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p38-MAPK signals survival by phosphorylation of caspase-8 and caspase-3 in human neutrophils.

Alvarado-Kristensson M, Melander F, Leandersson K, Rönnstrand L, Wernstedt C, Andersson T - J. Exp. Med. (2004)

Bottom Line: In in vitro experiments, immunoprecipitated active p38-MAPK phosphorylated and inhibited the activity of the active p20 subunits of caspase-8 and caspase-3.Phosphopeptide mapping revealed that these phosphorylations occurred on serine-364 and serine-150, respectively.Introduction of mutated (S150A), but not wild-type, TAT-tagged caspase-3 into primary neutrophils made the Fas-induced apoptotic response insensitive to p38-MAPK inhibition.

View Article: PubMed Central - PubMed

Affiliation: Division of Experimental Pathology, Lund University, U-MAS, Entrance 78, Floor 3, SE-205 02 Malmö, Sweden. maria.alvarado-kristensson@exppat.mas.lu.se

ABSTRACT
Neutrophil apoptosis occurs both in the bloodstream and in the tissue and is considered essential for the resolution of an inflammatory process. Here, we show that p38-mitogen-activated protein kinase (MAPK) associates to caspase-8 and caspase-3 during neutrophil apoptosis and that p38-MAPK activity, previously shown to be a survival signal in these primary cells, correlates with the levels of caspase-8 and caspase-3 phosphorylation. In in vitro experiments, immunoprecipitated active p38-MAPK phosphorylated and inhibited the activity of the active p20 subunits of caspase-8 and caspase-3. Phosphopeptide mapping revealed that these phosphorylations occurred on serine-364 and serine-150, respectively. Introduction of mutated (S150A), but not wild-type, TAT-tagged caspase-3 into primary neutrophils made the Fas-induced apoptotic response insensitive to p38-MAPK inhibition. Consequently, p38-MAPK can directly phosphorylate and inhibit the activities of caspase-8 and caspase-3 and thereby hinder neutrophil apoptosis, and, in so doing, regulate the inflammatory response.

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Mutation of Ser-150 abolishes the effect of the inhibition of p38-MAPK in vivo. (A) Active phosphorylated p38-MAPK immunoprecipitates from freshly isolated neutrophils were incubated with [γ-32P]ATP and recombinant wild-type TAT–caspase-3 or mutated TAT–caspase-3 (S150A). The phosphopeptide mapping was performed as in Fig. 4. The plates were exposed on a PhosphorImager as well as to film. The indicated electrophoresis direction is from the anode to the cathode. (B) Active phosphorylated p38-MAPK (P-p38) was immunoprecipitated and incubated with recombinant wild-type TAT–caspase-3 or mutated TAT-caspase-3 (S150A) as substrates in the presence (shaded bars) or absence (unshaded bars) of ATP. Thereafter, the activities of TAT–caspase-3 or TAT–caspase-3 (S150A) were measured separately. The results are presented as percentage of the activities found in samples depleted of ATP. The data are expressed as mean ± SD of five separate experiments. (C) Fas-induced caspase-3 activity in control cells or cells loaded with TAT–caspase-3 or TAT–caspase-3 (S150A) after 3 h after activation of the Fas-receptor. Unshaded bars represent cells treated with 20 μM SB203580 (n = 4–6). (D) The effect of SB203580 in cells loaded with TAT-fusion proteins (n = 4). (E) The Fas-treated cells were incubated for 4 h in the presence or absence of TAT-fusion protein and SB203580 and subsequently stained with acridine orange and ethidium bromide to assess their nuclear morphology. The effects are presented as percentage of untreated control cells (n = 4–6). (F) Neutrophils incubated in absence, as control (C) or presence of wild-type (WT) TAT-caspase-3 or (S150A) mutated (M) TAT–caspase-3. Samples were taken for Western blot analysis with an anti–HA-Ab (HA).
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fig6: Mutation of Ser-150 abolishes the effect of the inhibition of p38-MAPK in vivo. (A) Active phosphorylated p38-MAPK immunoprecipitates from freshly isolated neutrophils were incubated with [γ-32P]ATP and recombinant wild-type TAT–caspase-3 or mutated TAT–caspase-3 (S150A). The phosphopeptide mapping was performed as in Fig. 4. The plates were exposed on a PhosphorImager as well as to film. The indicated electrophoresis direction is from the anode to the cathode. (B) Active phosphorylated p38-MAPK (P-p38) was immunoprecipitated and incubated with recombinant wild-type TAT–caspase-3 or mutated TAT-caspase-3 (S150A) as substrates in the presence (shaded bars) or absence (unshaded bars) of ATP. Thereafter, the activities of TAT–caspase-3 or TAT–caspase-3 (S150A) were measured separately. The results are presented as percentage of the activities found in samples depleted of ATP. The data are expressed as mean ± SD of five separate experiments. (C) Fas-induced caspase-3 activity in control cells or cells loaded with TAT–caspase-3 or TAT–caspase-3 (S150A) after 3 h after activation of the Fas-receptor. Unshaded bars represent cells treated with 20 μM SB203580 (n = 4–6). (D) The effect of SB203580 in cells loaded with TAT-fusion proteins (n = 4). (E) The Fas-treated cells were incubated for 4 h in the presence or absence of TAT-fusion protein and SB203580 and subsequently stained with acridine orange and ethidium bromide to assess their nuclear morphology. The effects are presented as percentage of untreated control cells (n = 4–6). (F) Neutrophils incubated in absence, as control (C) or presence of wild-type (WT) TAT-caspase-3 or (S150A) mutated (M) TAT–caspase-3. Samples were taken for Western blot analysis with an anti–HA-Ab (HA).

