Genetic and Biochemical Approaches to Tyrosine Kinase Fu

酪氨酸激酶 Fu 的遗传和生化方法

• 批准号: 7146864
• 负责人: PAMELA SCHWARTZBERG
• 金额: --
• 依托单位: NATIONAL HUMAN GENOME RESEARCH INSTITUTE
• 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/7146864
• 关键词: T cell receptor; T lymphocyte; biological signal transduction; cell differentiation; cell proliferation; cellular immunity; cytokine; cytoskeleton; enzyme activity; gene expression; gene mutation; genetic disorder; genetically modified animals; human tissue; hypogammaglobulinemia; immunodeficiency; immunogenetics; immunoregulation; laboratory mouse; leukocyte activation /transformation; molecular genetics; protein structure function; protein tyrosine kinase; sex linked trait; tissue /cell culture

Summary: Our laboratory studies signal transduction in the immune system involving tyrosine phosphorylation and non-receptor tyrosine kinases, molecules required for intracellular signaling pathways involved in normal cellular growth and differentiation as well as the abnormal growth and development involved in cancer. Mutations affecting such signaling pathways have been found to give rise to multiple human disorders including several primary immunodeficiencies. Using a combination of genetics, protein biochemistry and cell biology, our goals are to understand how these molecules contribute to normal function of cells in the immune system. Our goals include understanding how manipulation of these pathways can help in the understanding of diseases of the immune system and in the development of therapeutics for disease. A large portion of our work is focused on studies of the Tec family of tyrosine kinases. Mutations affecting the prototypical member of this family, Btk, are responsible for the human genetic disorder X-linked agammmaglobulimemia. Btk is required for normal B cell development and function. We have shown that mutation of Tec family kinases expressed in T cells can also severely impair T lymphocyte function in mice and influence responses to infections in vivo. Previous studies have demonstrated that the Tec kinases are critical for antigen receptor induced activation of phospholipase-c gamma, a key enzyme required for Ca++ mobilization. Over the last two years, we have described and studies a newly discovered role forTec kinases in regulation of the actin cytoskeleton in T lymphocytes. We have recently reported altered activation of Cdc42, a Rho family GTPase that is an upstream activator of the Wiskott Aldrich Syndrome Protein (WASp) in cells deficient for the Tec kinase Itk. and altered subcellular localization of the guanine nucleotide exchange factor Vav. Mutations affecting both WASp and Vav have been implicated in primary immunodeficiencies. Using genetic and biochemical methods, we have recently extended our findings to help determine the interactions between Itk and these key regulators of the T cell actin cytoskeleton organization and have demonstrated a key role for Tec kinases in activated of lymphocyte adhesion. Our results place the Tec kinases as critical regulators of the actin cytoskeleton, cell adhesion and migration and suggest that cytoskeletal defects may contribute to the phenotypes associated with Tec kinase deficiency including those seen in X-linked agammaglobulinemia. In continuing work, we are also examining the effects of mutation of Tec kinases on T cell responses in vivo. We had previously found that mutation of the Tec kinases alters the balance of T helper cell differentiation and cytokine production. We are now extending these studies using a combination of transgenic and gene-targeted mice to alter expression of the Tec kinases in T lymphocytes. Additionally, we are examining the role of related signaling molecules including WASp in T helper cell differentiation and in vivo responses to infection. As an extension of these studies, we are examining other signaling molecules involved in T helper cell differentiation including SAP, a small SH2 containing adaptor protein, mutations of which are associated with the genetic disorder X-linked proliferative syndrome (XLP). SAP binds to and helps recruit the tyrosine kinase Fyn to the intracellular tails of SLAM and related co-stimulatory receptors. We had previously generated mice deficient in SAP and have found that upon challenge with infectious agents, these mice recapitulated features of XLP, including increased T cell activation and decreased antibody production. In the last year, we have extended these findings to demonstrate a critical role of SAP in responses to immunization. We further showed that the impaired antibody responses in these mice is secondary to a defect in CD4+ T cells, ie SAP deficient T cells fail to provide an essential signal to B cells for generating long-term antibody responses, a critical step for the development of successful immunization and immune responses, the hallmark of successful vaccine development. Understanding the cellular interactions and signals that are defective in these mice is therefore of high importance or understanding the requirements for successful vaccine development. To understand the defect in T cells in the SAP-deficient mice, we have continued to examine T cell function and biochemistry of T cell activation in cells from SAP-deficient mice. We have found that T cells from SAP deficient mice show increased Th1 cytokine production (IFN-g) and dramatic defects in Th2 cytokine production (IL-4, 5, and 10) in response to TCR stimulation. We have further demonstrated that the defect in IL-4 secretion in SAP-deficient T cells is independent of increased IFN-gamma production, but is secondary to decreased TCR-induced PKC-theta recruitment, Bcl-10 phosphorylation, IkappaB-alpha degradation and nuclear NF-kappaB1/p50 levels. Re-expression of wildtype (WT), but not a Fyn-binding mutant of SAP rescued the defects in both PKC-theta recruitment and IL-4 production in SAP-deficient cells, demonstrating that this phenotype requires SAP-Fyn recruitment. Moreover, SLAM engagement increased TCR-mediated PKC-theta recruitment, nuclear p50 levels and IL-4 production in WT but not SAP-deficient T cells, suggesting a potential new role for SLAM in T cell signaling. Our data indicates that a SAP/Fyn pathway is required for the efficient recruitment of PKC-theta/Bcl-10 as well as proper patterns of activation of NF-kappaB, and suggests a potentially novel pathway of T helper 2 (IL-4) cytokine regulation. Nonetheless, recednt studies suggest that the humoral defects may involve distinct signaling pathways. These findings provide a molecular framework for probing T cell function and immune cell dysregulation in this complex disorder.

