Molecular Mechanism of Lymphocyte Immuneactivation and S

淋巴细胞免疫激活及S的分子机制

• 批准号: 6839809
• 负责人: BARBARA L RELLAHAN
• 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/6839809
• 关键词: B lymphocyte; T lymphocyte; biological signal transduction; cellular immunity; leukocyte activation /transformation; phospholipase C; phosphorylation; protein tyrosine kinase

Crosslinking of the antigen (Ag) receptor by antigen or by anti-receptor antibodies induces signaling (Signal 1) via protein tyrosine kinase activation, the phosphorylation and activation of phospholipase C gamma-1 (PLCg1), and the activation of Ras and related molecules. The activation of PLCg1, in particular, mediates phosphoinositide (PI) hydrolysis which in turn controls calcium mobilization and protein kinase C activation, obligatory events in the activation of T- or B-lymphocytes. Furthermore, certain co-stimulatory molecules (e.g., CD2, CD28) responsible for a second required activation signal (Signal 2) also affect PLCg1 activation. The proper physiologic combination of Signal 1 and 2 induces immune activation, while an unbalanced signal will fail to activate cells or induce tolerance. The laboratory is concerned with establishing the mechanisms by which immune receptors regulate PLCg1 activation. While both of the src homology 2 (SH2) domains of PLCg1are required for full in vivo activation, the amino SH2 (SH2N) domain has been shown to be responsible for antigen-induced binding to LAT in T cells, thereby facilitating PLCg1 tyrosine phosphorylation and membrane translocation. Recently, we identified an intra-molecular association between the SH2C domain and the amino terminal split pleckstrin homology (sPHN) domain that sequesters it from associating with other proteins and promotes a structural configuration that inhibits PLCg1 enzymatic activity. The SH2C domain also appears to exert additional functions, including coupling the co-stimulatory signaling pathway to downstream effectors. Hence, the SH2C domain may be at the crossroads of Signal 1 and 2. Moreover data from series of PLCg1 variants with both rearranged and tandem SH2 domains indicated that the SH2 domains of PLCg1 do not have redundant functions. Data from a series of tyrosine (Y) to phenylalanine mutants identified two tyrosine residues that are critical for Ag receptor-induced activation of PLCg1. Mutation of either Y led to a significant decrease in PLCg1 activation, while mutation of both residues resulted in an almost complete defect in PLCg1 activation as measured by the Ag receptor-induced calcium response. Lastly, mammalian expression constructs for a catalytically inactive cytosolic and a catalytically inactive raft targeted (palmitoylated) PLCg1 were generated with the rationale of investigating possible non-enzymatic adaptor functions of PLCg1 and the role of localization in signal propagation. Both constructs were phosphorylated to the same degree as their catalytically active counterpart and both acted as dominant negatives in TCR induced PLCg1 activation although the raft-targeted construct demonstrated a stronger inhibitory potency than its cytosolic counterpart. The cellular proto-oncogene, c-Cbl, is an adapter that associates with numerous signaling proteins, including PLCg1, involved in signal transduction by distinct receptors. c-Cbl is a major target of tyrosine phosphorylation after TCR engagement. The laboratory used over-expression of c-Cbl in a thymoma and a B-cell line to defined the sub-domain structure and post-translational modifications of c-Cbl that are required for its ability to regulate signal transduction pathways. This work demonstrated, among other things, that binding of c-Cbl by the SH3 and SH2C domains of PLCg1 is required for c-Cbl to regulate the activation of PLCg1. This is in contrast to the sub-domain requirements of c-Cbl-mediated regulation of tyrosine kinase targets (such as the ErbB2 receptor) and therefore a new model of c-Cbl regulation of non-tyrosine kinase targets was proposed. In addition, the laboratory investigated the mechanism of action of an oncogenic form of c-Cbl, 70Z/3 Cbl, and identified a unique activation pathway that is triggered by this