Carter R H, Park D J, Rhee S G, Fearon D T
Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205.
Proc Natl Acad Sci U S A. 1991 Apr 1;88(7):2745-9. doi: 10.1073/pnas.88.7.2745.
Ligation of membrane IgM on B lymphocytes causes activation of a protein-tyrosine kinase(s) (PTK) and of phospholipase C (PLC). To determine whether these are elements of a common signal-transduction pathway, the effect of three PTK inhibitors on the rise in intracellular free Ca2+ concentration [( Ca2+]i) in human B-lymphoblastoid cell lines was assessed. Tyrphostin completely suppressed the increase in [Ca2+]i and the generation of inositol phosphates induced by ligation of membrane immunoglobulin (mIg) M. Herbimycin and genistein reduced by 30% and 50%, respectively, the rise in [Ca2+]i caused by optimal ligation of mIgM, and they abolished it in cells activated by suboptimal ligation of mIgM. Tyrphostin had no effect on the capacity of aluminum fluoride to increase [Ca2+]i. To determine whether a function of PTK is the phosphorylation of PLC, immunoprecipitates obtained with anti-phosphotyrosine from detergent lysates of B-lymphoblastoid cells were assayed for PLC activity. Ligation of mIgM increased immunoprecipitable PLC activity 2-fold by 90 sec and 4-fold by 30 min. Specific immunoprecipitation and Western blot analysis identified tyrosine phosphorylation of the gamma 1 isoform of PLC after 60 sec of stimulation. Activation of PLC in B cells by mIgM requires PTK function and is associated with tyrosine phosphorylation of PLC-gamma 1, suggesting a mechanism of PLC activation similar to that described for certain receptor PTKs.
B淋巴细胞上膜IgM的连接会导致一种蛋白酪氨酸激酶（PTK）和磷脂酶C（PLC）的激活。为了确定这些是否是共同信号转导途径的组成部分，评估了三种PTK抑制剂对人B淋巴母细胞系细胞内游离Ca2+浓度[Ca2+]i升高的影响。酪氨酸磷酸化抑制剂完全抑制了[Ca2+]i的升高以及膜免疫球蛋白（mIg）M连接诱导的肌醇磷酸的生成。除莠霉素和染料木黄酮分别使mIgM最佳连接引起的[Ca2+]i升高降低了30%和50%，并且在mIgM次优连接激活的细胞中使其消失。酪氨酸磷酸化抑制剂对氟化铝升高[Ca2+]i的能力没有影响。为了确定PTK的功能是否是PLC的磷酸化，用抗磷酸酪氨酸从B淋巴母细胞的去污剂裂解物中获得的免疫沉淀物检测了PLC活性。mIgM的连接在90秒时使可免疫沉淀的PLC活性增加了2倍，在30分钟时增加了4倍。特异性免疫沉淀和蛋白质印迹分析确定在刺激60秒后PLC的γ1同工型发生了酪氨酸磷酸化。mIgM在B细胞中激活PLC需要PTK功能，并且与PLC-γ1的酪氨酸磷酸化相关，这表明PLC激活的机制类似于某些受体PTK所描述的机制。
