Deans J P, Schieven G L, Shu G L, Valentine M A, Gilliland L A, Aruffo A, Clark E A, Ledbetter J A
Department of Microbiology, University of Washington, Seattle 98195.
J Immunol. 1993 Nov 1;151(9):4494-504.
CD20 is a B cell-specific 35/37 kDa integral membrane protein which modulates proliferation and differentiation of normal resting B cells when stimulated by CD20 antibodies. An increase in c-myc mRNA levels occurs within hours after treatment of resting B cells with CD20 mAb; however earlier events in the CD20 signal transduction pathway have not been described. Here we demonstrate that anti-CD20 mediated induction of c-myc mRNA is inhibited by the tyrosine kinase inhibitor herbimycin A, that CD20 is associated with both tyrosine and serine kinase activity, and that tyrosine phosphorylation of multiple substrates is induced within minutes upon ligation of CD20 with mAb. Association of the tyrosine and serine kinases with CD20 was stable in lysis buffer containing 1% NP40 and 0.25% deoxycholate. Under the same conditions, antibodies against several other B cell surface molecules failed to co-precipitate tyrosine kinase activity, however, a serine kinase was precipitated by the anti-CD19 mAb, B43. Both phospholipase C-gamma 1 and -gamma 2 were phosphorylated on tyrosine after cross-linking of CD20-bound mAb, and this correlated with increases in intracellular calcium that were partially resistant to depletion of extracellular calcium with EGTA. The pattern of tyrosine phosphorylated proteins observed in whole cell lysates after anti-CD20 cross-linking appeared to be a subset of those induced by anti-IgM; however, differences in phosphoproteins induced by anti-IgM and anti-CD20 were detected using a fynSH2-fusion protein.
CD20是一种B细胞特异性的35/37 kDa整合膜蛋白，当受到CD20抗体刺激时，它可调节正常静息B细胞的增殖和分化。在用CD20单克隆抗体处理静息B细胞后的数小时内，c-myc mRNA水平会升高；然而，CD20信号转导途径中更早发生的事件尚未见报道。在此我们证明，酪氨酸激酶抑制剂赫比霉素A可抑制抗CD20介导的c-myc mRNA诱导，CD20与酪氨酸激酶和丝氨酸激酶活性均相关，并且在用单克隆抗体连接CD20后数分钟内即可诱导多种底物的酪氨酸磷酸化。酪氨酸激酶和丝氨酸激酶与CD20的结合在含有1% NP40和0.25%脱氧胆酸盐的裂解缓冲液中是稳定的。在相同条件下，针对其他几种B细胞表面分子的抗体未能共沉淀酪氨酸激酶活性，然而，抗CD19单克隆抗体B43可沉淀一种丝氨酸激酶。在用与CD20结合的单克隆抗体交联后，磷脂酶C-γ1和-γ2的酪氨酸位点发生磷酸化，这与细胞内钙的增加相关，而细胞内钙的增加部分抵抗EGTA对细胞外钙的耗竭。抗CD20交联后在全细胞裂解物中观察到的酪氨酸磷酸化蛋白模式似乎是抗IgM诱导的蛋白模式的一个子集；然而，使用fynSH2融合蛋白检测到抗IgM和抗CD20诱导的磷蛋白存在差异。
