MOLECULAR MECHANISM OF LYMPHOCYTE ACTIVATION

淋巴细胞激活的分子机制

• 批准号: 6293789
• 负责人: EZIO BONVINI
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6293789
• 关键词: T cell receptor; T lymphocyte; antigen receptors; biological signal transduction; chimeric proteins; clone cells; enzyme activity; enzyme structure; gene expression; intermolecular interaction; leukocyte activation /transformation; major histocompatibility complex; phospholipase C; phosphoproteins; phosphorylation; protein structure function; receptor binding; transfection

Perturbation of the T- or B-cell antigen receptor (TCR or BCR) by antigen or by anti-receptor antibodies induces protein tyrosine kinase activation, the phosphorylation and activation of phospholipase Cgamma-1 (PLCg1), and the activation of small G-proteins, such as Ras. The activation of PLCg1, in particular, mediates phosphoinositide (PI) hydrolysis which in turn controls Ca2+ mobilization and protein kinase C activation, obligatory events in the activation of T- or B-lymphocytes. The laboratory is concerned with establishing the mechanisms by which immune receptors regulate PLCg1. PLCg1 has two src-homology (SH) 2 domains that bind tyrosine-phosphorylated proteins and one SH3 domain that interacts with proline-rich polipeptides. These domains play a role in coupling proteins to other regulatory molecules. Mutational analysis demonstrated that the amino terminal SH2 (SH2N) domain was required and sufficient for TCR- or BCR-induced tyrosine phosphorylation of PLCg1, and for the association of PLCg1 with the T cell adapter, LAT, or the B cell adapter, BLNK. PLCg1 membrane translocation required the SH2N domain and was decreased by mutation of the SH3 domain. BCR-induced PI hydrolysis was abrogated by mutation of either the SH2N or the carboxyl-terminal SH2 (SH2C) domain and was decreased by mutation of the SH3 domain. The cellular proto-oncogene, Cbl, is an adapter that associates with numerous signaling proteins, including PLCg1, involved in signal transduction by distinct receptors. Cbl is a major target of tyrosine phosphorylation after TCR engagement. We have found that over-expression of Cbl reduced TCR-induced AP1 (a Ras-dependent transcription factor) and NF-AT (a Ca2+-dependent transcription factor) reporter activities, suggesting that Cbl could act at a point shared by both the Ras and PLCg1/Ca2+ pathways. Cbl over-expression decreased TCR-induced PI-hydrolysis, but had no effect on TCR-induced tyrosine phosphorylation of PLCg1. An SH3 binding site and the aa. 655-906 region were required for Cbl-mediated regulation of PLCg1. Over-expression of an oncogenic Cbl mutant, 70Z/3, did not affect TCR-induced NF-AT activation and enhanced PI-hydrolysis. Wild type Cbl and the 70Z/3 Cbl were similarly able to bind PLCg1, indicating that the interaction with PLCg1 does not directly inhibit its activity. These data support a sequential model of immune receptor-induced PLCg1 activation where the earliest event is the engagement of the SH2N domain by a phosphoprotein that recruits PLCg1 in an activation complex within the membrane with ensuing phosphorylation, while the SH3 domain contributes additional membrane anchoring. Up-regulation of PLCg1 activity requires translocation and phosphorylation and is abrogated or reduced by disruption of the SH2N or SH3 domains, respectively. The SH2C domain is exclusively required for receptor-induced PLCg1 activation. Cbl regulates receptor-induced PLCg1 activity by affecting, in an SH3 domain-dependent manner, a point downstream of or independent of PLCg1 phosphorylation.

抗原或抗受体抗体对 T 或 B 细胞抗原受体 (TCR 或 BCR) 的干扰会诱导蛋白酪氨酸激酶激活、磷脂酶 Cgamma-1 (PLCg1) 磷酸化和激活，以及小 G 蛋白（如 Ras）的激活。 PLCg1 的激活尤其介导磷酸肌醇 (PI) 水解，进而控制 Ca2+ 动员和蛋白激酶 C 激活，这是 T 或 B 淋巴细胞激活中的必然事件。该实验室致力于建立免疫受体调节 PLCg1 的机制。 PLCg1 具有两个结合酪氨酸磷酸化蛋白的 src 同源 (SH) 2 结构域和一个与富含脯氨酸的多肽相互作用的 SH3 结构域。这些结构域在蛋白质与其他调节分子的偶联中发挥作用。突变分析表明，氨基末端 SH2 (SH2N) 结构域是 TCR 或 BCR 诱导的 PLCg1 酪氨酸磷酸化以及 PLCg1 与 T 细胞接头 LAT 或 B 细胞接头 BLNK 关联所必需的且足够的。 PLCg1 膜易位需要 SH2N 结构域，并且通过 SH3 结构域的突变而减少。 BCR 诱导的 PI 水解可通过 SH2N 或羧基末端 SH2 (SH2C) 结构域的突变而消除，并通过 SH3 结构域的突变而减少。细胞原癌基因 Cbl 是一种接头蛋白，与许多信号蛋白相关，包括 PLCg1，参与不同受体的信号转导。 Cbl 是 TCR 参与后酪氨酸磷酸化的主要靶标。我们发现 Cbl 的过度表达降低了 TCR 诱导的 AP1（一种 Ras 依赖性转录因子）和 NF-AT（一种 Ca2+ 依赖性转录因子）报告基因活性，表明 Cbl 可以在 Ras 和 PLCg1/Ca2+ 途径共享的点上发挥作用。 Cbl 过表达降低了 TCR 诱导的 PI 水解，但对 TCR 诱导的 PLCg1 酪氨酸磷酸化没有影响。 SH3 结合位点和 aa。 Cbl 介导的 PLCg1 调节需要 655-906 区域。致癌 Cbl 突变体 70Z/3 的过度表达不会影响 TCR 诱导的 NF-AT 激活和增强的 PI 水解。野生型Cbl和70Z/3 Cbl同样能够结合PLCg1，表明与PLCg1的相互作用不会直接抑制其活性。这些数据支持免疫受体诱导的 PLCg1 激活的顺序模型，其中最早的事件是 SH2N 结构域与磷蛋白的结合，该磷蛋白在膜内的激活复合物中招募 PLCg1，并随后磷酸化，而 SH3 结构域则提供额外的膜锚定。 PLCg1 活性的上调需要易位和磷酸化，并且分别通过 SH2N 或 SH3 结构域的破坏而消除或减少。 SH2C 结构域是受体诱导的 PLCg1 激活所必需的。 Cbl 通过以 SH3 结构域依赖性方式影响 PLCg1 磷酸化下游或独立的点来调节受体诱导的 PLCg1 活性。