Molecular Mechanism of Lymphocyte Activation

抗原或抗受体抗体对T细胞或B细胞抗原受体（TCR或BCR）的扰动诱导蛋白酪氨酸激酶活化、磷脂酶C γ-1（PLCg 1）的磷酸化和活化以及小G蛋白（如Ras）的活化。PLCg 1的激活，特别是介导磷酸肌醇（PI）水解，这反过来又控制Ca 2+动员和蛋白激酶C激活，在T-或B-淋巴细胞的激活中的强制性事件。该实验室致力于建立免疫受体调节PLCg 1的机制。 突变分析表明，PLC g1氨基末端SH 2（SH 2N）结构域是TCR或BCR诱导的PLC g1酪氨酸磷酸化所必需的，并且足以使PLC g1与T细胞适配器LAT或B细胞适配器BLNK结合。PLCg 1膜转位需要SH 2N结构域，并通过SH 3结构域的突变而减少。 BCR诱导的PI水解被废除的突变的SH 2N或羧基末端SH 2（SH 2C）结构域和减少突变的SH 3结构域。脂筏，专门的膜微区，在磷酸化和激活PLCg 1的作用也demonstarted。细胞原癌基因Cbl是一种衔接子，与许多信号蛋白（包括PLCg 1）相关，参与不同受体的信号转导。Cbl是TCR接合后酪氨酸磷酸化的主要靶标。我们已经发现，Cbl的过表达降低TCR诱导的AP 1（Ras依赖性转录因子）和NF-AT（Ca 2+依赖性转录因子）报告子活性，表明Cbl可以在Ras和PLC g1/Ca 2+途径共享的点起作用。Cbl过表达降低TCR诱导的PI水解，但对TCR诱导的PLCg 1酪氨酸磷酸化没有影响。在p53 ast中，致癌Cbl突变体70 Z/3的过度表达增强了PI水解。这些数据支持免疫受体诱导的PLCg 1激活的顺序模型，其中最早的事件是SH 2N结构域通过磷蛋白的参与，该磷蛋白在膜内的激活复合物中招募PLCg 1，随后磷酸化，而SH 3结构域有助于额外的膜锚定。PLCg 1活性的上调需要易位和磷酸化，并且分别通过SH 2N或SH 3结构域的破坏而被废除或减少。SH 2C结构域是受体诱导的PLCg 1激活所必需的。Cbl通过影响受体诱导的PLCg 1活性，以SH 3结构域依赖的方式，下游或独立于PLCg 1磷酸化的点。

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.