Mentions: To directly test the role of caspase phosphorylation in the regulation of apoptosis in intact human neutrophils, we subcloned human procaspase-8 and procaspase-3 into TAT-HA vectors. TAT-HA–tagged caspase-8 exhibited no in vitro activity, probably due to a conformational change, whereas TAT-HA–tagged caspase-3 did. Consequently, we also prepared a TAT-HA–tagged mutated caspase-3 in which serine-150 was replaced with an alanine residue, and this protein was used together with the wild-type TAT-HA–tagged caspase-3. Despite overexposure, we could not detect any phosphorylation of the TAT-tagged mutated caspase-3 (Fig. 6 A). Furthermore, protease activity measurements of wild-type TAT-tagged caspase-3 and mutated TAT-tagged caspase-3 (S150A) revealed that, in contrast to wild type, the activity of the mutated caspase-3 is not modified by the presence of immunoprecipitated active p38-MAPK (Fig. 6 B). In vitro experiments revealed that caspase-3 (S150A) has a lower protease activity than wild-type caspase-3 (unpublished data). For this reason, and also to avoid any misinterpretations due to a difference in the loading of the two TAT-tagged proteins, we studied the effects of p38-MAPK inhibition (SB203580) on apoptosis in primary neutrophils preincubated with either wild-type or mutated caspase-3. The results show that caspase-3 activity is significantly increased by p38-MAPK inhibition in nontreated control cells and cells loaded with wild-type caspase-3, but not mutated caspase-3 (Fig. 6 C). The difference in protease activity between cells loaded with wild-type or mutated caspase-3 and incubated with the p38-MAPK inhibitor SB203580 is even clearer if we subtract the endogenous caspase-3 activity and express the activities as percentage of untreated cells (Fig. 6 D). These data nicely correlate with our findings that p38-MAPK inhibition (SB203580) increased the percentage of apoptotic cells (as revealed by acridine orange and ethidium bromide costaining) if they were preincubated with wild-type caspase-3, but not with the mutated caspase-3 (Fig. 6 E). These observations strongly support the idea that p38-MAPK counteracts neutrophil apoptosis by phosphorylation and inactivation of caspases.


p38-MAPK signals survival by phosphorylation of caspase-8 and caspase-3 in human neutrophils.

