Hornung M, Lindemann D, Kraus C, Peters A, Berberich I
Institut für Virologie und Immunbiologie, Universität Würzburg, Germany.
Eur J Immunol. 1998 Nov;28(11):3812-23. doi: 10.1002/(SICI)1521-4141(199811)28:11<3812::AID-IMMU3812>3.0.CO;2-N.
Engagement of the antigen receptor on WEHI 231 murine B lymphoma cells leads to growth arrest and induction of apoptosis. Concomitant signaling through CD40 sustains proliferation and rescues the cells from apoptosis. At the molecular level, CD40 has been shown to activate nuclear factor kappaB (NF-kappaB) and stress-activated protein kinase (SAPK). The aim of our present study was to define the stretch of the CD40 cytoplasmic tail responsible for mediating these effects in WEHI 231 cells. Using recombinant retroviruses with the enhanced green fluorescent protein as selection marker we transduced WEHI 231 cells with chimeric molecules consisting of the extracellular and transmembrane region of human CD40 or rat CD4 and selected portions of the murine CD40 tail. Chimeric molecules with cytoplasmic fragments encompassing the "CD40 tumor necrosis factor-associated factor family member interacting motif" (TIM) were able to sustain growth and to uphold NF-kappaB activity as efficiently as the whole intracellular region of CD40. While the potential of the motif relative to the whole cytoplasmic tail was independent of the heterologous part of the chimeras it was strongly influenced by its distance to the membrane. Placing the 17-amino acid stretch of the motif too close to the membrane, i. e. only two or four amino acids apart, destroyed its capacity to mitigate the anti-IgM effect. Activation of SAPK through the chimeric molecules always correlated with their ability to activate NF-kappaB activity and to rescue the cells from apoptosis induced by antigen receptor ligation. Our data indicate that CD40-TIM carries most if not all of the information needed to deliver the signals responsible for sustaining growth in anti-IgM-stimulated WEHI 231 cells.
WEHI 231小鼠B淋巴瘤细胞上抗原受体的激活会导致生长停滞并诱导细胞凋亡。通过CD40的伴随信号传导可维持细胞增殖并使细胞免于凋亡。在分子水平上，CD40已被证明可激活核因子κB（NF-κB）和应激激活蛋白激酶（SAPK）。我们当前研究的目的是确定负责在WEHI 231细胞中介导这些效应的CD40胞质尾部区域。我们使用带有增强型绿色荧光蛋白作为选择标记的重组逆转录病毒，用由人CD40或大鼠CD4的细胞外和跨膜区域以及小鼠CD40尾部的选定部分组成的嵌合分子转导WEHI 231细胞。具有包含“CD40肿瘤坏死因子相关因子家族成员相互作用基序”（TIM）的胞质片段的嵌合分子能够像CD40的整个细胞内区域一样有效地维持生长并保持NF-κB活性。虽然该基序相对于整个胞质尾部的潜能与嵌合体的异源部分无关，但它受到其与膜距离的强烈影响。将该基序的17个氨基酸片段放置得离膜太近，即仅相隔两个或四个氨基酸，会破坏其减轻抗IgM效应的能力。通过嵌合分子激活SAPK总是与其激活NF-κB活性以及使细胞免于抗原受体连接诱导的凋亡的能力相关。我们的数据表明，CD40-TIM携带了在抗IgM刺激的WEHI 231细胞中传递维持生长所需信号的大部分（如果不是全部）信息。
