Melamed I, Wang G, Roifman C M
Division of Immunology/Allergy, Hospital for Sick Children, Toronto, Ontario, Canada.
J Immunol. 1992 Jul 1;149(1):169-74.
Ligation of the Ag receptor on B cells is associated with a rapid increase in phosphorylation on tyrosine residues of multiple substrates. One of the substrates is the phosphoinositide-specific phospholipase C-gamma 1. Because activation of phospholipase C-gamma 1 seems to be dependent on tyrosine phosphorylation, it is assumed that the two signaling pathways, phosphatidylinositol turnover and tyrosine phosphorylation, might be linked. However, since the Ag receptor does not possess a kinase domain, it remains unclear how these signaling pathways are regulated by the Ag receptor. Previous studies have proposed the existence of a receptor-coupled G protein that regulates inositol phosphate production in B cells. We confirm that phosphoinositide turnover is regulated by a pertussis toxin (PT)-sensitive G protein, most probably by controlling phosphorylation of phospholipase C-gamma 1. We show that treatment of permeabilized B cells with a nonhydrolyzable GTP analogue guanosine 5'-[3-thio]triphosphate induced an increase in tyrosine phosphorylation of multiple substrates that are identical to the proteins phosphorylated after anti-IgM stimulation. Furthermore, binding of the inactive form of G proteins with guanosine 5'-[2-thio]-triphosphate blocked anti-IgM induced tyrosine phosphorylation in permeabilized B cells. The results indicate that an Ag receptor-coupled G protein controls protein tyrosine kinase activity. We show that this G protein is sensitive to PT because tyrosine phosphorylation mediated by the Ag receptor was inhibited by this toxin in a concentration-dependent manner. Similar concentrations of PT also blocked tyrosine phosphorylation on phospholipase C-gamma 1 and generation of inositol phosphates. Preincubation of intact B cells with PT resulted in inhibition of c-fos mRNA expression and DNA synthesis in anti-IgM stimulated B cells, indicating that post-transcriptional events are also controlled by the Ag-receptor coupled G protein. We conclude that Ag receptor-associated protein tyrosine kinase activity is regulated by a G protein. This PT-sensitive G protein also regulates phosphorylation and activation of phospholipase C-gamma 1 as well as later events in B cell activation such as c-fos mRNA expression and proliferation.
B细胞上Ag受体的连接与多种底物酪氨酸残基磷酸化的快速增加有关。其中一种底物是磷酸肌醇特异性磷脂酶C-γ1。由于磷脂酶C-γ1的激活似乎依赖于酪氨酸磷酸化，因此推测磷脂酰肌醇代谢和酪氨酸磷酸化这两条信号通路可能相互关联。然而，由于Ag受体不具有激酶结构域，目前尚不清楚这些信号通路是如何由Ag受体调控的。先前的研究提出存在一种受体偶联G蛋白，它调节B细胞中肌醇磷酸的产生。我们证实磷酸肌醇代谢受百日咳毒素（PT）敏感的G蛋白调节，很可能是通过控制磷脂酶C-γ1的磷酸化来实现的。我们发现，用不可水解的GTP类似物鸟苷5'-[3-硫代]三磷酸处理通透化的B细胞会导致多种底物酪氨酸磷酸化增加，这些底物与抗IgM刺激后磷酸化的蛋白质相同。此外，G蛋白的无活性形式与鸟苷5'-[2-硫代]-三磷酸的结合阻断了通透化B细胞中抗IgM诱导的酪氨酸磷酸化。结果表明，一种Ag受体偶联G蛋白控制蛋白酪氨酸激酶活性。我们发现这种G蛋白对PT敏感，因为Ag受体介导的酪氨酸磷酸化被该毒素以浓度依赖的方式抑制。相似浓度的PT也阻断了磷脂酶C-γ1上的酪氨酸磷酸化以及肌醇磷酸的生成。用PT对完整B细胞进行预孵育会抑制抗IgM刺激的B细胞中c-fos mRNA表达和DNA合成，表明转录后事件也受Ag受体偶联G蛋白的控制。我们得出结论，Ag受体相关的蛋白酪氨酸激酶活性受G蛋白调节。这种PT敏感的G蛋白还调节磷脂酶C-γ1的磷酸化和激活以及B细胞激活中的后续事件，如c-fos mRNA表达和增殖。
