Functional analysis of Syk substrates in B cell receptor signaling

B细胞对抗原的反应能力依赖于通过B细胞抗原受体（BCR）复合物传递的信号。胞浆蛋白酪氨酸激酶（PTKs）的激活是对BCR交联最早可测量的生化反应。初始事件导致次级信号的产生，包括Ras活化、磷脂酰肌醇3-激酶（PI-3K）活化、磷脂酶C (PLC)-γ - 2活化。虽然关于bcr相关的ptk与下游效应物之间的关系已经了解了很多，但这些ptk调节下游事件的分子机制仍不清楚。与受体酪氨酸激酶类似，人们认为许多信号分子直接结合BCR复合物细胞质结构域磷酸化的酪氨酸残基。然而，这似乎并不是下游信号通路耦合机制的一个特征。人们的注意力反而集中在了适配蛋白上。基于我们之前的证据，在bcr相关的ptk中，Syk是PLC-γ2激活所必需的，我们纯化了由Syk介导的酪氨酸磷酸化蛋白。在多个纯化蛋白中，pp80的4个内部肽序列与人和小鼠BLNK非常同源。为了研究BLNK的功能，我们采用基因靶向的方法建立了BLNK缺乏的DT40 B细胞。与野生型DT40细胞相比，blnk缺陷细胞中未检测到PLC-γ2活化。综上所述，我们得出结论，BLNK在BCR信号传导中作为一个适配器分子，这是将Syk偶联到PLC-γ2激活所必需的。

The ability of B cells to respond to antigen relies on signals transmitted through the B cell antigen receptor (BCR) complex. Activation of cytoplasmic protein tyrosine kinases (PTKs) is the earliest measurable biochemical response to BCR cross-linking. The initial event leads to the generation of secondary signals including Ras activation, phosphatidylinositol 3-kinase (PI-3K) activation, phospholipase C (PLC)-γ2 activation.While much has been learned as to the relationship between the BCR-associated PTKs and downstream effectors, the molecular mechanism by which these PTKs regulate downstream events remains unclear. Analogous to receptor tyrosine kinases, it has been thought that many signaling molecules directly bind phosphorylated tyrosine residues on the cytoplamic domains of the BCR complex. However, this does not appear to be a feature of the coupling mechanism to downstream signaling pathways. Attention instead has focused on adaptor proteins.Based upon our previous evidence that Syk, among the BCR-associated PTKs, is essential for PLC-γ2 activation, we purified tyrosine-phosphorylated proteins mediated by Syk. Among several purified proteins, four internal peptide sequences obtained from microsequencing of pp80 were very homologous to those of human and mouse BLNK. To address the function of BLNK, we established DT40 B cells deficient in BLNK by genetargeting method. In contrast to wild-type DT40 cells, no PLC-γ2 activation was detected in BLNK-deficient cells. Taken together, we conclude that BLNK functions as an adaptor molecule in BCR signaling, which is required for coupling Syk to PLC-γ2 activation.