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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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Autoantigens are phosphorylated exclusively on serine residues during Fas-mediated apoptosis. Jurkat cells were labeled with 32P-orthophosphate, treated with the anti-Fas monoclonal antibody 7C11, and solubilized using NP40 lysis buffer after 2.5 h. Proteins were then  precipitated with autoimmune serum, separated on a 12% SDS–polyacrylamide gel, transferred to PVDF, and exposed for autoradiography. Individual phosphoproteins were localized on the membrane, excised, and  subjected to acid hydrolysis. Phosphoamino acids were separated by twodimensional electrophoresis in pH 1.9 buffer in the horizontal dimension,  followed by pH 3.5 buffer in the vertical dimension before autoradiographic analysis. Individual proteins correspond to those described in Table 1 as follows: (A) patient 1, pp200; (B) patient 1, pp54; (C) patient 7,  pp46; (D) patient 11, pp42; (E) patient 3, pp34; (F) patient 8, pp23; and  (G) patient 11, pp17. Migration of phosphoaminoacid standards are labeled with circles as follows: phosphoserine (pS), phosphothreonine (pT),  phosphotyrosine (pY).
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Figure 4: Autoantigens are phosphorylated exclusively on serine residues during Fas-mediated apoptosis. Jurkat cells were labeled with 32P-orthophosphate, treated with the anti-Fas monoclonal antibody 7C11, and solubilized using NP40 lysis buffer after 2.5 h. Proteins were then precipitated with autoimmune serum, separated on a 12% SDS–polyacrylamide gel, transferred to PVDF, and exposed for autoradiography. Individual phosphoproteins were localized on the membrane, excised, and subjected to acid hydrolysis. Phosphoamino acids were separated by twodimensional electrophoresis in pH 1.9 buffer in the horizontal dimension, followed by pH 3.5 buffer in the vertical dimension before autoradiographic analysis. Individual proteins correspond to those described in Table 1 as follows: (A) patient 1, pp200; (B) patient 1, pp54; (C) patient 7, pp46; (D) patient 11, pp42; (E) patient 3, pp34; (F) patient 8, pp23; and (G) patient 11, pp17. Migration of phosphoaminoacid standards are labeled with circles as follows: phosphoserine (pS), phosphothreonine (pT), phosphotyrosine (pY).

Mentions: Since both tyrosine kinases and serine/threonine kinases have been implicated in signaling Fas-mediated apoptosis (16, 17, 19, 25, 27–30), we subjected all seven phosphoprotein autoantigens to phosphoaminoacid analysis. In each case, phosphorylation was restricted to serine residues (Fig. 4, A–G), implicating one or more serine/threonine protein kinases in the phosphorylation of these autoantigens.


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)

Autoantigens are phosphorylated exclusively on serine residues during Fas-mediated apoptosis. Jurkat cells were labeled with 32P-orthophosphate, treated with the anti-Fas monoclonal antibody 7C11, and solubilized using NP40 lysis buffer after 2.5 h. Proteins were then  precipitated with autoimmune serum, separated on a 12% SDS–polyacrylamide gel, transferred to PVDF, and exposed for autoradiography. Individual phosphoproteins were localized on the membrane, excised, and  subjected to acid hydrolysis. Phosphoamino acids were separated by twodimensional electrophoresis in pH 1.9 buffer in the horizontal dimension,  followed by pH 3.5 buffer in the vertical dimension before autoradiographic analysis. Individual proteins correspond to those described in Table 1 as follows: (A) patient 1, pp200; (B) patient 1, pp54; (C) patient 7,  pp46; (D) patient 11, pp42; (E) patient 3, pp34; (F) patient 8, pp23; and  (G) patient 11, pp17. Migration of phosphoaminoacid standards are labeled with circles as follows: phosphoserine (pS), phosphothreonine (pT),  phosphotyrosine (pY).
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Related In: Results  -  Collection

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Figure 4: Autoantigens are phosphorylated exclusively on serine residues during Fas-mediated apoptosis. Jurkat cells were labeled with 32P-orthophosphate, treated with the anti-Fas monoclonal antibody 7C11, and solubilized using NP40 lysis buffer after 2.5 h. Proteins were then precipitated with autoimmune serum, separated on a 12% SDS–polyacrylamide gel, transferred to PVDF, and exposed for autoradiography. Individual phosphoproteins were localized on the membrane, excised, and subjected to acid hydrolysis. Phosphoamino acids were separated by twodimensional electrophoresis in pH 1.9 buffer in the horizontal dimension, followed by pH 3.5 buffer in the vertical dimension before autoradiographic analysis. Individual proteins correspond to those described in Table 1 as follows: (A) patient 1, pp200; (B) patient 1, pp54; (C) patient 7, pp46; (D) patient 11, pp42; (E) patient 3, pp34; (F) patient 8, pp23; and (G) patient 11, pp17. Migration of phosphoaminoacid standards are labeled with circles as follows: phosphoserine (pS), phosphothreonine (pT), phosphotyrosine (pY).
Mentions: Since both tyrosine kinases and serine/threonine kinases have been implicated in signaling Fas-mediated apoptosis (16, 17, 19, 25, 27–30), we subjected all seven phosphoprotein autoantigens to phosphoaminoacid analysis. In each case, phosphorylation was restricted to serine residues (Fig. 4, A–G), implicating one or more serine/threonine protein kinases in the phosphorylation of these autoantigens.

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