Limits...
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.

Show MeSH

Related in: MedlinePlus

Caspase-8 and caspase-3 are coimmunoprecipitated with p38-MAPK. Neutrophils were incubated with anti-Fas Ab for the indicated times and lysed. (A) Samples were immunoprecipitated with an anti–p38-MAPKα or an isotype-matched control (C) antibody and subsequently assessed by Western blotting. The blots were either analyzed with a mixture of two antibodies respectively directed against caspase-8 (both the proform, pC8, and the active form, C8) and caspase-3 (both the proform, pC3, and the active form, C3), sequentially stripped, and reprobed with antibodies against p38-MAPKα (p38α). (B) Samples were immunoprecipitated with an anti-phospho–p38-MAPK or an isotype-matched control (C) antibody and subsequently assessed by Western blotting. The blots were sequentially analyzed with the antibodies against caspase-8, caspase-3, phospho–p38-MAPK (P-p38), p38-MAPKα (p38α), and p38-MAPKδ (p38δ). The blots in A and B are representative of at least seven separate experiments.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2211830&req=5

fig1: Caspase-8 and caspase-3 are coimmunoprecipitated with p38-MAPK. Neutrophils were incubated with anti-Fas Ab for the indicated times and lysed. (A) Samples were immunoprecipitated with an anti–p38-MAPKα or an isotype-matched control (C) antibody and subsequently assessed by Western blotting. The blots were either analyzed with a mixture of two antibodies respectively directed against caspase-8 (both the proform, pC8, and the active form, C8) and caspase-3 (both the proform, pC3, and the active form, C3), sequentially stripped, and reprobed with antibodies against p38-MAPKα (p38α). (B) Samples were immunoprecipitated with an anti-phospho–p38-MAPK or an isotype-matched control (C) antibody and subsequently assessed by Western blotting. The blots were sequentially analyzed with the antibodies against caspase-8, caspase-3, phospho–p38-MAPK (P-p38), p38-MAPKα (p38α), and p38-MAPKδ (p38δ). The blots in A and B are representative of at least seven separate experiments.

Mentions: Initially, we detected procaspase-8 and procaspase-3 (Fig. 1 A), but not procaspase-9 (not depicted), in immunoprecipitates of total p38α-MAPK, the most abundant p38-MAPK isoform in neutrophils (30). We also specifically immunoprecipitated active p38-MAPK from anti–Fas-treated cells, which showed a time-dependent increase in association of active p38-MAPK with the active p20 subunits of caspase-8 and caspase-3, levels of which were low or undetectable at time zero (Fig. 1 B). In immunoprecipitates obtained with an agarose-conjugated, isotype-matched control antibody, we could not detect any caspase-8 or caspase-3 (Fig. 1, A and B, top two blots). These findings imply intimate and regulatory interactions between these caspases and active p38-MAPK.


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)

Caspase-8 and caspase-3 are coimmunoprecipitated with p38-MAPK. Neutrophils were incubated with anti-Fas Ab for the indicated times and lysed. (A) Samples were immunoprecipitated with an anti–p38-MAPKα or an isotype-matched control (C) antibody and subsequently assessed by Western blotting. The blots were either analyzed with a mixture of two antibodies respectively directed against caspase-8 (both the proform, pC8, and the active form, C8) and caspase-3 (both the proform, pC3, and the active form, C3), sequentially stripped, and reprobed with antibodies against p38-MAPKα (p38α). (B) Samples were immunoprecipitated with an anti-phospho–p38-MAPK or an isotype-matched control (C) antibody and subsequently assessed by Western blotting. The blots were sequentially analyzed with the antibodies against caspase-8, caspase-3, phospho–p38-MAPK (P-p38), p38-MAPKα (p38α), and p38-MAPKδ (p38δ). The blots in A and B are representative of at least seven separate experiments.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Caspase-8 and caspase-3 are coimmunoprecipitated with p38-MAPK. Neutrophils were incubated with anti-Fas Ab for the indicated times and lysed. (A) Samples were immunoprecipitated with an anti–p38-MAPKα or an isotype-matched control (C) antibody and subsequently assessed by Western blotting. The blots were either analyzed with a mixture of two antibodies respectively directed against caspase-8 (both the proform, pC8, and the active form, C8) and caspase-3 (both the proform, pC3, and the active form, C3), sequentially stripped, and reprobed with antibodies against p38-MAPKα (p38α). (B) Samples were immunoprecipitated with an anti-phospho–p38-MAPK or an isotype-matched control (C) antibody and subsequently assessed by Western blotting. The blots were sequentially analyzed with the antibodies against caspase-8, caspase-3, phospho–p38-MAPK (P-p38), p38-MAPKα (p38α), and p38-MAPKδ (p38δ). The blots in A and B are representative of at least seven separate experiments.
Mentions: Initially, we detected procaspase-8 and procaspase-3 (Fig. 1 A), but not procaspase-9 (not depicted), in immunoprecipitates of total p38α-MAPK, the most abundant p38-MAPK isoform in neutrophils (30). We also specifically immunoprecipitated active p38-MAPK from anti–Fas-treated cells, which showed a time-dependent increase in association of active p38-MAPK with the active p20 subunits of caspase-8 and caspase-3, levels of which were low or undetectable at time zero (Fig. 1 B). In immunoprecipitates obtained with an agarose-conjugated, isotype-matched control antibody, we could not detect any caspase-8 or caspase-3 (Fig. 1, A and B, top two blots). These findings imply intimate and regulatory interactions between these caspases and active p38-MAPK.

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