MOLECULAR MECHANISM OF T LYMPHOCYTE ACTIVATION--ROLO OF PROTOONCOGENE, CB1

实验室致力于确定细胞的作用。 癌基因CBL在T淋巴细胞活化中的作用。而CBL的功能是 未知，它是酪氨酸磷酸化的主要靶点 多种受体的结合，包括T细胞的抗原受体 B淋巴细胞、Fc受体、EGF和GM-CSF受体， 提示CBL参与了信号转导途径 由这些不同的受体类型触发。此外，CBL员工 含有许多被认为在调节过程中活跃的蛋白质 受体介导的激活事件。这些蛋白质包括适配器 分子(Grb2，Nck，P85-PI3K和Crk)，蛋白酪氨酸激酶(Lck， Fyn、ZAP70和SYK)，以及其他效应分子，如PLCG1。在T中 淋巴细胞。我们已经发现CBL的过度表达显著地 降低TCR交联激活AP1(RAS)的能力 和核因子-AT(PLC/Ca)激活通路。以AP1为例 激活时，CBL似乎在RAS水平上起作用 激活或RAS激活的上游。CBL对核因子-AT无影响 PMA和离子霉素刺激细胞时的活性提示 CBL可能直接调节PLCG1的激活。此外， CBL与适配器Grb2的关联性被发现在 T细胞激活，而胍交换的关联 TCR交联后，GRb2的SOS因子增加。因为Grb2 被认为介导了SOS的激活依赖的易位，从 细胞质到膜，在那里它与RAS相互作用并激活RAS， 我们目前正在调查CBL是否会影响RAS的激活 通过调节SOS与Grb2相互作用的能力。这是一种 通过分析CBL过度表达如何影响SOS的关联来完成 通过产生和研究CBL突变体的功能 它们不再能够与Grb2相互作用。这些和其他CBL 突变体也将被用来确定CBL 调节PLC/Ca途径。我们的实验室已经证明，CBL可以直接 与PLC交互。我们实验室生成的表位标记的PLC构建 将用于分析CBL对激活依赖PLC的影响 磷酸化和易位。长期计划是产生一个CBL 缺陷T细胞株完全阐明CBL在T细胞中的作用 发信号。Cbl在该品系中的表达可与野生型 决定CBL结构/功能的CBL类型和突变形式 不同CBL区域之间的关系。

The laboratory is concened with identifing the role of the cellular oncogene, cbl, in T lymphocyte activation. While the function of cbl is unknown, it is a major target of tyrosine phosphorylation after the engagement of multiple receptors, including the antigen receptors of T- and B-lymphocytes, Fc-receptors, and receptors for EGF and GM-CSF, suggesting that cbl is involved in the signal transduction pathways triggered by these distinct receptor types. In addition, cbl associates with numerous proteins which are thought to be active in the regulation of receptor mediated activation events. These proteins include adapter molecules (Grb2, nck, p85-PI3K and crk), protein tyrosine kinases (lck, fyn, Zap70 and syk), and other effector molecules, such as PLCg1. In T lymphocytes. we have found that over-expression of cbl significantly reduced the abiltiy of TCR cross-linking to activate both the AP1 (Ras) and the NF-AT (PLC/Ca++) activation pathways. In the case of AP1 activation, cbl appeared to be acting either at the level of Ras activation or upstream of Ras activation. Cbl had no affect on NF-AT activity when cells were stimulated with PMA and ionomycin suggesting that cbl may directly regulate PLCg1 activation. In addition, the association of cbl with the adaptor, Grb2, was found to decrease after T cell activation, whereas the association of the guanidine exchange factor, Sos, with Grb2 increased after TCR cross-linking. Because Grb2 is thought to mediate an activation dependent translocation of Sos, from the cytoplasm to the membrane where it interacts with and activates Ras, we are currently investigating whether cbl may influence Ras activation by regulating the ability of Sos to interact with Grb2. This is being done by analyzing how cbl over-expression affects the association of Sos with Grb2 and by generating and investigating the function of cbl mutants which are no longer able to interact with Grb2. These and additional cbl mutants will also be used to determine the mechanism by which cbl regulates the PLC/Ca++ pathway. Our lab has shown that cbl can directly interact with PLC. Epitope-tagged PLC constructs generated in our lab will be used to analyze the affect cbl has on activation-dependent PLC phosphorylation and translocation. Long range plans are to generate a cbl deficient T cell line to fully elucidate the role of cbl in T cell signaling. Cbl expression in this line can be reconstituted with wild type and mutant forms of cbl to determine the structure/function relationship of different cbl regions.