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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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Human autoimmune sera precipitate phosphoproteins from apoptotic Jurkat  cell lysates. (A) Jurkat cells were  labeled with 32P-orthophosphate, treated with the anti-Fas  monoclonal antibody 7C11, and  lysed either before (odd numbered lanes) or 2.5 h after (even  numbered lanes) the addition of  antibodies. Proteins were then  precipitated using the indicated  autoimmune serum, separated on  a 12% SDS–polyacrylamide gel,  transferred to nitrocellulose, and  exposed for autoradiography.  Arrows point to new phosphoproteins in the anti-Fas–treated  lanes. (B) The identical experiment with 35S-labeled cells. Patient numbers are located above  each figure and correspond to  those in Table 1. Lane numbers  appear beneath the corresponding lane. The relative migration  of molecular size markers in kilodaltons are indicated on the left  side of the gel.
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Figure 1: Human autoimmune sera precipitate phosphoproteins from apoptotic Jurkat cell lysates. (A) Jurkat cells were labeled with 32P-orthophosphate, treated with the anti-Fas monoclonal antibody 7C11, and lysed either before (odd numbered lanes) or 2.5 h after (even numbered lanes) the addition of antibodies. Proteins were then precipitated using the indicated autoimmune serum, separated on a 12% SDS–polyacrylamide gel, transferred to nitrocellulose, and exposed for autoradiography. Arrows point to new phosphoproteins in the anti-Fas–treated lanes. (B) The identical experiment with 35S-labeled cells. Patient numbers are located above each figure and correspond to those in Table 1. Lane numbers appear beneath the corresponding lane. The relative migration of molecular size markers in kilodaltons are indicated on the left side of the gel.

Mentions: Jurkat cells metabolically labeled with 32P-orthophosphate were cultured for 2.5 h in the absence or presence of a monoclonal antibody reactive with Fas (anti-7C11), solubilized in NP40 lysis buffer, and immunoprecipitated using the indicated autoimmune or control sera. Immunoprecipitates were separated on a 12% SDS–polyacrylamide gel, transferred to nitrocellulose, and subjected to autoradiograpy. Fig. 1 A shows that 9/12 ANA+ autoimmune sera, representing 9/10 SLE or SLE overlap patients, precipitated at least one new phosphoprotein from cells undergoing Fas-mediated apoptosis compared to untreated cells. The phosphorylation of these proteins did not result from a nonspecific, general increase in kinase activity after Fas engagement, as 32P-labeled,whole cell extracts prepared from untreated and apoptotic cells were identical when compared on SDS-PAGE gels (data not shown). The individual phosphoproteins precipitated by several of the patient sera is strikingly similar in profile, but variable in intensity of phosphorylation. For example, serum from patients 1, 2, 3, 4, 8, 11, and 12 precipitates a protein of ∼54 kD (pp54) that is weakly phosphorylated in untreated cell lysates and strongly phosphorylated in lysates from apoptotic cells. Similarly, a 34-kD protein (pp34) was precipitated using serum derived from patients 3, 8, 11, and 12; and a doublet of ∼42 kD (pp42) was precipitated using serum derived from patients 3, 8, 11, and 12. None of these phosphoproteins were precipitated using ANA (−) sera derived from patients 13 or 14, nor using sera derived from 12 healthy control patients or 4 additional patients with RA (data not shown). The level of phosphorylation of pp42, pp34, and pp17 differed significantly between patients (patients 3, 8, 11, and 12) and was independent of the ANA titer as detected by immunfluorescence (Table 1), suggesting that these phosphoproteins may be novel and independent of the major proteins responsible for the immunfluorescence detectable as an ANA. In addition to the phosphoproteins described above, three other new phosphoproteins can be seen as bands migrating at the following positions: 17 kD doublet (pp17; patients 1, 4, 5, 8, and 11), 23 kD (pp23; patients 3, 8, and 11), and 46 kD (pp46; patient 7). A seventh protein migrating between 96 and 200 kD (pp200) was observed for patient 1 (Fig. 2, A–C).


