Stetler D A, Jacob S T
J Biol Chem. 1984 Nov 25;259(22):13629-32.
Purified RNA polymerase I was phosphorylated by the endogenous protein kinase or dephosphorylated by alkaline phosphatase and used as antigen in a radioimmunoassay with sera from systemic lupus erythematosus patients or serum from an immunized rabbit. Enzyme incubated in the absence of ATP or phosphatase served as control. Three to seven times more of the autoantibodies in the patients' sera reacted with phosphorylated RNA polymerase I than with control enzyme. The reactivity of the dephosphorylated enzyme with lupus autoantibodies was only 50-60% of that observed with control enzyme. Neither phosphorylation nor dephosphorylation of the enzyme had an effect on its reaction with the rabbit antibodies. The effect of phosphorylation on the reaction of each RNA polymerase I subunit (S1-S8; Mr = 190,000-17,000) with the patients' antibodies was determined by an immunoblot procedure following resolution of the subunits on polyacrylamide gels. Prior phosphorylation of the enzyme resulted in a dramatic increase in binding of each patient's antibodies to all polymerase subunits with the exception of S4. Anti-S4 antibody was not detected with either phosphorylated or control enzyme. Strikingly, antibodies in each patients' sera reacted with S6 only after its phosphorylation. Similarly, anti-S5 antibodies in the serum of one patient were only detected with phosphorylated RNA polymerase I. The present data suggest that at least a significant fraction of the anti-RNA polymerase I autoantibodies in the sera of systemic lupus erythematosus patients might be directed against phosphorylated sites on the enzyme and that phosphorylation may have a role in the production of this and other autoimmunogenic nuclear components which are hallmarks of this disease.
纯化的RNA聚合酶I被内源性蛋白激酶磷酸化或被碱性磷酸酶去磷酸化，然后用作放射免疫分析中的抗原，与系统性红斑狼疮患者的血清或免疫兔的血清反应。在无ATP或磷酸酶的情况下孵育的酶用作对照。患者血清中自身抗体与磷酸化RNA聚合酶I反应的量比与对照酶反应的量多3至7倍。去磷酸化酶与狼疮自身抗体的反应性仅为对照酶的50-60%。酶的磷酸化和去磷酸化对其与兔抗体的反应均无影响。通过在聚丙烯酰胺凝胶上分离亚基后进行免疫印迹程序，确定了磷酸化对每个RNA聚合酶I亚基（S1-S8；Mr = 190,000-17,000）与患者抗体反应的影响。酶的预先磷酸化导致每个患者的抗体与除S4之外的所有聚合酶亚基的结合显著增加。无论是磷酸化酶还是对照酶都未检测到抗S4抗体。令人惊讶的是，每个患者血清中的抗体仅在S6磷酸化后才与S6反应。同样，一名患者血清中的抗S5抗体仅在磷酸化RNA聚合酶I中检测到。目前的数据表明，系统性红斑狼疮患者血清中至少相当一部分抗RNA聚合酶I自身抗体可能针对该酶上的磷酸化位点，并且磷酸化可能在这种以及其他自身免疫原性核成分的产生中起作用，而这些成分是该疾病的标志。
