Luton F, Buferne M, Davoust J, Schmitt-Verhulst A M, Boyer C
Centre d'Immunologie INSERM-CNRS de Marseille-Luminy, France.
J Immunol. 1994 Jul 1;153(1):63-72.
Triggering of the TCR/CD3 complex can lead to its internalization and modulation from the cell surface. In the present study, we address the question of the dependence of internalization on protein tyrosine kinase (PTK) activation. With use of an activating anti-clonotypic (anti-Ti) mAb on a CTL clone, we have shown that the PTK inhibitors genistein and tyrphostin 25 delayed anti-Ti-induced internalization, but did not affect fluid phase protein uptake or transferrin receptor cycling. Confocal microscopy with use of fluorescent anti-Ti mAb revealed that the inhibition of TCR internalization corresponded to the induction of large patches that were localized in cell membrane areas depleted of polymerized actin, the formation of which was dependent on the combined action of the anti-Ti mAb and the PTK inhibitors. In contrast to the effect of these PTK inhibitors, depletion of Src-like PTKs by T cell pretreatment with herbimycin A led to an increased rate of anti-Ti-induced internalization. Internalization induced by the monovalent Fab fraction of anti-Ti mAb was similarly affected by the PTK inhibitors, although the extent of induced internalization was less by approximately one-half. An analysis of substrates phosphorylated in kinase assays on TCR/CD3 immunoprecipitates of the CTL, which were activated by anti-Ti mAb in both the absence and presence of genistein, identified protein bands in which phosphorylation or association with CD3 was inhibited in the presence of genistein. Thus, a genistein-sensitive PTK activity seems to control ligand-induced TCR/CD3 complex redistribution and internalization.
TCR/CD3复合物的激活可导致其从细胞表面内化和调节。在本研究中，我们探讨了内化对蛋白酪氨酸激酶（PTK）激活的依赖性问题。通过在CTL克隆上使用激活型抗克隆型（抗Ti）单克隆抗体，我们发现PTK抑制剂染料木黄酮和 tyrphostin 25延迟了抗Ti诱导的内化，但不影响液相蛋白摄取或转铁蛋白受体循环。使用荧光抗Ti单克隆抗体的共聚焦显微镜显示，TCR内化的抑制对应于大斑块的诱导，这些斑块位于聚合肌动蛋白耗尽的细胞膜区域，其形成依赖于抗Ti单克隆抗体和PTK抑制剂的联合作用。与这些PTK抑制剂的作用相反，用除草菌素A对T细胞进行预处理以耗尽Src样PTK，导致抗Ti诱导的内化速率增加。抗Ti单克隆抗体的单价Fab片段诱导的内化同样受到PTK抑制剂的影响，尽管诱导内化的程度大约降低了一半。对CTL的TCR/CD3免疫沉淀物进行激酶分析时磷酸化的底物进行分析，这些沉淀物在不存在和存在染料木黄酮的情况下均由抗Ti单克隆抗体激活，确定了在存在染料木黄酮时磷酸化或与CD3结合受到抑制的蛋白条带。因此，一种对染料木黄酮敏感的PTK活性似乎控制着配体诱导的TCR/CD3复合物的重新分布和内化。
