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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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Autoantigen phosphorylation coincides with or precedes the  onset of DNA fragmentation in apoptotic Jurkat cells. Jurkat cells were  triggered to undergo apoptosis and harvested at the indicated times. Each  time point represents a total of 1 million cells. The DNA was prepared as  described in the Materials and Methods, separated on a 0.8% agarose gel  and visualized by staining with ethidium bromide before UV exposure.  (A) Anti-Fas treatment; (B) gamma irradiation; (C) UV irradiation; (D)  anti-CD3 treatment. The time, in hours, from initial exposure to each  stimulus is indicated at the top of each lane. The relative migration of  molecular size markers in kilobases is indicated on the right side of each  panel.
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Figure 3: Autoantigen phosphorylation coincides with or precedes the onset of DNA fragmentation in apoptotic Jurkat cells. Jurkat cells were triggered to undergo apoptosis and harvested at the indicated times. Each time point represents a total of 1 million cells. The DNA was prepared as described in the Materials and Methods, separated on a 0.8% agarose gel and visualized by staining with ethidium bromide before UV exposure. (A) Anti-Fas treatment; (B) gamma irradiation; (C) UV irradiation; (D) anti-CD3 treatment. The time, in hours, from initial exposure to each stimulus is indicated at the top of each lane. The relative migration of molecular size markers in kilobases is indicated on the right side of each panel.

Mentions: The results shown in Fig. 1 indicate that autoimmune sera preferentially precipitate proteins phosphorylated in response to Fas ligation. To determine whether these proteins are also phosphorylated during apoptosis triggered by stimuli other than Fas ligation, selected patient sera were used to precipitate 32P-labeled Jurkat lysates prepared from cells subjected to apoptotic stimuli or an activation stimulus for various times. This kinetic analysis reveals that phosphorylation of autoantigens is induced between 1 and 2.5 h after Fas ligation (Fig. 2 A), between 2.5 and 4.5 h after gamma irradiation (Fig. 2 B), and between 1 and 2.5 h after UV irradiation (Fig. 2 C). Individual autoantisera precipitate a similar cadre of phosphoproteins regardless of the apoptotic trigger. In contrast, ligation of the T cell receptor complex using a monoclonal antibody reactive with CD3, a stimulus that induces IL-2 production and enhances proliferation in these cells (data not shown), induced neither new protein phosphorylation, nor DNA fragmentation over the course of this experiment (Figs. 2 D and 3 D). Control sera derived from an individual without autoimmune disease did not precipitate phosphoproteins from apoptotic lysates, nor from lysates prepared from CD3-stimulated cells (Fig. 2, A–D, right). The kinetics of DNA fragmentation induced by apoptotic or activation stimuli was also determined. As shown in Fig. 3, A–D, the onset of DNA fragmentation is approximately coincident with the phosphorylation of autoantigens regardless of the apoptotic stimulus.


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)

Autoantigen phosphorylation coincides with or precedes the  onset of DNA fragmentation in apoptotic Jurkat cells. Jurkat cells were  triggered to undergo apoptosis and harvested at the indicated times. Each  time point represents a total of 1 million cells. The DNA was prepared as  described in the Materials and Methods, separated on a 0.8% agarose gel  and visualized by staining with ethidium bromide before UV exposure.  (A) Anti-Fas treatment; (B) gamma irradiation; (C) UV irradiation; (D)  anti-CD3 treatment. The time, in hours, from initial exposure to each  stimulus is indicated at the top of each lane. The relative migration of  molecular size markers in kilobases is indicated on the right side of each  panel.
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Related In: Results  -  Collection

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Figure 3: Autoantigen phosphorylation coincides with or precedes the onset of DNA fragmentation in apoptotic Jurkat cells. Jurkat cells were triggered to undergo apoptosis and harvested at the indicated times. Each time point represents a total of 1 million cells. The DNA was prepared as described in the Materials and Methods, separated on a 0.8% agarose gel and visualized by staining with ethidium bromide before UV exposure. (A) Anti-Fas treatment; (B) gamma irradiation; (C) UV irradiation; (D) anti-CD3 treatment. The time, in hours, from initial exposure to each stimulus is indicated at the top of each lane. The relative migration of molecular size markers in kilobases is indicated on the right side of each panel.
Mentions: The results shown in Fig. 1 indicate that autoimmune sera preferentially precipitate proteins phosphorylated in response to Fas ligation. To determine whether these proteins are also phosphorylated during apoptosis triggered by stimuli other than Fas ligation, selected patient sera were used to precipitate 32P-labeled Jurkat lysates prepared from cells subjected to apoptotic stimuli or an activation stimulus for various times. This kinetic analysis reveals that phosphorylation of autoantigens is induced between 1 and 2.5 h after Fas ligation (Fig. 2 A), between 2.5 and 4.5 h after gamma irradiation (Fig. 2 B), and between 1 and 2.5 h after UV irradiation (Fig. 2 C). Individual autoantisera precipitate a similar cadre of phosphoproteins regardless of the apoptotic trigger. In contrast, ligation of the T cell receptor complex using a monoclonal antibody reactive with CD3, a stimulus that induces IL-2 production and enhances proliferation in these cells (data not shown), induced neither new protein phosphorylation, nor DNA fragmentation over the course of this experiment (Figs. 2 D and 3 D). Control sera derived from an individual without autoimmune disease did not precipitate phosphoproteins from apoptotic lysates, nor from lysates prepared from CD3-stimulated cells (Fig. 2, A–D, right). The kinetics of DNA fragmentation induced by apoptotic or activation stimuli was also determined. As shown in Fig. 3, A–D, the onset of DNA fragmentation is approximately coincident with the phosphorylation of autoantigens regardless of the apoptotic stimulus.

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