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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–dependent phosphorylations of procaspase-8 and procaspase-3 in intact cells. Neutrophils were incubated with anti-Fas Ab for the indicated times and lysed. (A) Samples were taken for Western blot analysis with an anti-phospho–p38-MAPK (P-p38) antibody. The blot shown is representative of at least eight separate experiments. (B) Alternatively, lysate samples were analyzed for IETDase (C8) and DEVDase (C3) activities (n = 6). To adjust for differences between blood batches, the caspase activities measured after 2 h were defined as 100%, and values at other time points were compared with that level. Caspase-3 immunoprecipitates were obtained from freshly isolated or 32P-labeled neutrophils after the indicated periods of exposure to anti-Fas Ab. (C) Unlabeled neutrophils were lysed, and the immunoprecipitates were immunoblotted with an anti–phospho-serine Ab, stripped, and reprobed with a mixture of anti–caspase-8 (detecting both the proform, pC8, and the active form) and anti–caspase-3 (detecting both the proform, pC3, and the active form) Abs and thereafter with an anti-phospho–p38-MAPK (P-p38) Ab. (D) The 32P-labeled neutrophils were lysed, and immunoprecipitates were analyzed by gel electrophoresis and blotted. The blots were developed with a PhosphorImager and subsequently analyzed with a mixture of the anti–caspase-8 and the anti–caspase-3 Abs, stripped, and reprobed with the anti-phospho–p38-MAPK Ab. The blots and the autoradiogram in C and D are representative of at least three separate experiments.
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fig2: p38-MAPK–dependent phosphorylations of procaspase-8 and procaspase-3 in intact cells. Neutrophils were incubated with anti-Fas Ab for the indicated times and lysed. (A) Samples were taken for Western blot analysis with an anti-phospho–p38-MAPK (P-p38) antibody. The blot shown is representative of at least eight separate experiments. (B) Alternatively, lysate samples were analyzed for IETDase (C8) and DEVDase (C3) activities (n = 6). To adjust for differences between blood batches, the caspase activities measured after 2 h were defined as 100%, and values at other time points were compared with that level. Caspase-3 immunoprecipitates were obtained from freshly isolated or 32P-labeled neutrophils after the indicated periods of exposure to anti-Fas Ab. (C) Unlabeled neutrophils were lysed, and the immunoprecipitates were immunoblotted with an anti–phospho-serine Ab, stripped, and reprobed with a mixture of anti–caspase-8 (detecting both the proform, pC8, and the active form) and anti–caspase-3 (detecting both the proform, pC3, and the active form) Abs and thereafter with an anti-phospho–p38-MAPK (P-p38) Ab. (D) The 32P-labeled neutrophils were lysed, and immunoprecipitates were analyzed by gel electrophoresis and blotted. The blots were developed with a PhosphorImager and subsequently analyzed with a mixture of the anti–caspase-8 and the anti–caspase-3 Abs, stripped, and reprobed with the anti-phospho–p38-MAPK Ab. The blots and the autoradiogram in C and D are representative of at least three separate experiments.

Mentions: Neutrophils were lysed, and cell debris was removed by centrifugation, as described previously (11). The remaining supernatant was precleared with protein-G plus agarose (Oncogene Research Products). Thereafter, 40 μl agarose conjugated with an anti-phospho–p38-MAPK (Thr180/Tyr182) IgG1 mAb (New England BioLabs, Inc.), an anti–p38-MAPK IgG mAb (Santa Cruz Biotechnology, Inc.), or, as controls, with anti–c-Myc IgG1 mAb (ClONTECH Laboratories, Inc.), anti-Fyn IgG1 Ab, or nonimmune anti–rabbit IgG (Santa Cruz Biotechnology, Inc.) were added to the samples. The samples were incubated under rotation at 4°C overnight. Alternatively, 20 μl agarose, conjugated with an anti–caspase-3 polyclonal IgG Ab (Santa Cruz Biotechnology, Inc.) was added to the samples, which were incubated as aforementioned for 2 h. The immunoprecipitates were washed four times with lysis buffer and these, or lysates of intact cells, were boiled in sample buffer (11), after which the proteins were separated by SDS-PAGE and electrophoretically transferred to nitrocellulose membranes. It is worth mentioning that in the anti–caspase-3 immunoprecipitates, we observed a low recovery of the active p20 kD of the caspases (Fig. 2). A possible explanation for this finding is a much larger abundance of procaspases resulting in a preferential immunoprecipitation of these proforms. The membranes were analyzed with an anti–caspase-8 polyclonal Ab (Chemicon or Santa Cruz Biotechnology, Inc.), an anti–caspase-3 polyclonal IgG Ab (BD Biosciences or Santa Cruz Biotechnology, Inc.), an anti-p38 MAPKα IgG Ab, a MAPKδ IgG mAb, or an HA IgG2a mAb (F-7) (Santa Cruz Biotechnology, Inc.), an anti–caspase-9 polyclonal IgG Ab (BD Biosciences), an anti–phospho-p38 MAPK (Thr180/Tyr182) IgG1 mAb (New England BioLabs, Inc.), or the anti–phospho-serine IgMκ mAb 16B4 (BIOMOL Research Laboratories, Inc.). Before immunoblotting, the membranes that contained radioactive labels were analyzed using a PhosphorImager. As indicated in the figure legends, certain blots were stripped and reprobed according to the instructions of the manufacturers.


