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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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Autoimmune serum precipitates a serine kinase activity from apoptotic Jurkat lysates. Jurkat cells cultured in the absence (odd numbered  lanes) or presence (even numbered lanes) of anti-Fas were solubilized in NP40 lysis buffer after 2.5 h, and precipitated using 3.5 μl of serum derived from  the indicated patient. Individual precipitates were subjected to an in vitro kinase reaction at 30°C for 30 min, separated on an SDS–polyacrylamide gel,  transferred to nitrocellulose, and subjected to autoradiographic exposure for 2 min. (A) In vitro kinase reaction. Serum derived from the patient number  indicated at the top of the figure corresponds to patients described in Table 1. The relative migration of molecular size markers in kilodaltons is indicated  on the right side of the panel. (B) Kinetics of kinase activation after Fas ligation as measured using the in vitro kinase reaction performed on immunoprecipitates prepared using serum derived from patient 7. The time in minutes from initial exposure to anti-Fas is indicated at the top of each lane. The position of pp46 is indicated with an arrow on the left side of the panel. (C) Phosphoamino acid analysis of the in vitro phosphorylated 46-kD protein. Migration of phosphoamino acid standards are labeled with circles as follows: phosphoserine (pS), phosphothreonine (pT), phosphotyrosine (pY).
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Figure 5: Autoimmune serum precipitates a serine kinase activity from apoptotic Jurkat lysates. Jurkat cells cultured in the absence (odd numbered lanes) or presence (even numbered lanes) of anti-Fas were solubilized in NP40 lysis buffer after 2.5 h, and precipitated using 3.5 μl of serum derived from the indicated patient. Individual precipitates were subjected to an in vitro kinase reaction at 30°C for 30 min, separated on an SDS–polyacrylamide gel, transferred to nitrocellulose, and subjected to autoradiographic exposure for 2 min. (A) In vitro kinase reaction. Serum derived from the patient number indicated at the top of the figure corresponds to patients described in Table 1. The relative migration of molecular size markers in kilodaltons is indicated on the right side of the panel. (B) Kinetics of kinase activation after Fas ligation as measured using the in vitro kinase reaction performed on immunoprecipitates prepared using serum derived from patient 7. The time in minutes from initial exposure to anti-Fas is indicated at the top of each lane. The position of pp46 is indicated with an arrow on the left side of the panel. (C) Phosphoamino acid analysis of the in vitro phosphorylated 46-kD protein. Migration of phosphoamino acid standards are labeled with circles as follows: phosphoserine (pS), phosphothreonine (pT), phosphotyrosine (pY).

Mentions: A cascade of stress-activated serine/threonine kinases has been implicated in signaling apoptotic cell death (16, 17, 19, 25, 31). Individual kinases within this cascade are regulated, in part, by phosphorylation. It is therefore possible that stress-activated kinases may be recognized directly by sera derived from patients with autoimmune disease, or may be recruited during apoptosis to preexisting complexes. To test this possibility, lysates from untreated or anti-Fas–treated Jurkat cells were precipitated with individual patient sera, and subjected to an in vitro kinase assay as described (25). Five sera were chosen to encompass all seven phosphoproteins that had been identified in the initial screen using in vivo–labeled apoptotic Jurkat cells (Fig. 1 A and Table 1). In addition, sera from a healthy control patient and patient 6, whose serum is monospecific for the Ro protein, were included for comparison. Fig. 5 A shows that 4/5 ANA + patient sera (i.e., patients 3, 7, 8, and 11) precipitate a kinase whose activity is increased in apoptotic cell extracts compared to untreated cell extracts. The healthy control patient and patient 6 were devoid of kinase activity in this assay. Phosphoproteins migrating at 34 kD (Fig. 5, lanes 4, 6, 8, and 10), 23 kD (lanes 4, 6, 8, and 10), and 46 kD (lane 6) were identified in this assay. The relative migration of these phosphoproteins is similar to that of prominant phosphoproteins identified in the in vivo phosphorylation assay shown in Fig. 1 A. The kinetics with which the kinase (precipitated using serum from patient 7) was activated after Fas ligation was correlated with the induction of DNA fragmentation in the experiment shown in Fig. 5 B. In this experiment, Jurkat cells were cultured in the presence of anti-Fas monoclonal antibodies for the indicated times before processing for DNA fragmentation and in vitro kinase activity. The first appearance of pp46 in the in vitro kinase assay was observed at 90 min (Fig. 5 B), while DNA fragmentation was first observed 120 min after Fas ligation (data not shown). Phosphoamino acid analysis of pp46 showed that the in vitro phosphorylation of pp46 is restricted to serine residues (Fig. 5 C), consistent with the in vivo results shown in Fig. 4 C. A similar kinetic analysis targeting pp34 and pp23 using serum from patient 11 (Fig. 5 A, lanes 9 and 10) gave similar results (data not shown). These results are consistent with the less rigorous time courses presented in Figs. 2 and 3, and suggest that a serine/threonine kinase activated by Fas stimulation is present in the immunoprecipitates from patients 7 and 11 at a time that precedes the onset of DNA fragmentation.


