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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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Related in: MedlinePlus

p38-MAPK–induced phosphorylations of caspase-8 and caspase-3 in vitro. (A and B) Active phosphorylated p38-MAPK immunoprecipitates from freshly isolated neutrophils were incubated with [γ-32P]ATP and recombinant caspase-8 or -3 in the absence or presence of the p38-MAPK inhibitor SB203580. As controls, the same reaction was performed in the absence of caspases but in the presence of either purified proteins from E. coli transformed with an empty vector (C) or BSA. All blots were first developed with a PhosphorImager, cut, and analyzed with (A) the anti–caspase-8 (pC8 and C8) Ab or (B) the anti–caspase-3 (pC3 and C3) Ab, and, lastly, stripped and reprobed with the anti-phospho–p38-MAPK (P-p38) Ab. The illustrated autoradiograms and blots are representative of at least seven separate experiments. (C) Active phosphorylated p38-MAPK (P-p38) was immunoprecipitated and incubated with recombinant caspase-8 and caspase-3 as substrates, in the presence or absence of ATP and under the same conditions as aforementioned. Thereafter, the activities of caspase-8 and caspase-3 were measured separately. The results are presented as percentage of the activities found in samples incubated in the same way but with an immunoprecipitate obtained using an isotype-matched control antibody. The data are expressed as mean ± SEM of seven separate experiments. The substrates, recombinant procaspase-8 (pC8) in D and recombinant procaspase-3 (pC3) in E, were incubated in the presence of ATP and either the immunoprecipitated active phosphorylated p38-MAPK (P-p38) or an immunoprecipitate obtained using an isotype-matched control antibody (Control). Thereafter, the in vitro amounts of the procaspases (pC8 and pC3) and caspases (C8 and C3), after incubations in the presence of (D) immunoprecipitated active Fas (FasR; n = 3) or (E) active caspase-8 (n = 5), were analyzed by Western blotting.
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fig3: p38-MAPK–induced phosphorylations of caspase-8 and caspase-3 in vitro. (A and B) Active phosphorylated p38-MAPK immunoprecipitates from freshly isolated neutrophils were incubated with [γ-32P]ATP and recombinant caspase-8 or -3 in the absence or presence of the p38-MAPK inhibitor SB203580. As controls, the same reaction was performed in the absence of caspases but in the presence of either purified proteins from E. coli transformed with an empty vector (C) or BSA. All blots were first developed with a PhosphorImager, cut, and analyzed with (A) the anti–caspase-8 (pC8 and C8) Ab or (B) the anti–caspase-3 (pC3 and C3) Ab, and, lastly, stripped and reprobed with the anti-phospho–p38-MAPK (P-p38) Ab. The illustrated autoradiograms and blots are representative of at least seven separate experiments. (C) Active phosphorylated p38-MAPK (P-p38) was immunoprecipitated and incubated with recombinant caspase-8 and caspase-3 as substrates, in the presence or absence of ATP and under the same conditions as aforementioned. Thereafter, the activities of caspase-8 and caspase-3 were measured separately. The results are presented as percentage of the activities found in samples incubated in the same way but with an immunoprecipitate obtained using an isotype-matched control antibody. The data are expressed as mean ± SEM of seven separate experiments. The substrates, recombinant procaspase-8 (pC8) in D and recombinant procaspase-3 (pC3) in E, were incubated in the presence of ATP and either the immunoprecipitated active phosphorylated p38-MAPK (P-p38) or an immunoprecipitate obtained using an isotype-matched control antibody (Control). Thereafter, the in vitro amounts of the procaspases (pC8 and pC3) and caspases (C8 and C3), after incubations in the presence of (D) immunoprecipitated active Fas (FasR; n = 3) or (E) active caspase-8 (n = 5), were analyzed by Western blotting.

Mentions: We used an in vitro assay to confirm that caspase-8 and caspase-3 are direct substrates of p38-MAPK. Active p38-MAPK immunoprecipitated from freshly isolated neutrophils was incubated with recombinant hamster caspase-3 (not depicted) as well as human caspase-8 or caspase-3 (Fig. 3, A and B) in the presence of γ-[32P]ATP. Both the procaspases and the 20-kD monomers of the active heterodimers of all three caspases were phosphorylated by the active p38-MAPK in the absence but not in the presence of SB203580.


