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

Identification of phosphorylation sites on caspase-8 and caspase-3. (A) Active phosphorylated p38-MAPK immunoprecipitates from freshly isolated neutrophils were incubated with [γ-32P]ATP and recombinant procaspase-8 or procaspase-3. The proteins were separated by SDS–gel electrophoresis, and the separated proteins were digested in situ with trypsin. The obtained phosphopeptides were separated on cellulose TLC glass plates (elect.), followed by ascending chromatography (chrom.). The indicated electrophoresis direction is from the anode to the cathode. The plates were analyzed in a PhosphorImager as well as exposed to an X-ray film. (B) The phosphopeptides from caspase-8 or caspase-3 were eluted from the TLC plates and subjected to two-dimensional phosphoamino acid analysis. The locations of the phosphoamino acids (top) were compared with that of phosphoamino acid markers (bottom) as follows: serine (S), threonine (T), and tyrosine (Y). The phosphopeptides obtained from A were subjected to amino acid sequencing (C), and the radioactivity released in each cycle was measured by spotting onto TLC plates and exposure on a Fuji image analyzer. The phosphorylated serine residues, 364 for caspase-8 (C) and 150 for caspase-3 (C), are indicated in the sequence of the putative fragment from caspase-8 and caspase-3, respectively. The illustrated phosphomapping is representative of three experiments.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2211830&req=5

fig4: Identification of phosphorylation sites on caspase-8 and caspase-3. (A) Active phosphorylated p38-MAPK immunoprecipitates from freshly isolated neutrophils were incubated with [γ-32P]ATP and recombinant procaspase-8 or procaspase-3. The proteins were separated by SDS–gel electrophoresis, and the separated proteins were digested in situ with trypsin. The obtained phosphopeptides were separated on cellulose TLC glass plates (elect.), followed by ascending chromatography (chrom.). The indicated electrophoresis direction is from the anode to the cathode. The plates were analyzed in a PhosphorImager as well as exposed to an X-ray film. (B) The phosphopeptides from caspase-8 or caspase-3 were eluted from the TLC plates and subjected to two-dimensional phosphoamino acid analysis. The locations of the phosphoamino acids (top) were compared with that of phosphoamino acid markers (bottom) as follows: serine (S), threonine (T), and tyrosine (Y). The phosphopeptides obtained from A were subjected to amino acid sequencing (C), and the radioactivity released in each cycle was measured by spotting onto TLC plates and exposure on a Fuji image analyzer. The phosphorylated serine residues, 364 for caspase-8 (C) and 150 for caspase-3 (C), are indicated in the sequence of the putative fragment from caspase-8 and caspase-3, respectively. The illustrated phosphomapping is representative of three experiments.

Mentions: We further examined the phosphorylation of recombinant caspase-8 and caspase-3 by active p38-MAPK immunoprecipitates from freshly isolated neutrophils in the presence of γ-[32P]ATP. Phosphorylated procaspase-8 and procaspase-3 were separated by gel electrophoresis and transferred to nitrocellulose membranes before tryptic digestion in situ and two-dimensional phosphopeptide mapping. As shown in Fig. 4 A, p38-MAPK phosphorylates caspase-8 on a single peptide fragment, whereas it phosphorylates caspase-3 on two peptide fragments. These different phosphopeptides were scraped off the TLC plates and subjected to two-dimensional phosphoamino acid analysis. All three phosphopeptides were phosphorylated on serine residues (Fig. 4 B). The phosphorylated serine residue in the phosphopeptide that originated from caspase-8 (Fig. 4 A) was located at position 3, as shown by an automated Edman degradation assay (Fig. 4 C). The phosphopeptides originating from caspase-3 (Fig. 4 A) had their phosphoserine residues located either at position 1 (weakest spot) or 3 (strongest spot) (Fig. 4 C). After compiling all the theoretical tryptic peptides derived from a complete digestion of procaspase-8 and procaspase-3 and comparing the possible tryptic peptides with homologous amino acid sequences in caspase-6, we concluded that the peptide from procaspase-8 containing Ser-364 and from procaspase-3 containing Ser-150 were likely to be the phosphorylated peptides. The peptide derived from procaspase-3, which contains Ser-150, is proceed by two arginine residues. A partial tryptic digestion of the arginines will generate two different phosphopeptides, as we detected in the TLC plates. Ser-364 and Ser-150 are present in regions that lie 11 amino acids upstream of the active sites of caspase-8 and caspase-3, respectively (Fig. 5 A). This region, containing either a Ser or a Thr residue, is conserved among different human caspases (1, 2, 4, 5, 7, and 9 but not 10 or 14). In addition, this region is also well conserved among other vertebrates (for example, caspase-6, -7, -8, -9, and -10 from Xenopus laevis) and invertebrates (for example, caspase-1 from Drosophila melanogaster). Interestingly, the three-dimensional structure of this region is also preserved among caspases (Fig. 5 B), implying a common regulatory region for caspases that is conserved among species.