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)

Autoimmune serum precipitates a serine kinase activity from apoptotic Jurkat lysates. Jurkat cells cultured in the absence (odd numbered  lanes) or presence (even numbered lanes) of anti-Fas were solubilized in NP40 lysis buffer after 2.5 h, and precipitated using 3.5 μl of serum derived from  the indicated patient. Individual precipitates were subjected to an in vitro kinase reaction at 30°C for 30 min, separated on an SDS–polyacrylamide gel,  transferred to nitrocellulose, and subjected to autoradiographic exposure for 2 min. (A) In vitro kinase reaction. Serum derived from the patient number  indicated at the top of the figure corresponds to patients described in Table 1. The relative migration of molecular size markers in kilodaltons is indicated  on the right side of the panel. (B) Kinetics of kinase activation after Fas ligation as measured using the in vitro kinase reaction performed on immunoprecipitates prepared using serum derived from patient 7. The time in minutes from initial exposure to anti-Fas is indicated at the top of each lane. The position of pp46 is indicated with an arrow on the left side of the panel. (C) Phosphoamino acid analysis of the in vitro phosphorylated 46-kD protein. Migration of phosphoamino acid standards are labeled with circles as follows: phosphoserine (pS), phosphothreonine (pT), phosphotyrosine (pY).
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Figure 5: Autoimmune serum precipitates a serine kinase activity from apoptotic Jurkat lysates. Jurkat cells cultured in the absence (odd numbered lanes) or presence (even numbered lanes) of anti-Fas were solubilized in NP40 lysis buffer after 2.5 h, and precipitated using 3.5 μl of serum derived from the indicated patient. Individual precipitates were subjected to an in vitro kinase reaction at 30°C for 30 min, separated on an SDS–polyacrylamide gel, transferred to nitrocellulose, and subjected to autoradiographic exposure for 2 min. (A) In vitro kinase reaction. Serum derived from the patient number indicated at the top of the figure corresponds to patients described in Table 1. The relative migration of molecular size markers in kilodaltons is indicated on the right side of the panel. (B) Kinetics of kinase activation after Fas ligation as measured using the in vitro kinase reaction performed on immunoprecipitates prepared using serum derived from patient 7. The time in minutes from initial exposure to anti-Fas is indicated at the top of each lane. The position of pp46 is indicated with an arrow on the left side of the panel. (C) Phosphoamino acid analysis of the in vitro phosphorylated 46-kD protein. Migration of phosphoamino acid standards are labeled with circles as follows: phosphoserine (pS), phosphothreonine (pT), phosphotyrosine (pY).
Mentions: A cascade of stress-activated serine/threonine kinases has been implicated in signaling apoptotic cell death (16, 17, 19, 25, 31). Individual kinases within this cascade are regulated, in part, by phosphorylation. It is therefore possible that stress-activated kinases may be recognized directly by sera derived from patients with autoimmune disease, or may be recruited during apoptosis to preexisting complexes. To test this possibility, lysates from untreated or anti-Fas–treated Jurkat cells were precipitated with individual patient sera, and subjected to an in vitro kinase assay as described (25). Five sera were chosen to encompass all seven phosphoproteins that had been identified in the initial screen using in vivo–labeled apoptotic Jurkat cells (Fig. 1 A and Table 1). In addition, sera from a healthy control patient and patient 6, whose serum is monospecific for the Ro protein, were included for comparison. Fig. 5 A shows that 4/5 ANA + patient sera (i.e., patients 3, 7, 8, and 11) precipitate a kinase whose activity is increased in apoptotic cell extracts compared to untreated cell extracts. The healthy control patient and patient 6 were devoid of kinase activity in this assay. Phosphoproteins migrating at 34 kD (Fig. 5, lanes 4, 6, 8, and 10), 23 kD (lanes 4, 6, 8, and 10), and 46 kD (lane 6) were identified in this assay. The relative migration of these phosphoproteins is similar to that of prominant phosphoproteins identified in the in vivo phosphorylation assay shown in Fig. 1 A. The kinetics with which the kinase (precipitated using serum from patient 7) was activated after Fas ligation was correlated with the induction of DNA fragmentation in the experiment shown in Fig. 5 B. In this experiment, Jurkat cells were cultured in the presence of anti-Fas monoclonal antibodies for the indicated times before processing for DNA fragmentation and in vitro kinase activity. The first appearance of pp46 in the in vitro kinase assay was observed at 90 min (Fig. 5 B), while DNA fragmentation was first observed 120 min after Fas ligation (data not shown). Phosphoamino acid analysis of pp46 showed that the in vitro phosphorylation of pp46 is restricted to serine residues (Fig. 5 C), consistent with the in vivo results shown in Fig. 4 C. A similar kinetic analysis targeting pp34 and pp23 using serum from patient 11 (Fig. 5 A, lanes 9 and 10) gave similar results (data not shown). These results are consistent with the less rigorous time courses presented in Figs. 2 and 3, and suggest that a serine/threonine kinase activated by Fas stimulation is present in the immunoprecipitates from patients 7 and 11 at a time that precedes the onset of DNA fragmentation.

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