Alvarado-Kristensson M, Melander F, Leandersson K, Rönnstrand L, Wernstedt C, Andersson T - J. Exp. Med. (2004)

Mutation of Ser-150 abolishes the effect of the inhibition of p38-MAPK in vivo. (A) Active phosphorylated p38-MAPK immunoprecipitates from freshly isolated neutrophils were incubated with [γ-32P]ATP and recombinant wild-type TAT–caspase-3 or mutated TAT–caspase-3 (S150A). The phosphopeptide mapping was performed as in Fig. 4. The plates were exposed on a PhosphorImager as well as to film. The indicated electrophoresis direction is from the anode to the cathode. (B) Active phosphorylated p38-MAPK (P-p38) was immunoprecipitated and incubated with recombinant wild-type TAT–caspase-3 or mutated TAT-caspase-3 (S150A) as substrates in the presence (shaded bars) or absence (unshaded bars) of ATP. Thereafter, the activities of TAT–caspase-3 or TAT–caspase-3 (S150A) were measured separately. The results are presented as percentage of the activities found in samples depleted of ATP. The data are expressed as mean ± SD of five separate experiments. (C) Fas-induced caspase-3 activity in control cells or cells loaded with TAT–caspase-3 or TAT–caspase-3 (S150A) after 3 h after activation of the Fas-receptor. Unshaded bars represent cells treated with 20 μM SB203580 (n = 4–6). (D) The effect of SB203580 in cells loaded with TAT-fusion proteins (n = 4). (E) The Fas-treated cells were incubated for 4 h in the presence or absence of TAT-fusion protein and SB203580 and subsequently stained with acridine orange and ethidium bromide to assess their nuclear morphology. The effects are presented as percentage of untreated control cells (n = 4–6). (F) Neutrophils incubated in absence, as control (C) or presence of wild-type (WT) TAT-caspase-3 or (S150A) mutated (M) TAT–caspase-3. Samples were taken for Western blot analysis with an anti–HA-Ab (HA).
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fig6: Mutation of Ser-150 abolishes the effect of the inhibition of p38-MAPK in vivo. (A) Active phosphorylated p38-MAPK immunoprecipitates from freshly isolated neutrophils were incubated with [γ-32P]ATP and recombinant wild-type TAT–caspase-3 or mutated TAT–caspase-3 (S150A). The phosphopeptide mapping was performed as in Fig. 4. The plates were exposed on a PhosphorImager as well as to film. The indicated electrophoresis direction is from the anode to the cathode. (B) Active phosphorylated p38-MAPK (P-p38) was immunoprecipitated and incubated with recombinant wild-type TAT–caspase-3 or mutated TAT-caspase-3 (S150A) as substrates in the presence (shaded bars) or absence (unshaded bars) of ATP. Thereafter, the activities of TAT–caspase-3 or TAT–caspase-3 (S150A) were measured separately. The results are presented as percentage of the activities found in samples depleted of ATP. The data are expressed as mean ± SD of five separate experiments. (C) Fas-induced caspase-3 activity in control cells or cells loaded with TAT–caspase-3 or TAT–caspase-3 (S150A) after 3 h after activation of the Fas-receptor. Unshaded bars represent cells treated with 20 μM SB203580 (n = 4–6). (D) The effect of SB203580 in cells loaded with TAT-fusion proteins (n = 4). (E) The Fas-treated cells were incubated for 4 h in the presence or absence of TAT-fusion protein and SB203580 and subsequently stained with acridine orange and ethidium bromide to assess their nuclear morphology. The effects are presented as percentage of untreated control cells (n = 4–6). (F) Neutrophils incubated in absence, as control (C) or presence of wild-type (WT) TAT-caspase-3 or (S150A) mutated (M) TAT–caspase-3. Samples were taken for Western blot analysis with an anti–HA-Ab (HA).
Mentions: To directly test the role of caspase phosphorylation in the regulation of apoptosis in intact human neutrophils, we subcloned human procaspase-8 and procaspase-3 into TAT-HA vectors. TAT-HA–tagged caspase-8 exhibited no in vitro activity, probably due to a conformational change, whereas TAT-HA–tagged caspase-3 did. Consequently, we also prepared a TAT-HA–tagged mutated caspase-3 in which serine-150 was replaced with an alanine residue, and this protein was used together with the wild-type TAT-HA–tagged caspase-3. Despite overexposure, we could not detect any phosphorylation of the TAT-tagged mutated caspase-3 (Fig. 6 A). Furthermore, protease activity measurements of wild-type TAT-tagged caspase-3 and mutated TAT-tagged caspase-3 (S150A) revealed that, in contrast to wild type, the activity of the mutated caspase-3 is not modified by the presence of immunoprecipitated active p38-MAPK (Fig. 6 B). In vitro experiments revealed that caspase-3 (S150A) has a lower protease activity than wild-type caspase-3 (unpublished data). For this reason, and also to avoid any misinterpretations due to a difference in the loading of the two TAT-tagged proteins, we studied the effects of p38-MAPK inhibition (SB203580) on apoptosis in primary neutrophils preincubated with either wild-type or mutated caspase-3. The results show that caspase-3 activity is significantly increased by p38-MAPK inhibition in nontreated control cells and cells loaded with wild-type caspase-3, but not mutated caspase-3 (Fig. 6 C). The difference in protease activity between cells loaded with wild-type or mutated caspase-3 and incubated with the p38-MAPK inhibitor SB203580 is even clearer if we subtract the endogenous caspase-3 activity and express the activities as percentage of untreated cells (Fig. 6 D). These data nicely correlate with our findings that p38-MAPK inhibition (SB203580) increased the percentage of apoptotic cells (as revealed by acridine orange and ethidium bromide costaining) if they were preincubated with wild-type caspase-3, but not with the mutated caspase-3 (Fig. 6 E). These observations strongly support the idea that p38-MAPK counteracts neutrophil apoptosis by phosphorylation and inactivation of caspases.

