Ren C L, Fu S M, Geha R S
Division of Immunology, Children's Hospital, Boston, MA.
Immunol Invest. 1994 Nov;23(6-7):437-48. doi: 10.3109/08820139409066838.
CD40 is a 47kD glycoprotein expressed on all B cells that plays an important role in B cell development and activation. Previous investigations have focused on signal transduction events in activated B cells and B cell lines, and little information is available regarding CD40 signal transduction in resting, normal B cells. Because CD40 plays a critical role in the regulation and activation of resting B cells, we studied the signaling mechanisms involved in functional responses to CD40 ligation in these cells. Treatment of dense tonsil B cells with either protein tyrosine kinase (PTK) or protein kinase C (PKC) inhibitors, but not with an inhibitor of cyclic nucleotide dependent kinases, resulted in abrogation of CD40-mediated cell adhesion, suggesting a role for both PTK and PKC in CD40-mediated B cell adhesion. Direct evidence for CD40-mediated PTK activation was demonstrated by increased tyrosine phosphorylation as detected by anti-phosphotyrosine Western blots of cell lysates prepared from dense resting tonsil B cells stimulated with biotinylated anti-CD40 monoclonal antibody plus avidin. CD40 engagement also resulted in PKC activation, as detected by translocation of cytosolic PKC activity to the membrane-bound compartment. CD40-mediated PKC translocation was dependent on PTK activation, whereas PTK activity induced by CD40 ligation was independent of PKC activity, suggesting that PTK activation precedes PKC activation in resting B cells stimulated with anti-CD40 mAb. The results of our experiments identify PTK and PKC activation as components of CD40 signal transduction in normal, resting B cells and establish a functional role for these events.
CD40是一种在所有B细胞上表达的47kD糖蛋白，在B细胞发育和激活中起重要作用。以往的研究主要集中在活化B细胞和B细胞系中的信号转导事件，而关于静息正常B细胞中CD40信号转导的信息较少。由于CD40在静息B细胞的调节和激活中起关键作用，我们研究了这些细胞中对CD40配体功能反应所涉及的信号传导机制。用蛋白酪氨酸激酶（PTK）或蛋白激酶C（PKC）抑制剂处理密集扁桃体B细胞，但不用环核苷酸依赖性激酶抑制剂处理，导致CD40介导的细胞粘附被废除，这表明PTK和PKC在CD40介导的B细胞粘附中都起作用。通过对用生物素化抗CD40单克隆抗体加抗生物素蛋白刺激的密集静息扁桃体B细胞制备的细胞裂解物进行抗磷酸酪氨酸免疫印迹检测，发现酪氨酸磷酸化增加，从而证明了CD40介导的PTK激活的直接证据。CD40结合还导致PKC激活，这可通过胞质PKC活性向膜结合区室的转位来检测。CD40介导的PKC转位依赖于PTK激活，而CD40配体诱导的PTK活性独立于PKC活性，这表明在用抗CD40单克隆抗体刺激的静息B细胞中，PTK激活先于PKC激活。我们的实验结果确定PTK和PKC激活是正常静息B细胞中CD40信号转导的组成部分，并确立了这些事件的功能作用。