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)

p38-MAPK–induced phosphorylations of caspase-8 and caspase-3 in vitro. (A and B) Active phosphorylated p38-MAPK immunoprecipitates from freshly isolated neutrophils were incubated with [γ-32P]ATP and recombinant caspase-8 or -3 in the absence or presence of the p38-MAPK inhibitor SB203580. As controls, the same reaction was performed in the absence of caspases but in the presence of either purified proteins from E. coli transformed with an empty vector (C) or BSA. All blots were first developed with a PhosphorImager, cut, and analyzed with (A) the anti–caspase-8 (pC8 and C8) Ab or (B) the anti–caspase-3 (pC3 and C3) Ab, and, lastly, stripped and reprobed with the anti-phospho–p38-MAPK (P-p38) Ab. The illustrated autoradiograms and blots are representative of at least seven separate experiments. (C) Active phosphorylated p38-MAPK (P-p38) was immunoprecipitated and incubated with recombinant caspase-8 and caspase-3 as substrates, in the presence or absence of ATP and under the same conditions as aforementioned. Thereafter, the activities of caspase-8 and caspase-3 were measured separately. The results are presented as percentage of the activities found in samples incubated in the same way but with an immunoprecipitate obtained using an isotype-matched control antibody. The data are expressed as mean ± SEM of seven separate experiments. The substrates, recombinant procaspase-8 (pC8) in D and recombinant procaspase-3 (pC3) in E, were incubated in the presence of ATP and either the immunoprecipitated active phosphorylated p38-MAPK (P-p38) or an immunoprecipitate obtained using an isotype-matched control antibody (Control). Thereafter, the in vitro amounts of the procaspases (pC8 and pC3) and caspases (C8 and C3), after incubations in the presence of (D) immunoprecipitated active Fas (FasR; n = 3) or (E) active caspase-8 (n = 5), were analyzed by Western blotting.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2211830&req=5

fig3: p38-MAPK–induced phosphorylations of caspase-8 and caspase-3 in vitro. (A and B) Active phosphorylated p38-MAPK immunoprecipitates from freshly isolated neutrophils were incubated with [γ-32P]ATP and recombinant caspase-8 or -3 in the absence or presence of the p38-MAPK inhibitor SB203580. As controls, the same reaction was performed in the absence of caspases but in the presence of either purified proteins from E. coli transformed with an empty vector (C) or BSA. All blots were first developed with a PhosphorImager, cut, and analyzed with (A) the anti–caspase-8 (pC8 and C8) Ab or (B) the anti–caspase-3 (pC3 and C3) Ab, and, lastly, stripped and reprobed with the anti-phospho–p38-MAPK (P-p38) Ab. The illustrated autoradiograms and blots are representative of at least seven separate experiments. (C) Active phosphorylated p38-MAPK (P-p38) was immunoprecipitated and incubated with recombinant caspase-8 and caspase-3 as substrates, in the presence or absence of ATP and under the same conditions as aforementioned. Thereafter, the activities of caspase-8 and caspase-3 were measured separately. The results are presented as percentage of the activities found in samples incubated in the same way but with an immunoprecipitate obtained using an isotype-matched control antibody. The data are expressed as mean ± SEM of seven separate experiments. The substrates, recombinant procaspase-8 (pC8) in D and recombinant procaspase-3 (pC3) in E, were incubated in the presence of ATP and either the immunoprecipitated active phosphorylated p38-MAPK (P-p38) or an immunoprecipitate obtained using an isotype-matched control antibody (Control). Thereafter, the in vitro amounts of the procaspases (pC8 and pC3) and caspases (C8 and C3), after incubations in the presence of (D) immunoprecipitated active Fas (FasR; n = 3) or (E) active caspase-8 (n = 5), were analyzed by Western blotting.
Mentions: We used an in vitro assay to confirm that caspase-8 and caspase-3 are direct substrates of p38-MAPK. Active p38-MAPK immunoprecipitated from freshly isolated neutrophils was incubated with recombinant hamster caspase-3 (not depicted) as well as human caspase-8 or caspase-3 (Fig. 3, A and B) in the presence of γ-[32P]ATP. Both the procaspases and the 20-kD monomers of the active heterodimers of all three caspases were phosphorylated by the active p38-MAPK in the absence but not in the presence of SB203580.

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