Regulation of B cell antigen receptor-mediated signaling by SHP-1

利用小鼠未成熟B细胞系WEHI-231，我们研究了SHP-1和SHP-1相关分子，即含有两个SH2结构域的胞浆蛋白酪氨酸磷酸酶(PTP)如何调节B细胞抗原受体(BCR)介导的信号转导事件，特别是在细胞增殖、分化和细胞凋亡中发挥重要作用的MAPK通路。(1)将编码PTP活性的野生型SHP-1(SHP-1-wt)或突变型SHP-1(SHP-1-CS)的Cys替换为PTP结构域中的Ser，分别导入WEHI-231细胞。然后，使用针对磷酸化形式的MAPK的抗体，通过Western blotting检测MAPK家族每个成员的活性。SHP-1-wt和SHP-1-CS转染组ERK活性与未转染组基本相同，而抗-Ig M诱导的JNK和p38活性均高于对照组…更多。值得注意的是，抗IgM刺激后的JNK活性增强最为显著。此外，SHP-1-wt转基因细胞还能增强抗IgM诱导的细胞凋亡。(2)我们先前已经证明SHP-1与SLP-76密切相关，SLP-76是一种C端含有SH2结构域的白细胞特异性蛋白。接下来，将编码野生型和突变型SLP-76基因的cDNA导入WEHI-231细胞，并分析其对抗IgM诱导的MAPK活性的影响。未转染组和转染组的三个MAPK成员的活性程度差异不大。相反，SH2结构域不能结合磷酸化Tyr残基的突变型SLP-76可显著抑制抗IgM诱导的细胞死亡。(3)当SHP-1-CS编码的基因被导入细胞后，抗IgM刺激后几种蛋白的酪氨酸磷酸化增强。我们聚焦于一种磷酸化程度最高的蛋白质作为SHP-1底物的候选蛋白，并对其性质进行了表征。较少

By using mouse immature B cell line, WEHI-231, we have investigated how SHP-1 and SHP-1-associated molecules, cytoplasmic proteintyrosine phosphatase (PTP) containing two SH2 domains, regulate B cell antigen receptor (BCR)-mediated signaling event, especially MAP kinase (MAPK) pathways which play important roles in cell proliferation, differentiation and apoptotic cell death. The results obtained are follows ;(1) WEHI-231 cells were transfected with cDNA encoding wild-type SHP-1 (SHP-1-wt) or mutant SHP-1 (SHP-1-CS) which lacks PTP activity by substituting Cys to Ser in PTP domain. Then, the activity of each member of MAPK family was measured by Western blotting using antibodies against phosphorylated forms of MAPKs. ERK activity in SHP-1-wt or SHP-1-CS transfectant after the stimulation with anti-IgM Ab was almost identical to that in untransfected control cells, whereas anti-IgM-induced JNK and p38 activities in both transfectants were enhanced when compared to those in control cells … More . It should be noted that enhancement of JNK activity after anti-IgM stimulation was most prominent. In addition, SHP-1-wt transfectant showed enhanced anti-IgM-induced apoptotic cell death.(2) We have previously showen that SHP-1 is constitutively associated with SLP-76, leukocyte specific protein containing a SH2 domain at its C terminus. Next, WEHI-231 cells were transfected with cDNA encoding wild-type or mutant forms of SLP-76 and anti-IgM-induced MAPK activation was analyzed in these transfectants. There are little differences in the degree of activities of three MAPK members between untransfected and transfected cells. In contrast, anti-IgM-induced cell death was significantly suppressed in the cells transfected with mutant SLP-76 whose SH2 domain fails to bind phosphorylated Tyr residues.(3) When cells were transfected with SHP-1-CS-encoding cDNA, tyrosine phosphorylation of several proteins were enhanced after anti-IgM stimulation. We focused on one protein whose phosphorylation was most enhanced as a candidate for the SHP-1 substrate and characterized its nature. Less