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

基于BLNK/SLP-65是B细胞胞质蛋白酪氨酸磷酸酶SHP-1的生理底物的研究结果，我们重点研究了B细胞受体(BCR)介导的信号转导受SHP-1和接头蛋白相互作用调控的分子机制。结果如下：(1)在表达一种缺乏磷酸酶活性的SHP-1-C/S的WEHI-231细胞中，BCR诱导的JNK的激活显著增强，并抑制了细胞的凋亡。(2)在Vav、Nck、TRAF2和HPK1等可能通过BLNK调节JNK激活的候选蛋白中，发现Nck接头蛋白与酪氨酸磷酸化的BLNK有关，这种关联在表达SHP-1-C/S的细胞中更为明显。具有SH2结构域突变的两种NCK突变形式不能与磷酸化的BLNK结合，提示这种联系是通过NCK的SH2结构域介导的。此外，NCK的SH2突变体的表达抑制了BCR诱导的JNK活性的增强。(3)NCK SH2突变体或显性阴性形式的MKK4与SHP-1-C/S共表达逆转了对BCR诱导的细胞凋亡的抑制，表明JNK活性负调控凋亡途径。(4)SLP-76是一种结构上与BLNK相关的适配蛋白，已报道优先在T细胞、NK细胞和巨噬细胞中表达，因此对B细胞的生理作用知之甚少。我们发现SLP-76在所有受试的小鼠B细胞系和脾B细胞中都表达，SHP-1在WEHI-231和小鼠成熟B细胞系BAL-76中去磷酸化SLP-76。

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.