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–dependent phosphorylations of procaspase-8 and procaspase-3 in intact cells. Neutrophils were incubated with anti-Fas Ab for the indicated times and lysed. (A) Samples were taken for Western blot analysis with an anti-phospho–p38-MAPK (P-p38) antibody. The blot shown is representative of at least eight separate experiments. (B) Alternatively, lysate samples were analyzed for IETDase (C8) and DEVDase (C3) activities (n = 6). To adjust for differences between blood batches, the caspase activities measured after 2 h were defined as 100%, and values at other time points were compared with that level. Caspase-3 immunoprecipitates were obtained from freshly isolated or 32P-labeled neutrophils after the indicated periods of exposure to anti-Fas Ab. (C) Unlabeled neutrophils were lysed, and the immunoprecipitates were immunoblotted with an anti–phospho-serine Ab, stripped, and reprobed with a mixture of anti–caspase-8 (detecting both the proform, pC8, and the active form) and anti–caspase-3 (detecting both the proform, pC3, and the active form) Abs and thereafter with an anti-phospho–p38-MAPK (P-p38) Ab. (D) The 32P-labeled neutrophils were lysed, and immunoprecipitates were analyzed by gel electrophoresis and blotted. The blots were developed with a PhosphorImager and subsequently analyzed with a mixture of the anti–caspase-8 and the anti–caspase-3 Abs, stripped, and reprobed with the anti-phospho–p38-MAPK Ab. The blots and the autoradiogram in C and D are representative of at least three separate experiments.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: p38-MAPK–dependent phosphorylations of procaspase-8 and procaspase-3 in intact cells. Neutrophils were incubated with anti-Fas Ab for the indicated times and lysed. (A) Samples were taken for Western blot analysis with an anti-phospho–p38-MAPK (P-p38) antibody. The blot shown is representative of at least eight separate experiments. (B) Alternatively, lysate samples were analyzed for IETDase (C8) and DEVDase (C3) activities (n = 6). To adjust for differences between blood batches, the caspase activities measured after 2 h were defined as 100%, and values at other time points were compared with that level. Caspase-3 immunoprecipitates were obtained from freshly isolated or 32P-labeled neutrophils after the indicated periods of exposure to anti-Fas Ab. (C) Unlabeled neutrophils were lysed, and the immunoprecipitates were immunoblotted with an anti–phospho-serine Ab, stripped, and reprobed with a mixture of anti–caspase-8 (detecting both the proform, pC8, and the active form) and anti–caspase-3 (detecting both the proform, pC3, and the active form) Abs and thereafter with an anti-phospho–p38-MAPK (P-p38) Ab. (D) The 32P-labeled neutrophils were lysed, and immunoprecipitates were analyzed by gel electrophoresis and blotted. The blots were developed with a PhosphorImager and subsequently analyzed with a mixture of the anti–caspase-8 and the anti–caspase-3 Abs, stripped, and reprobed with the anti-phospho–p38-MAPK Ab. The blots and the autoradiogram in C and D are representative of at least three separate experiments.
Mentions: Neutrophils were lysed, and cell debris was removed by centrifugation, as described previously (11). The remaining supernatant was precleared with protein-G plus agarose (Oncogene Research Products). Thereafter, 40 μl agarose conjugated with an anti-phospho–p38-MAPK (Thr180/Tyr182) IgG1 mAb (New England BioLabs, Inc.), an anti–p38-MAPK IgG mAb (Santa Cruz Biotechnology, Inc.), or, as controls, with anti–c-Myc IgG1 mAb (ClONTECH Laboratories, Inc.), anti-Fyn IgG1 Ab, or nonimmune anti–rabbit IgG (Santa Cruz Biotechnology, Inc.) were added to the samples. The samples were incubated under rotation at 4°C overnight. Alternatively, 20 μl agarose, conjugated with an anti–caspase-3 polyclonal IgG Ab (Santa Cruz Biotechnology, Inc.) was added to the samples, which were incubated as aforementioned for 2 h. The immunoprecipitates were washed four times with lysis buffer and these, or lysates of intact cells, were boiled in sample buffer (11), after which the proteins were separated by SDS-PAGE and electrophoretically transferred to nitrocellulose membranes. It is worth mentioning that in the anti–caspase-3 immunoprecipitates, we observed a low recovery of the active p20 kD of the caspases (Fig. 2). A possible explanation for this finding is a much larger abundance of procaspases resulting in a preferential immunoprecipitation of these proforms. The membranes were analyzed with an anti–caspase-8 polyclonal Ab (Chemicon or Santa Cruz Biotechnology, Inc.), an anti–caspase-3 polyclonal IgG Ab (BD Biosciences or Santa Cruz Biotechnology, Inc.), an anti-p38 MAPKα IgG Ab, a MAPKδ IgG mAb, or an HA IgG2a mAb (F-7) (Santa Cruz Biotechnology, Inc.), an anti–caspase-9 polyclonal IgG Ab (BD Biosciences), an anti–phospho-p38 MAPK (Thr180/Tyr182) IgG1 mAb (New England BioLabs, Inc.), or the anti–phospho-serine IgMκ mAb 16B4 (BIOMOL Research Laboratories, Inc.). Before immunoblotting, the membranes that contained radioactive labels were analyzed using a PhosphorImager. As indicated in the figure legends, certain blots were stripped and reprobed according to the instructions of the manufacturers.

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