Increased ubiquitination and reduced expression of LCK in T lymphocytes from patients with systemic lupus erythematosus
Increased ubiquitination and reduced expression of LCK in T lymphocytes from patients with systemic lupus erythematosus
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DOI：
10.1002/art.10978
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发表时间：
2003-05-01
影响因子：
--
通讯作者：
Isenberg, DA
中科院分区：
文献类型：
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作者：
Jury, EC;Kabouridis, PS;Isenberg, DA
Objective.探讨系统性红斑狼疮（SLE）患者T淋巴细胞近端信号转导及脂筏组成的调控。研究了50例SLE患者T淋巴细胞中脂筏相关信号分子的表达、磷酸化和降解，并与28例健康对照和22例类风湿关节炎患者进行了比较。通过超离心分离来自T细胞的脂质筏和非筏组分。蛋白质在脂筏和nonraft馏分进行了分析，通过蛋白质印迹和探测磷酸酪氨酸活性和LCK，LAT，和CD3E。进行免疫沉淀实验以评估T细胞裂解物中的蛋白质泛素化。流式细胞术检测T细胞表型和细胞内LCK水平。LCK是T细胞活化的重要信号分子，与对照组相比，活动性SLE患者T淋巴细胞的脂筏和非脂筏组分均显著降低，且这种降低与治疗无关。为了确定LCK降低的可能原因，我们探讨了T淋巴细胞的慢性激活导致LCK降解的可能性。结果显示，SLE患者T细胞中蛋白质泛素化，特别是LCK泛素化增加。然而，我们的研究结果表明，泛素化的增加是独立的T细胞活化。SLE患者T细胞脂筏中LCK减少。这种减少似乎是独立的激活，并可能与异常的泛素介导的调节机制。
Objective. To explore regulation of proximal signaling and composition of lipid rafts in T lymphocytes from patients with systemic lupus erythematosus (SLE).Methods. The expression, phosphorylation, and degradation of lipid raft-associated signaling molecules in T lymphocytes from 50 patients with SLE compared with 28 healthy controls and 22 rheumatoid arthritis patients were investigated. Lipid raft and nonraft fractions from T cells were isolated by ultracentrifugation. Proteins in the lipid raft and nonraft fractions were analyzed by Western blotting and probed for phosphotyrosine activity and for LCK, LAT, and CD3E. Immunoprecipitation experiments were performed to assess protein ubiquitination in T cell lysates. T cell phenotype and levels of intracellular LCK were determined by flow cytometry.Results. LCK, an essential signaling molecule for T cell activation, was significantly reduced in both lipid raft and nonraft fractions of, T lymphocytes from patients with active SLE compared with controls, and the reduction was independent of treatment. To identify the likely causes of reduced LCK, we explored the possibility that chronic activation of T lymphocytes underlies LCK degradation. The results revealed an increase in protein ubiquitination, and specifically LCK ubiquitination, in T cells from SLE patients. However, our findings suggest that the increase in ubiquitination is independent of T cell activation.Conclusion. LCK is reduced in T cell lipid rafts from patients with SLE. This reduction appears to be independent of activation and may be associated with abnormal ubiquitin-mediated regulation mechanisms.