Bottom Line: In in vitro experiments, immunoprecipitated active p38-MAPK phosphorylated and inhibited the activity of the active p20 subunits of caspase-8 and caspase-3.Phosphopeptide mapping revealed that these phosphorylations occurred on serine-364 and serine-150, respectively.Introduction of mutated (S150A), but not wild-type, TAT-tagged caspase-3 into primary neutrophils made the Fas-induced apoptotic response insensitive to p38-MAPK inhibition.

View Article: PubMed Central - PubMed

Affiliation: Division of Experimental Pathology, Lund University, U-MAS, Entrance 78, Floor 3, SE-205 02 Malmö, Sweden. maria.alvarado-kristensson@exppat.mas.lu.se

ABSTRACT
Neutrophil apoptosis occurs both in the bloodstream and in the tissue and is considered essential for the resolution of an inflammatory process. Here, we show that p38-mitogen-activated protein kinase (MAPK) associates to caspase-8 and caspase-3 during neutrophil apoptosis and that p38-MAPK activity, previously shown to be a survival signal in these primary cells, correlates with the levels of caspase-8 and caspase-3 phosphorylation. In in vitro experiments, immunoprecipitated active p38-MAPK phosphorylated and inhibited the activity of the active p20 subunits of caspase-8 and caspase-3. Phosphopeptide mapping revealed that these phosphorylations occurred on serine-364 and serine-150, respectively. Introduction of mutated (S150A), but not wild-type, TAT-tagged caspase-3 into primary neutrophils made the Fas-induced apoptotic response insensitive to p38-MAPK inhibition. Consequently, p38-MAPK can directly phosphorylate and inhibit the activities of caspase-8 and caspase-3 and thereby hinder neutrophil apoptosis, and, in so doing, regulate the inflammatory response.

Show MeSH
Related in: MedlinePlus