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Proteins phosphorylated during stress-induced apoptosis are common targets for autoantibody production in patients with systemic lupus erythematosus.

Utz PJ, Hottelet M, Schur PH, Anderson P - J. Exp. Med. (1997)

Bottom Line: None of these phosphoproteins were included in precipitates prepared using sera from patients with diseases that are not associated with autoantibody production or using serum from rheumatoid arthritis patients.Serum from four patients precipitated a serine/threonine kinase from apoptotic cell lysates that phosphorylates proteins of 23-, 34-, and 46-kD in in vitro kinase assays.Our results suggest that proteins phosphorylated during apoptosis may be preferred targets for autoantibody production in patients with SLE.

View Article: PubMed Central - PubMed

Affiliation: Division of Tumor Immunology, Dana Farber Cancer Institute, Boston, Massachusetts, USA.

ABSTRACT
Proteins cleaved by interleukin-1 beta converting enzyme family proteases during apoptosis are common targets for autoantibody production in patients with systemic lupus erythematosus (SLE). We have tested the possibility that proteins phosphorylated in cells undergoing apoptosis are also targets for autoantibody production in patients with autoimmune disease. Sera from 9/12 patients containing antinuclear antibodies (10/12 meeting diagnostic criteria for SLE or a lupus overlap syndrome), precipitated new phosphoproteins from lysates derived from Jurkat T cells treated with apoptotic stimuli (i.e., Fas-ligation, gamma irradiation, ultraviolet irradiation), but not with an activation (i.e., CD3-ligation) stimulus. Sera derived from individual patients precipitated different combinations of seven distinct serine-phosphorylated proteins. None of these phosphoproteins were included in precipitates prepared using sera from patients with diseases that are not associated with autoantibody production or using serum from rheumatoid arthritis patients. Protein phosphorylation precedes, or is coincident with, the induction of DNA fragmentation, and is not observed when apoptosis is inhibited by overexpression of bcl-2. Serum from four patients precipitated a serine/threonine kinase from apoptotic cell lysates that phosphorylates proteins of 23-, 34-, and 46-kD in in vitro kinase assays. Our results suggest that proteins phosphorylated during apoptosis may be preferred targets for autoantibody production in patients with SLE.

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In vivo phosphorylation of pp46 correlates with the induction of apoptosis and is inhibited in Jurkat cells overexpressing bcl-2. Jurkat transformants (bcl-2, left) or Jurkat control transformants (neo, right)  were labeled with 32P-orthophosphate, subjected to the indicated apoptotic stimulus, solubilized in NP40 lysis buffer, and precipitated using serum derived from patient 7 before electrophoretic separation. (A) AntiFas treatment; (B) gamma irradiation; (C) UV irradiation. The relative  migration of molecular size markers in kilodaltons is indicated on the  right side of each panel. The time, in hours, from initial exposure to each  stimulus is indicated at the top of each lane.
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Figure 6: In vivo phosphorylation of pp46 correlates with the induction of apoptosis and is inhibited in Jurkat cells overexpressing bcl-2. Jurkat transformants (bcl-2, left) or Jurkat control transformants (neo, right) were labeled with 32P-orthophosphate, subjected to the indicated apoptotic stimulus, solubilized in NP40 lysis buffer, and precipitated using serum derived from patient 7 before electrophoretic separation. (A) AntiFas treatment; (B) gamma irradiation; (C) UV irradiation. The relative migration of molecular size markers in kilodaltons is indicated on the right side of each panel. The time, in hours, from initial exposure to each stimulus is indicated at the top of each lane.

Mentions: We next asked whether the phosphorylation of pp46 could be blocked by overexpression of the bcl-2 protein, which has been shown to efficiently block apoptosis induced by multiple apoptotic stimuli, including gamma and UV irradiation (32–35). In Fig. 6, Jurkat T cells stably transformed with either bcl-2 (left) or empty vector (right) were labeled with 32P-orthophosphate and subjected to Fas ligation, gamma irradiation, or UV irradiation. Cells were solubilized at the indicated times and lysates were precipitated using serum derived from patient 7. While phosphorylation of pp46 is rapidly induced in Jurkat (neo) control cells in response to gamma irradiation (Fig. 6 B, right), pp46 is absent from Jurkat (bcl-2) transformants treated with this same stimulus (Fig. 6 B, left). Qualitatively similar results are seen with UV irradiation (Fig. 6 C), although a small amount of pp46 is observed in Jurkat (bcl-2) transformants beginning at 4.5 h. Overexpression of bcl-2 effectively inhibited apoptosis in response to these triggers, as judged by the induction of DNA fragmentation (data not shown). In contrast, phosphorylation of pp46 after Fas ligation was relatively unaffected by overexpression of bcl-2 (Fig. 6 A). The induction of DNA fragmentation after Fas ligation was similarly unaffected by overexpression of bcl-2 in these cells (data not shown), supporting the correlation between phosphorylation of pp46 and the induction of apoptosis. A similar inhibitory effect of bcl-2 after gamma and UV irradiation but not anti-Fas treatment, on the phosphorylation of pp54, pp34, and pp17 (Fig. 1 A and Table 1) recognized by serum from patient 11, was also observed (data not shown). Taken together, these results demonstrate that the in vivo phosphorylation of all four autoantigens that were tested correlated with the induction of apoptosis, and is downstream of the inhibitory effects of bcl-2.


