Whisler R L, Liu B Q, Newhouse Y G, Walters J D, Breckenridge M B, Grants I S
Department of Internal Medicine, College of Medicine, Ohio State University, Columbus 43210.
Lymphokine Cytokine Res. 1991 Dec;10(6):463-73.
Protein phosphorylation is considered an early cellular mechanism of signal transduction by surface immunoglobulins (sIg) and other receptors of B cells. Using intact human peripheral blood B cells of young subjects labeled with orthophosphate, increased phosphorylation levels of serine/threonine and tyrosine substrates were demonstrated on indicator phosphoproteins corresponding to the CD20 isoforms and microtubule-associated protein 2 kinase after cross-linking sIg and costimulation with phorbol diesters. By contrast, stimulated B cells from certain elderly subjects displayed substantial alterations in the phosphorylation patterns of serine/threonine or tyrosine indicator phosphoproteins. Also, age-related impairments in sIg stimulated mobilization of cytosolic protein kinase C (PKC) enzymatic activity and in cytosolic calcium [Ca2+]i responses of B cells were observed with the altered phosphorylation reactions. Comparison of the substrate phosphorylation profiles to the proliferative responses of stimulated B cells from individual elderly subjects suggested a model of signal transduction in which differing stimuli have different dependencies on phosphorylation reactions. Diminished proliferative responses after sIg ligation coincided with decreased phosphorylations of either tyrosine or serine/threonine indicator substrates. However, the decreased proliferative responses of B cells from elderly subjects with substantial reductions of tyrosine phosphorylation after sIg ligation were enhanced by the direct stimulation of serine/threonine kinase activity with phorbol diesters or CD40 ligation. Experiments with kinase inhibitors evaluated the relative dependency of different B cell stimuli on tyrosine and serine/threonine phosphorylation reactions. The proliferative responses of normal B cells to sIg ligation were quite sensitive to the tyrosine kinase inhibitor genistein whereas those observed following costimulations with phorbol diesters or CD40 ligation were more resistant. However, treatment of B cells with H7, an inhibitor of PKC activity, led to a more uniform reduction of B-cell responses after different stimuli. Results from RNase protection assays of c-myc expression also suggested that different B-cell stimuli might utilize distinct intracellular signaling pathways. Both the type of stimuli and mode of sIg ligation were important in determining the stimulated levels of c-myc mRNA expression. Thus, the current findings suggest that age-related defects are present in human B cell signaling pathways as reflected by tyrosine and serine/threonine phosphorylation reactions. Also, these age-related defects can coexist with altered mobilization of PKC enzymatic activity and with alterations in [Ca2+]i and proliferative responses.
蛋白质磷酸化被认为是表面免疫球蛋白（sIg）和B细胞其他受体进行信号转导的早期细胞机制。使用用正磷酸盐标记的年轻受试者完整的人外周血B细胞，在交联sIg并用佛波酯进行共刺激后，对应于CD20亚型和微管相关蛋白2激酶的指示性磷蛋白上的丝氨酸/苏氨酸和酪氨酸底物的磷酸化水平增加。相比之下，来自某些老年受试者的受刺激B细胞在丝氨酸/苏氨酸或酪氨酸指示性磷蛋白的磷酸化模式上表现出显著变化。此外，随着磷酸化反应的改变，观察到sIg刺激的胞质蛋白激酶C（PKC）酶活性动员和B细胞的胞质钙[Ca2+]i反应中与年龄相关的损伤。将底物磷酸化谱与个体老年受试者受刺激B细胞的增殖反应进行比较，提出了一种信号转导模型，其中不同的刺激对磷酸化反应有不同的依赖性。sIg连接后增殖反应减弱与酪氨酸或丝氨酸/苏氨酸指示性底物的磷酸化减少相一致。然而，sIg连接后酪氨酸磷酸化大幅降低的老年受试者B细胞的增殖反应减弱，可通过用佛波酯直接刺激丝氨酸/苏氨酸激酶活性或CD40连接来增强。用激酶抑制剂进行的实验评估了不同B细胞刺激对酪氨酸和丝氨酸/苏氨酸磷酸化反应的相对依赖性。正常B细胞对sIg连接的增殖反应对酪氨酸激酶抑制剂染料木黄酮相当敏感，而在用佛波酯或CD40连接进行共刺激后观察到的增殖反应则更具抗性。然而，用PKC活性抑制剂H7处理B细胞，导致不同刺激后B细胞反应更均匀地降低。c-myc表达的核糖核酸酶保护分析结果也表明，不同的B细胞刺激可能利用不同的细胞内信号通路。刺激类型和sIg连接方式在决定c-myc mRNA表达的刺激水平方面都很重要。因此，目前的研究结果表明，酪氨酸和丝氨酸/苏氨酸磷酸化反应反映出人类B细胞信号通路中存在与年龄相关的缺陷。此外，这些与年龄相关的缺陷可能与PKC酶活性的动员改变、[Ca2+]i和增殖反应的改变同时存在。