摘要：我们的实验室研究免疫系统中涉及酪氨酸磷酸化和非受体酪氨酸激酶的信号转导，这些分子是参与正常细胞生长和分化以及癌症异常生长和发育的细胞内信号传导途径所需的分子。已发现影响此类信号传导途径的突变会引起多种人类疾病，包括几种原发性免疫缺陷。结合遗传学、蛋白质生物化学和细胞生物学，我们的目标是了解这些分子如何促进免疫系统细胞的正常功能。我们的目标包括了解操纵这些途径如何有助于了解免疫系统疾病和开发疾病疗法。 我们的大部分工作集中在酪氨酸激酶 Tec 家族的研究上。影响该家族典型成员 Btk 的突变是导致人类遗传性疾病 X 连锁无球蛋白血症的原因。 Btk 是正常 B 细胞发育和功能所必需的。我们已经证明，T 细胞中表达的 Tec 家族激酶的突变也会严重损害小鼠的 T 淋巴细胞功能，并影响体内对感染的反应。先前的研究表明，Tec 激酶对于抗原受体诱导的磷脂酶-c γ 激活至关重要，磷脂酶-c γ 是 Ca++ 动员所需的关键酶。在过去的两年中，我们描述并研究了新发现的 Tec 激酶在 T 淋巴细胞肌动蛋白细胞骨架调节中的作用。我们最近报道了 Cdc42 的激活发生改变，Cdc42 是一种 Rho 家族 GTP 酶，是 Tec 激酶 Itk 缺陷细胞中 Wiskott Aldrich 综合征蛋白 (WASp) 的上游激活剂。并改变鸟嘌呤核苷酸交换因子 Vav 的亚细胞定位。影响 WASp 和 Vav 的突变与原发性免疫缺陷有关。使用遗传和生化方法，我们最近扩展了我们的研究结果，以帮助确定 Itk 与 T 细胞肌动蛋白细胞骨架组织的这些关键调节因子之间的相互作用，并证明了 Tec 激酶在激活淋巴细胞粘附中的关键作用。我们的结果将 Tec 激酶作为肌动蛋白细胞骨架、细胞粘​​附和迁移的关键调节因子，并表明细胞骨架缺陷可能导致与 Tec 激酶缺乏相关的表型，包括 X 连锁无丙种球蛋白血症中所见的表型。 在后续工作中，我们还在研究 Tec 激酶突变对体内 T 细胞反应的影响。我们之前发现 Tec 激酶的突变会改变 T 辅助细胞分化和细胞因子产生的平衡。我们现在正在扩展这些研究，结合使用转基因和基因靶向小鼠来改变 T 淋巴细胞中 Tec 激酶的表达。此外，我们正在研究相关信号分子（包括 WASp）在 T 辅助细胞分化和体内感染反应中的作用。 作为这些研究的延伸，我们正在检查参与 T 辅助细胞分化的其他信号分子，包括 SAP，一种含有 SH2 的小接头蛋白，其突变与遗传性疾病 X 连锁增殖综合征 (XLP) 相关。 SAP 结合并帮助将酪氨酸激酶 Fyn 招募到 SLAM 和相关共刺激受体的细胞内尾部。我们之前培育了 SAP 缺陷小鼠，并发现在受到感染因子的攻击后，这些小鼠重现了 XLP 的特征，包括 T 细胞激活增加和抗体产生减少。去年，我们扩展了这些发现，以证明 SAP 在免疫接种中的关键作用。我们进一步表明，这些小鼠中抗体反应受损是继发于 CD4+ T 细胞缺陷的，即 SAP 缺陷 T 细胞无法向 B 细胞提供产生长期抗体反应的重要信号，这是成功免疫和免疫反应发展的关键步骤，也是成功疫苗开发的标志。因此，了解这些小鼠中存在缺陷的细胞相互作用和信号非常重要，或者了解成功开发疫苗的要求非常重要。为了了解 SAP 缺陷小鼠中 T 细胞的缺陷，我们继续检查 SAP 缺陷小鼠细胞中的 T 细胞功能和 T 细胞活化的生物化学。我们发现 SAP 缺陷小鼠的 T 细胞响应 TCR 刺激，Th1 细胞因子生成 (IFN-g) 增加，而 Th2 细胞因子生成 (IL-4、5 和 10) 显着缺陷。我们进一步证明 SAP 缺陷 T 细胞中 IL-4 分泌的缺陷与 IFN-γ 产生的增加无关，而是继发于 TCR 诱导的 PKC-theta 招募、Bcl-10 磷酸化、IkappaB-alpha 降解和核 NF-kappaB1/p50 水平降低。 SAP 野生型 (WT) 的重新表达（而非 Fyn 结合突变体）挽救了 SAP 缺陷细胞中 PKC-theta 募集和 IL-4 产生的缺陷，表明该表型需要 SAP-Fyn 募集。此外，SLAM 参与增加了 WT 中 TCR 介导的 PKC-theta 募集、核 p50 水平和 IL-4 产生，但 SAP 缺陷 T 细胞没有增加，这表明 SLAM 在 T 细胞信号传导中具有潜在的新作用。我们的数据表明，SAP/Fyn 通路是 PKC-theta/Bcl-10 的有效募集以及 NF-kappaB 激活的正确模式所必需的，并提出了 T 辅助细胞 2 (IL-4) 细胞因子调节的潜在新通路。尽管如此，最近的研究表明体液缺陷可能涉及不同的信号通路。这些发现为探索这种复杂疾病中的 T 细胞功能和免疫细胞失调提供了分子框架。