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)

Identification of phosphorylation sites on caspase-8 and caspase-3. (A) Active phosphorylated p38-MAPK immunoprecipitates from freshly isolated neutrophils were incubated with [γ-32P]ATP and recombinant procaspase-8 or procaspase-3. The proteins were separated by SDS–gel electrophoresis, and the separated proteins were digested in situ with trypsin. The obtained phosphopeptides were separated on cellulose TLC glass plates (elect.), followed by ascending chromatography (chrom.). The indicated electrophoresis direction is from the anode to the cathode. The plates were analyzed in a PhosphorImager as well as exposed to an X-ray film. (B) The phosphopeptides from caspase-8 or caspase-3 were eluted from the TLC plates and subjected to two-dimensional phosphoamino acid analysis. The locations of the phosphoamino acids (top) were compared with that of phosphoamino acid markers (bottom) as follows: serine (S), threonine (T), and tyrosine (Y). The phosphopeptides obtained from A were subjected to amino acid sequencing (C), and the radioactivity released in each cycle was measured by spotting onto TLC plates and exposure on a Fuji image analyzer. The phosphorylated serine residues, 364 for caspase-8 (C) and 150 for caspase-3 (C), are indicated in the sequence of the putative fragment from caspase-8 and caspase-3, respectively. The illustrated phosphomapping is representative of three experiments.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Identification of phosphorylation sites on caspase-8 and caspase-3. (A) Active phosphorylated p38-MAPK immunoprecipitates from freshly isolated neutrophils were incubated with [γ-32P]ATP and recombinant procaspase-8 or procaspase-3. The proteins were separated by SDS–gel electrophoresis, and the separated proteins were digested in situ with trypsin. The obtained phosphopeptides were separated on cellulose TLC glass plates (elect.), followed by ascending chromatography (chrom.). The indicated electrophoresis direction is from the anode to the cathode. The plates were analyzed in a PhosphorImager as well as exposed to an X-ray film. (B) The phosphopeptides from caspase-8 or caspase-3 were eluted from the TLC plates and subjected to two-dimensional phosphoamino acid analysis. The locations of the phosphoamino acids (top) were compared with that of phosphoamino acid markers (bottom) as follows: serine (S), threonine (T), and tyrosine (Y). The phosphopeptides obtained from A were subjected to amino acid sequencing (C), and the radioactivity released in each cycle was measured by spotting onto TLC plates and exposure on a Fuji image analyzer. The phosphorylated serine residues, 364 for caspase-8 (C) and 150 for caspase-3 (C), are indicated in the sequence of the putative fragment from caspase-8 and caspase-3, respectively. The illustrated phosphomapping is representative of three experiments.
Mentions: We further examined the phosphorylation of recombinant caspase-8 and caspase-3 by active p38-MAPK immunoprecipitates from freshly isolated neutrophils in the presence of γ-[32P]ATP. Phosphorylated procaspase-8 and procaspase-3 were separated by gel electrophoresis and transferred to nitrocellulose membranes before tryptic digestion in situ and two-dimensional phosphopeptide mapping. As shown in Fig. 4 A, p38-MAPK phosphorylates caspase-8 on a single peptide fragment, whereas it phosphorylates caspase-3 on two peptide fragments. These different phosphopeptides were scraped off the TLC plates and subjected to two-dimensional phosphoamino acid analysis. All three phosphopeptides were phosphorylated on serine residues (Fig. 4 B). The phosphorylated serine residue in the phosphopeptide that originated from caspase-8 (Fig. 4 A) was located at position 3, as shown by an automated Edman degradation assay (Fig. 4 C). The phosphopeptides originating from caspase-3 (Fig. 4 A) had their phosphoserine residues located either at position 1 (weakest spot) or 3 (strongest spot) (Fig. 4 C). After compiling all the theoretical tryptic peptides derived from a complete digestion of procaspase-8 and procaspase-3 and comparing the possible tryptic peptides with homologous amino acid sequences in caspase-6, we concluded that the peptide from procaspase-8 containing Ser-364 and from procaspase-3 containing Ser-150 were likely to be the phosphorylated peptides. The peptide derived from procaspase-3, which contains Ser-150, is proceed by two arginine residues. A partial tryptic digestion of the arginines will generate two different phosphopeptides, as we detected in the TLC plates. Ser-364 and Ser-150 are present in regions that lie 11 amino acids upstream of the active sites of caspase-8 and caspase-3, respectively (Fig. 5 A). This region, containing either a Ser or a Thr residue, is conserved among different human caspases (1, 2, 4, 5, 7, and 9 but not 10 or 14). In addition, this region is also well conserved among other vertebrates (for example, caspase-6, -7, -8, -9, and -10 from Xenopus laevis) and invertebrates (for example, caspase-1 from Drosophila melanogaster). Interestingly, the three-dimensional structure of this region is also preserved among caspases (Fig. 5 B), implying a common regulatory region for caspases that is conserved among species.

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