Proteins phosphorylated during stress-induced apoptosis are common targets for autoantibody production in patients with systemic lupus erythematosus.

Utz PJ, Hottelet M, Schur PH, Anderson P - J. Exp. Med. (1997)

In vivo phosphorylation of pp46 correlates with the induction of apoptosis and is inhibited in Jurkat cells overexpressing bcl-2. Jurkat transformants (bcl-2, left) or Jurkat control transformants (neo, right)  were labeled with 32P-orthophosphate, subjected to the indicated apoptotic stimulus, solubilized in NP40 lysis buffer, and precipitated using serum derived from patient 7 before electrophoretic separation. (A) AntiFas treatment; (B) gamma irradiation; (C) UV irradiation. The relative  migration of molecular size markers in kilodaltons is indicated on the  right side of each panel. The time, in hours, from initial exposure to each  stimulus is indicated at the top of each lane.
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Related In: Results  -  Collection

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Figure 6: In vivo phosphorylation of pp46 correlates with the induction of apoptosis and is inhibited in Jurkat cells overexpressing bcl-2. Jurkat transformants (bcl-2, left) or Jurkat control transformants (neo, right) were labeled with 32P-orthophosphate, subjected to the indicated apoptotic stimulus, solubilized in NP40 lysis buffer, and precipitated using serum derived from patient 7 before electrophoretic separation. (A) AntiFas treatment; (B) gamma irradiation; (C) UV irradiation. The relative migration of molecular size markers in kilodaltons is indicated on the right side of each panel. The time, in hours, from initial exposure to each stimulus is indicated at the top of each lane.
Mentions: We next asked whether the phosphorylation of pp46 could be blocked by overexpression of the bcl-2 protein, which has been shown to efficiently block apoptosis induced by multiple apoptotic stimuli, including gamma and UV irradiation (32–35). In Fig. 6, Jurkat T cells stably transformed with either bcl-2 (left) or empty vector (right) were labeled with 32P-orthophosphate and subjected to Fas ligation, gamma irradiation, or UV irradiation. Cells were solubilized at the indicated times and lysates were precipitated using serum derived from patient 7. While phosphorylation of pp46 is rapidly induced in Jurkat (neo) control cells in response to gamma irradiation (Fig. 6 B, right), pp46 is absent from Jurkat (bcl-2) transformants treated with this same stimulus (Fig. 6 B, left). Qualitatively similar results are seen with UV irradiation (Fig. 6 C), although a small amount of pp46 is observed in Jurkat (bcl-2) transformants beginning at 4.5 h. Overexpression of bcl-2 effectively inhibited apoptosis in response to these triggers, as judged by the induction of DNA fragmentation (data not shown). In contrast, phosphorylation of pp46 after Fas ligation was relatively unaffected by overexpression of bcl-2 (Fig. 6 A). The induction of DNA fragmentation after Fas ligation was similarly unaffected by overexpression of bcl-2 in these cells (data not shown), supporting the correlation between phosphorylation of pp46 and the induction of apoptosis. A similar inhibitory effect of bcl-2 after gamma and UV irradiation but not anti-Fas treatment, on the phosphorylation of pp54, pp34, and pp17 (Fig. 1 A and Table 1) recognized by serum from patient 11, was also observed (data not shown). Taken together, these results demonstrate that the in vivo phosphorylation of all four autoantigens that were tested correlated with the induction of apoptosis, and is downstream of the inhibitory effects of bcl-2.

Bottom Line: None of these phosphoproteins were included in precipitates prepared using sera from patients with diseases that are not associated with autoantibody production or using serum from rheumatoid arthritis patients.Serum from four patients precipitated a serine/threonine kinase from apoptotic cell lysates that phosphorylates proteins of 23-, 34-, and 46-kD in in vitro kinase assays.Our results suggest that proteins phosphorylated during apoptosis may be preferred targets for autoantibody production in patients with SLE.

View Article: PubMed Central - PubMed

Affiliation: Division of Tumor Immunology, Dana Farber Cancer Institute, Boston, Massachusetts, USA.

ABSTRACT
Proteins cleaved by interleukin-1 beta converting enzyme family proteases during apoptosis are common targets for autoantibody production in patients with systemic lupus erythematosus (SLE). We have tested the possibility that proteins phosphorylated in cells undergoing apoptosis are also targets for autoantibody production in patients with autoimmune disease. Sera from 9/12 patients containing antinuclear antibodies (10/12 meeting diagnostic criteria for SLE or a lupus overlap syndrome), precipitated new phosphoproteins from lysates derived from Jurkat T cells treated with apoptotic stimuli (i.e., Fas-ligation, gamma irradiation, ultraviolet irradiation), but not with an activation (i.e., CD3-ligation) stimulus. Sera derived from individual patients precipitated different combinations of seven distinct serine-phosphorylated proteins. None of these phosphoproteins were included in precipitates prepared using sera from patients with diseases that are not associated with autoantibody production or using serum from rheumatoid arthritis patients. Protein phosphorylation precedes, or is coincident with, the induction of DNA fragmentation, and is not observed when apoptosis is inhibited by overexpression of bcl-2. Serum from four patients precipitated a serine/threonine kinase from apoptotic cell lysates that phosphorylates proteins of 23-, 34-, and 46-kD in in vitro kinase assays. Our results suggest that proteins phosphorylated during apoptosis may be preferred targets for autoantibody production in patients with SLE.

Show MeSH
Related in: MedlinePlus