Molecular Mechanism of Lymphocyte Activation

摘要：T细胞或B细胞抗原受体(TCR或BCR)被抗原或抗受体抗体干扰，可诱导蛋白酪氨酸激酶的激活、磷脂酶CGamma-1(PLCG1)的磷酸化和激活，以及小G蛋白的激活，如RAS。特别是PLCG1的激活，介导了磷脂酰肌醇(PI)的水解，进而控制着T或B淋巴细胞激活过程中的钙动员和蛋白激酶C的激活。该实验室致力于建立免疫受体调节PLCG1的机制。突变分析表明，PLCG1氨基末端SH2(SH2N)结构域是TCR或BCR诱导的PLCG1酪氨酸磷酸化所必需的，也是充分的，并与T细胞适配器LAT或B细胞适配器BLNK结合。PLCG1膜转位需要SH2N结构域，SH3结构域突变后PLCG1膜转位减少。BCR诱导的PI水解度可被SH2N或SH2(SH2C)结构域的突变所抑制，而被SH3结构域的突变所抑制。脂筏，特殊的膜微域，在PLCG1的磷酸化和激活中的作用也被证明。细胞原癌基因Cbl是一种与包括PLCG1在内的多种信号蛋白结合的接头，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点。我们发现，Cb1的过表达降低了TCR诱导的AP1(RAS依赖的转录因子)和NF-AT(钙依赖的转录因子)的报告活性，这表明Cb1可能在RAS和PLCG1/Ca~(2+)途径所共有的点起作用。Cbl过表达可降低TCR诱导的PI水解率，但对TCR诱导的PLCG1酪氨酸磷酸化无影响。相比之下，癌基因Cbl突变体70z/3的过表达促进了PI的水解性。这些数据支持免疫受体诱导PLCG1激活的顺序模型，其中最早的事件是SH2N结构域被一种磷蛋白结合，该磷蛋白在膜内的激活复合体中招募PLCG1并随后磷酸化，而SH3结构域则贡献了额外的膜锚定。PLCG1活性的上调需要移位和磷酸化，并分别通过SH2N或SH3结构域的破坏而被取消或降低。受体诱导的PLCG1激活只需要SH2C结构域。CBL通过以SH3结构域依赖的方式影响PLCG1磷酸化下游或不依赖于PLCG1的一个点来调节受体诱导的PLCG1活性。

Summary: 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. Mutational analysis demonstrated that PLCg1 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. A role for lipid rafts, specialized membrane microdomains, in phosphorylation and activation of PLCg1 was also demonstarted. 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. In constrast, over-expression of an oncogenic Cbl mutant, 70Z/3, enhanced PI-hydrolysis. 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.