Amsen D, Kruisbeek A, Bos J L, Reedquist K
Division of Immunology, The Netherlands Cancer Institute, Amsterdam.
Eur J Immunol. 2000 Oct;30(10):2832-41. doi: 10.1002/1521-4141(200010)30:10<2832::AID-IMMU2832>3.0.CO;2-D.
Signals mediated by activation of the small GTPase Ras play an essential role both in thymocyte development and in TCR-mediated activation of mature T cells. Given the critical requirement of Ras signaling pathways in thymocyte development, and recent indications that Rap1 may negatively regulate Ras-dependent signaling pathways, we examined the possible involvement of Rap1 in thymocyte TCR signaling. We find that Rap1 and proposed regulators of Rap1 (the proto-oncogene product Cbl, Crk family adaptor proteins, and the Rap1 guanine nucleotide exchange factor C3G) are expressed at equivalent levels in both double-negative and double-positive murine thymocytes. Rap1 was transiently activated following TCR stimulation of both total thymocytes and purified double-positive thymocytes, and this activation correlated with tyrosine phosphorylation of Cbl and Cbl association with CrkL. TCR-dependent Rap1 activation was enhanced by co-stimulation through CD28 and could be mimicked by treatment of thymocytes with phorbol ester and calcium. In contrast to mature peripheral T lymphocytes, Rap1 stimulation by CD3 ligation in thymocytes did not require intracellular calcium mobilization. Intriguingly, we found a clear elevation of activated Rap1 in thymocytes undergoing positive selection, suggesting a functional role for Rap1 in thymocyte development and selection.
由小GTP酶Ras激活介导的信号在胸腺细胞发育以及TCR介导的成熟T细胞激活过程中均发挥着至关重要的作用。鉴于Ras信号通路在胸腺细胞发育中的关键需求，以及近期有迹象表明Rap1可能对Ras依赖性信号通路起负向调节作用，我们研究了Rap1在胸腺细胞TCR信号传导中的可能作用。我们发现，Rap1及其假定的调节因子（原癌基因产物Cbl、Crk家族衔接蛋白以及Rap1鸟嘌呤核苷酸交换因子C3G）在双阴性和双阳性小鼠胸腺细胞中的表达水平相当。在TCR刺激总胸腺细胞和纯化的双阳性胸腺细胞后，Rap1会被短暂激活，且这种激活与Cbl的酪氨酸磷酸化以及Cbl与CrkL的结合相关。通过CD28共刺激可增强TCR依赖性Rap1激活，用佛波酯和钙处理胸腺细胞也可模拟这种激活。与成熟外周T淋巴细胞不同,胸腺细胞中CD3连接介导的Rap1刺激不需要细胞内钙动员。有趣的是，我们发现正在经历阳性选择的胸腺细胞中活化的Rap1明显升高，这表明Rap1在胸腺细胞发育和选择中具有功能性作用。