oncoprotein. These data support a model of immune receptor-induced PLCg1 activation whereby positive regulation is exerted by the scaffold-binding role of the SH2N domain which is regulated by Signal 1. The SH2C domain plays a complex role since it both negatively regulates PLCg1 activity through an intra-molecular association with the sPHN domain and positively influences PLCg1 activation through an, as of yet, unidentified mechanism which is influenced by both Signal 1 and 2. In addition both the SH2C and the SH3 domains of PLCg1 appear to be required for the c-Cbl-mediated negative feedback loop of PLCg1 activation induced by Signal 1. Molecular Mechanism of Lymphocyte Immuneactivation and Suppression. Perturbation of the antigen (Ag) receptor by antigen or by anti-receptor antibodies induces signaling (Signal 1) via protein tyrosine kinase activation, the phosphorylation and activation of phospholipase C gamma-1 (PLCg1), and the activation of Ras and related molecules. The activation of PLCg1, in particular, mediates phosphoinositide (PI) hydrolysis which in turn controls calcium mobilization and protein kinase C activation, obligatory events in the activation of T- or B-lymphocytes. Furthermore, certain co-stimulatory molecules (e.g., CD2, CD28) responsible for a second required activation signal (Signal 2) also affect PLCg1 activation. The proper physiologic combination of Signal 1 and 2 induces immune activation, while an unbalanced signal will fail to activate cells or induce tolerance. The laboratory is concerned with establishing the mechanisms by which immune receptors regulate PLCg1 activation. While both of the src homology 2 (SH2) domains of PLCg1are required for full in vivo activation, the amino SH2 (SH2N) domain has been shown to be responsible for antigen-induced binding to LAT in T cells, thereby facilitating PLCg1 tyrosine phosphorylation and membrane translocation. Recently, we identified an intra-molecular association between the SH2C domain and the amino terminal split pleckstrin homology (sPHN) domain that sequesters it from associating with other proteins and promotes a structural configuration that inhibits PLCg1 enzymatic activity. The SH2C domain also appears to exert additional functions, including coupling the co-stimulatory signaling pathway to downstream effectors. Hence, the SH2C domain may be at the crossroads of Signal 1 and 2. Moreover data from series of PLCg1 variants with both rearranged and tandem SH2 domains indicated that the SH2 domains of PLCg1 do not have redundant functions. Data from a series of tyrosine (Y) to phenylalanine mutants identified two tyrosine residues that are critical for Ag receptor-induced activation of PLCg1. Mutation of either Y led to a significant decrease in PLCg1 activation, while mutation of both residues resulted in an almost complete defect in PLCg1 activation as measured by the Ag receptor-induced calcium response. Lastly, mammalian expression constructs for a catalytically inactive cytosolic and a catalytically inactive raft targeted (palmitoylated) PLCg1 were generated with the rationale of investigating possible non-enzymatic adaptor functions of PLCg1 and the role of localization in signal propagation. Both constructs were phosphorylated to the same degree as their catalytically active counterpart and both acted as dominant negatives in TCR induced PLCg1 activation although the raft-targeted construct demonstrated a stronger inhibitory potency than its cytosolic counterpart. These data support a model of immune receptor-induced PLCg1 activation whereby positive regulation is exerted by the scaffold-binding role of the SH2N domain which is regulated by Signal 1. The SH2C domain plays a complex role since it both negatively regulates PLCg1 activity through an intra-molecular association with the sPHN domain and positively influences PLCg1 activation through an, as of yet, unidentified mechanism which is influenced by both Signal 1 and 2.

抗原或抗受体抗体将抗原（Ag）受体交联，通过蛋白酪氨酸激酶激活、磷脂酶C γ -1 （PLCg1）的磷酸化和激活以及Ras及相关分子的激活诱导信号传导（Signal 1）。特别是，PLCg1的激活介导磷酸肌苷（PI）水解，从而控制钙动员和蛋白激酶C活化，这是T淋巴细胞或b淋巴细胞活化的必要事件。此外，负责第二个所需激活信号（信号2）的某些共刺激分子（例如CD2、CD28）也会影响PLCg1的激活。信号1和信号2的合理生理组合可诱导免疫激活，而信号不平衡则无法激活细胞或诱导耐受。