Regulation of B cell activation and inactivation by the interaction between SHP-1 and adaptor molecules

我们研究了胞浆蛋白酪氨酸磷酸酶SHP-1调节B细胞受体介导的信号转导的分子机制，并获得了以下结果：1)通过底物捕捉法，我们在小鼠未成熟B细胞系WEHI-231细胞中鉴定了SLP-76为SHP-1的新底物。与以前的报道不同，我们的数据显示SLP-76在所有受试的小鼠B细胞系和正常脾B细胞中都有表达。我们进一步研究了SLP-76在B细胞分化过程中的表达是否受到调控，发现未成熟的B细胞表达相对较低的SLP-76，表现为过渡型B细胞表达增加约20%，过渡型B细胞和成熟B细胞表达增加约40%，表明SLP-76在B细胞发育过程中表达受调控。2)SHP-1去磷酸化SLP-76抑制其与Nck的结合，下调JNK的激活，并对细胞凋亡起积极作用。…中SLP-76的故障排除通过小干扰RNA使更多的WEHI-231细胞减弱JNK的激活，但对ERK或p38的激活几乎没有影响。3)研究表明，为了激活T细胞，SLP-76/GADS复合体需要移位到酪氨酸磷酸化的LAT上，并且在BLNK缺陷的细胞中，SLP-76和LAT都是BCR信号恢复所必需的。相反，我们发现，尽管WEHI-231缺乏LAT的表达，但SLP-76的单独表达至少足以激活BCR诱导的JNK。为了阐明SLP-76在没有LAT的情况下发挥作用的机制，我们首先确定了SLP-76的亚细胞定位。虽然WEHI-231不表达LAT，但SLP-76定位于膜组分，B细胞受体(BCR)交联后SLP-76表达增加。进一步的分析表明，SLP-76与Gads的结合是通过SLP-76和Gads的SH2结构域与酪氨酸磷酸化的CD22结合的。考虑到SHP-1在BCR连接时与CD22结合，我们的发现表明SHP-1对CD22上SLP-76的去磷酸化抑制了BCR诱导的JNK激活，从而导致了细胞凋亡。较少

We have examined the molecular mechanisms how SHP-1, a cytosolic protein tyrosine phoshatase, regulates B cell receptor-mediated signaling and obtained the following results.1)By using substrate trapping approach, we identified SLP-76 as a new substrates of SHP-1 in WEHI-231 cells, a murine immature B cell line. In contrast to the previous reports, our data demonstrated that SLP-76 is expressed in all murine B cell lines tested and in normal splenic B cells. We next examined whether SLP-76 expression in B cell is regulated during differentiation of B cells and found that immature B cells expressed relatively low amount of SLP-76, which showed 〜20% increase in transitional type 1 B cells and 〜40% increase in transitional type 2 and mature B cells, indicating the regulated expression of SLP-76 during B cell development.2)Dephosphorylation of SLP-76 by SHP-1 inhibits its association with Nck, downregulating JNK activation and exerting positive effect on apoptosis. Knock down of SLP-76 in … More WEHI-231 cells by small interfering RNA attenuated JNK activation but showed little effects on ERK or p38 activation.3)It has been shown that for T cell activation, SLP-76/Gads complex needs to be translocated to tyrosine-phosphorylated LAT and that both SLP-76 and LAT are required for the restoration of BCR-signaling in BLNK-deficient cells. Contrally, we found the expression of SLP-76 alone is sufficient at least for BCR-induced JNK activation, although WEHI-231 lacks LAT expression. To clarify mechanisms by which SLP-76 functions in the absence of LAT, we first determined the subcellular localization of SLP-76. Although WEHI-231 does not express LAT, SLP-76 localizes in membrane fraction, which increases following B cell receptor (BCR) crosslinking. Further analyses reveals that SLP-76 complexed with Gads is associated with tyrosine-phosphorylated CD22 through the SH2 domains of SLP-76 and Gads. Given that SHP-1 binds to CD22 upon BCR ligation, our findings suggest that dephosphorylation of SLP-76 recruited to CD22 by SHP-1 inhibits BCR-induced JNK activation, dictating apoptosis. Less