JNK1/2. (A) Fas-mediated apoptosis was sensitized by PD169316 but inhibited by SP600125 in si-38 cells but only by SP600125 in si-p38ß cells, SP600125, but not PD169316 exerted the sensitizing effect. Both inhibitors (SP600125 > PD169316) potentiated Fas-mediated apoptosis in si-C cells. Fas-mediated apoptosis in si-JNK1/2 cells was comparable to that induced by SP600126 in si-C cells and was inhibited by PD169316. (B) The potentiating effect of SP600125 on the reduction in m in response to Fas-activation was abrogated in both si-p38 cells and si-p38ß cells, but not in si-C cells. The reduction in m in Fas-activated si-JNK1/2 cells was attenuated by. PD169316. (C,D,E) Fas-mediated activation of the three caspases in si-p38 cells was observed in presence, but not in absence of PD169316 and/or SP600125. Caspases 8 and 3 were activated to a greater extent in si-p38ß and si-JNK1/2 cells. Caspase-9 activation was lesser in si-p38ß cells and greater in si-JNK1/2 cells than that seen in si-C cells, and was suppressed by PD169316. The sensitizing effect of SP600125 on caspase-9 was markedly lower in si-p38 and si-p38ß cells than in si-C cells.

View larger version (32K): [in a new window]   Fig. 11. Effect of inhibition of p38 and JNK on cellular localization and/or biochemical properties Bcl-2 family of proteins in Jurkat cells undergoing Fas-mediated apoptosis. Aliquots containing equal amounts of protein from cytosolic (C) and mitochondrial (M) fractions or whole cell lysates (L) were subjected to SDS-PAGE and immunoblot analysis. (A) Fas-induced translocation of Bax from the cytosol to the mitochondria (compare blots 1 and 2) and tBid (blot 3) is inhibited by PD169316 (lane 3) but enhanced by SP600125 (lane 4). Mitochondrial targeting of Bax, but not tBid, was inhibited in presence of both inhibitors (lane 5). The presence of Bak in the mitochondria (blot 4) was unaffected under all experimental conditions. The loss of Bad phosphorylation at Ser112 (blot 5) and Ser155 (blot 7) in response to Fas-activation is inhibited by PD169316, but not SP600125. By contrast, Ser136 phosphorylation was inhibited in presence, but not absence of, SP600125 (blot 6). Total Bad remained unchanged (blot 8). In cells treated with both inhibitors there was a significant, but not complete reversal of Bad phosphorylation at Ser112, Ser136 and Ser155 (lane 5, blots 5, 6 and 7). Bcl-2 phosphorylation at Ser70 was decreased in Fas-activated cells in presence of PD169316, but not SP600125 (blot 9) in the absence of a change in total Bcl-2 level (blot 10). The quality of mitochondrial preparations was established by immunoblot analysis for the marker protein Tom-20 (blot 11). (B) Mitochondrial presence of Bax in response to Fas-activation was inhibited in si-p38 and si-p38ß, but not si-JNK1/2 cells (compare blots 1 and 2). Cleavage of Bid into tBid (blot 3) and the mitochondrial presence of tBid (blot 4) occurred in si-p38ß and si-JNK1/2 cells, but not si-p38 cells. Fas-induced reduction in Bad Ser112 and Ser155 phosphorylation was inhibited in both si-38 and si-p38ß cells (blots 5 and 7) whereas Ser136 phosphorylation was decreased uniquely in si-JNK1/2 cells (blot 6). No change in the total level of Bad was seen in these cells (blot 8). Bcl-2 Ser70 phosphorylation was inhibited in uniquely in si-p38 cells (blot 9) in the absence of any change in total Bcl-2 level (blot 10). Data are representative of three experiments.

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