Molecular mechanisms for the regulation of BCR-mediated signal transduction by SHP-1 and adaptor proteins.

基于BLNK/SLP-65是B细胞中胞浆蛋白酪氨酸磷酸酶SHP-1的生理底物的研究结果，我们重点研究了SHP-1与衔接蛋白相互作用调控B细胞受体（BCR）介导的信号转导的分子机制。结果如下。(1)BCR诱导的JNK活化显著增强，并且在表达缺乏磷酸酶活性的SHP- 1形式（SHP-1-C/S）的WEHI-231细胞中细胞凋亡受到抑制。(2)在可能通过BLNK调节JNK活化的候选蛋白质如Vav、Nck、TRAF 2和HPK 1中，发现Nck衔接蛋白与酪氨酸磷酸化的BLNK相关联，并且这种关联在SHP-1-C/S表达细胞中更明显。Nck的两种SH 2结构域突变体均不能与磷酸化的BLNK结合，提示这种结合是通过Nck的SH 2结构域介导的。此外，Nck的SH 2突变体的表达抑制了BCR诱导的JNK激活的增强。(3)Nck SH 2突变体或MKK 4的显性负性形式与SHP-1-C/S的共表达逆转了对BCR诱导的细胞凋亡的抑制，表明JNK活性负性调节细胞凋亡途径。(4)SLP-76是一种结构上与BLNK相关的衔接蛋白，已报道其优先在T细胞、NK细胞和巨噬细胞中表达，因此对于其在B细胞中的生理作用知之甚少。我们发现SLP-76在所有测试的鼠B细胞系和脾B细胞中表达，并且SHP-1使WEHI-231和鼠成熟B细胞系BAL-76中的SLP-76去磷酸化。

Based on the previous findings showing that BLNK/SLP-65 is a physiological substrate of SHP-1, a cytosolic protein tyrosine phosphatase, in B cells, we focused on the molecular mechanisms how B cell receptor (BCR)-mediated signaling is regulated by the interaction between SHP-1 and adaptor proteins. The results ate as follows.(1) BCR-induced activation of JNK is significantly enhanced and apoptosis is suppressed in WEHI-231 cells expressing a form of SHP- 1 lacking phosphatase activity (SHP-1-C/S).(2) Among candidate proteins likely to regulate JNK activation through BLNK such as Vav, Nck, TRAF2 and HPK1, Nck adaptor protein was found to associate with tyrosine-phosphorylated BLNK and this association was more pronounced in SHP-1-C/S-expressing cells. Two mutant forms of Nck possessing mutation in SH2 domain failed to bind phosphorylated BLNK, suggesting that this association is mediated via SH2 domain of Nck. Furthermore, expression of SH2 mutants of Nck inhibited the augumentation of BCR-induced JNK activation.(3) Coexpression of Nck SH2 mutants or a dominant negative form of MKK4 with SHP-1-C/S reversed suppression of BCR-induced apoptosis, indicating that JNK activity negatively regulates apoptotic pathway.(4) SLP-76 is an adaptor protein, structurally related to BLNK, has been reported to be expressed preferentially in T cells, NK cells and macrophages so that little is known as for the physiological role in B cells. We found that SLP-76 is expressed in all murine B cell lines tested and splenic B cells and that SHP-1 dephosphorylates SLP-76 in WEHI-231 and murine mature B cell line, BAL-76.