Roifman C M, Chin K, Gazit A, Mills G B, Gilon C, Levitzki A
Division of Immunology and Allergy, Hospital for Sick Children, Toronto, Ontario, Canada.
J Immunol. 1991 May 1;146(9):2965-71.
Staphylococcus aureus Cowan I (SAC) is a potent mitogen for purified human B cells. By using Western blotting with antiphosphotyrosine antibodies, we demonstrated that the mitogenic effect of SAC is associated with rapid tyrosine phosphorylation of proteins of 45, 68, 75, 97, and 145 kDa. This tyrosine phosphorylation was detected within 30 s of the addition of SAC; it reached a maximum within 10 min, after which it declined gradually. In contrast to SAC, most soluble anti-IgM antibodies do not induce proliferation of isolated human B cells. As indicated by Western blotting, soluble anti-IgM antibodies induced a similar pattern of tyrosine phosphorylation, with the exception of the 68-kDa protein, which was the most heavily phosphorylated protein in SAC-treated cells. A similar but less intense 68-kDa band was also induced by mitogenic anti-IgM bound to beads. This suggested that tyrosine phosphorylation, especially of p68, may play an important role in B cell mitogenesis. To test this hypothesis, we determined the effect of specific tyrosine kinase inhibitors (tyrphostins) on SAC-induced tyrosine phosphorylation, oncogene expression, and B cell proliferation. The concentration dependencies of inhibition of these processes suggested that they were linked. Nonspecific toxic effects of the tyrphostins were ruled out by the demonstration that the tyrphostins did not alter cell viability and did not inhibit B cell proliferation induced by phorbol esters, which do not induce tyrosine phosphorylation. For maximal inhibition of SAC-induced cell proliferation, the tyrophostins needed to be added before or shortly after addition of SAC. Taken together, these data indicate that tyrosine phosphorylation is an obligatory early signal in B cell proliferation.
金黄色葡萄球菌考恩I株（SAC）是纯化人B细胞的一种强效促有丝分裂原。通过使用抗磷酸酪氨酸抗体进行蛋白质免疫印迹分析，我们证明SAC的促有丝分裂作用与45、68、75、97和145 kDa蛋白质的快速酪氨酸磷酸化有关。在加入SAC后30秒内即可检测到这种酪氨酸磷酸化；10分钟内达到最大值，之后逐渐下降。与SAC不同，大多数可溶性抗IgM抗体不会诱导分离的人B细胞增殖。如蛋白质免疫印迹分析所示，可溶性抗IgM抗体诱导了类似的酪氨酸磷酸化模式，但68 kDa蛋白质除外，该蛋白是SAC处理细胞中磷酸化程度最高的蛋白质。与珠子结合的促有丝分裂抗IgM也诱导出一条类似但强度较低的68 kDa条带。这表明酪氨酸磷酸化，尤其是p68的磷酸化，可能在B细胞有丝分裂中起重要作用。为了验证这一假设，我们确定了特异性酪氨酸激酶抑制剂（曲磷胺）对SAC诱导的酪氨酸磷酸化、癌基因表达和B细胞增殖的影响。这些过程抑制的浓度依赖性表明它们是相关联的。曲磷胺不会改变细胞活力，也不会抑制佛波酯诱导的B细胞增殖（佛波酯不会诱导酪氨酸磷酸化），从而排除了曲磷胺的非特异性毒性作用。为了最大程度地抑制SAC诱导的细胞增殖，需要在加入SAC之前或之后不久加入曲磷胺。综上所述，这些数据表明酪氨酸磷酸化是B细胞增殖中必不可少的早期信号。