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)

Human autoimmune sera precipitate phosphoproteins from apoptotic Jurkat  cell lysates. (A) Jurkat cells were  labeled with 32P-orthophosphate, treated with the anti-Fas  monoclonal antibody 7C11, and  lysed either before (odd numbered lanes) or 2.5 h after (even  numbered lanes) the addition of  antibodies. Proteins were then  precipitated using the indicated  autoimmune serum, separated on  a 12% SDS–polyacrylamide gel,  transferred to nitrocellulose, and  exposed for autoradiography.  Arrows point to new phosphoproteins in the anti-Fas–treated  lanes. (B) The identical experiment with 35S-labeled cells. Patient numbers are located above  each figure and correspond to  those in Table 1. Lane numbers  appear beneath the corresponding lane. The relative migration  of molecular size markers in kilodaltons are indicated on the left  side of the gel.
© Copyright Policy
Related In: Results  -  Collection

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Figure 1: Human autoimmune sera precipitate phosphoproteins from apoptotic Jurkat cell lysates. (A) Jurkat cells were labeled with 32P-orthophosphate, treated with the anti-Fas monoclonal antibody 7C11, and lysed either before (odd numbered lanes) or 2.5 h after (even numbered lanes) the addition of antibodies. Proteins were then precipitated using the indicated autoimmune serum, separated on a 12% SDS–polyacrylamide gel, transferred to nitrocellulose, and exposed for autoradiography. Arrows point to new phosphoproteins in the anti-Fas–treated lanes. (B) The identical experiment with 35S-labeled cells. Patient numbers are located above each figure and correspond to those in Table 1. Lane numbers appear beneath the corresponding lane. The relative migration of molecular size markers in kilodaltons are indicated on the left side of the gel.
Mentions: Jurkat cells metabolically labeled with 32P-orthophosphate were cultured for 2.5 h in the absence or presence of a monoclonal antibody reactive with Fas (anti-7C11), solubilized in NP40 lysis buffer, and immunoprecipitated using the indicated autoimmune or control sera. Immunoprecipitates were separated on a 12% SDS–polyacrylamide gel, transferred to nitrocellulose, and subjected to autoradiograpy. Fig. 1 A shows that 9/12 ANA+ autoimmune sera, representing 9/10 SLE or SLE overlap patients, precipitated at least one new phosphoprotein from cells undergoing Fas-mediated apoptosis compared to untreated cells. The phosphorylation of these proteins did not result from a nonspecific, general increase in kinase activity after Fas engagement, as 32P-labeled,whole cell extracts prepared from untreated and apoptotic cells were identical when compared on SDS-PAGE gels (data not shown). The individual phosphoproteins precipitated by several of the patient sera is strikingly similar in profile, but variable in intensity of phosphorylation. For example, serum from patients 1, 2, 3, 4, 8, 11, and 12 precipitates a protein of ∼54 kD (pp54) that is weakly phosphorylated in untreated cell lysates and strongly phosphorylated in lysates from apoptotic cells. Similarly, a 34-kD protein (pp34) was precipitated using serum derived from patients 3, 8, 11, and 12; and a doublet of ∼42 kD (pp42) was precipitated using serum derived from patients 3, 8, 11, and 12. None of these phosphoproteins were precipitated using ANA (−) sera derived from patients 13 or 14, nor using sera derived from 12 healthy control patients or 4 additional patients with RA (data not shown). The level of phosphorylation of pp42, pp34, and pp17 differed significantly between patients (patients 3, 8, 11, and 12) and was independent of the ANA titer as detected by immunfluorescence (Table 1), suggesting that these phosphoproteins may be novel and independent of the major proteins responsible for the immunfluorescence detectable as an ANA. In addition to the phosphoproteins described above, three other new phosphoproteins can be seen as bands migrating at the following positions: 17 kD doublet (pp17; patients 1, 4, 5, 8, and 11), 23 kD (pp23; patients 3, 8, and 11), and 46 kD (pp46; patient 7). A seventh protein migrating between 96 and 200 kD (pp200) was observed for patient 1 (Fig. 2, A–C).

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