Genetic/Biochemical Approaches-Tyrosine Kinase Function

遗传/生化方法-酪氨酸激酶功能

• 批准号: 6830364
• 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/6830364
• 关键词: B lymphocyte; T cell receptor; T lymphocyte; antibody formation; biological signal transduction; cell differentiation; cell proliferation; cellular immunity; enzyme activity; gene mutation; genetic manipulation; genetically modified animals; helper T lymphocyte; immunogenetics; immunoregulation; interferon gamma; interleukin 10; interleukin 4; interleukin 5; laboratory mouse; leukocyte activation /transformation; molecular genetics; protein tyrosine kinase; sex linked trait; tissue /cell culture; transcription factor

Our laboratory studies signal transduction 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 the formation and progression of cancer. Using a combination of genetics, cell biology and protein biochemistry, our work has concentrated on how these molecules contribute to normal function of cells in the immune system. Through these studies we hope to understand how manipulation of these pathways can be utlilized to develop therapeutics for disease. In recent years our work has concentrated on studies of the Tec family of tyrosine kinases, the prototypical member of which, Btk, is required for normal function of B cells. Mutation of Btk is responsible for the human genetic disorder X-linked agammmaglobulimemia. We have shown that mutation of Tec family kinases expressed in T cells can also severely impair T lymphocyte function in mice and has profound effects on responses to infections in vivo. In the last year, we have concentrated on biochemical defects associated with mutation of the Tec kinases in T lymphocytes. 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. We have now found that mutation of Tec kinases also impairs actin cytoskeletal reorganization and activation of WASP, the protein mutated in Wiskott-Aldrich Syndrome, a syndrome associated with defective cytoskeleton organization. We have further shown that the defect in WASP activation is secondary to altered activation of Cdc42, a Rho family GTPase and altered subcellular localization of the guanine nucleotide exchange factor Vav. Moreover, we have found that cells deficient in Tec kinases also show abnormal responses to chemokines, small molecules that direct cell polarization and cell trafficking in the immune system. 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. As an extension of these studies, we began examining other signaling molecules potentially involved in T helper cell differentiation including SAP, which is mutated in the genetic disorder X-linked proliferative syndrome (XLP). 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 IFN-g production, a Th1 response, and decreased antibody production. Last year, we showed that the impaired antibody response in these mice was 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 ability to develop a sustained antibody response is a hallmark of productive immunity and a standard for successful vaccine development. Thus, understanding the cellular interactions and signals leading to productive immunization is of high importance. To understand the defect in T cells in the Sap-deficient mice, we have examined T cell function, cytokine production 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. Given the role of Th2 cytokines in promoting B cell help for antibody production, the defect in Th2 cytokine production provides new insights into the mechanism of disease in XLP. We have further examined T cell activation and have found that SAP-deficient cells show defective activation of the NFKB transcription factor. These studies are revealing a new pathway for the regulation of T helper cell differentiation via NFKB. Furthermore, our results suggest that T helper cell misregulation may contribute to phenotypes associated with XLP.

我们的实验室研究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参与癌症形成和发展的异常生长和发展的细胞内信号传导途径所需的分子。使用遗传学，细胞生物学和蛋白质生物化学的组合，我们的工作集中在这些分子如何有助于免疫系统中细胞的正常功能。通过这些研究，我们希望了解如何操纵这些途径可以利用开发疾病的治疗方法。近年来，我们的工作集中在Tec家族的酪氨酸激酶的研究，其中的原型成员，Btk，所需的B细胞的正常功能。Btk的突变是导致人类遗传性疾病X连锁无丙种球蛋白血症的原因。我们已经表明，T细胞中表达的Tec家族激酶的突变也可以严重损害小鼠中的T淋巴细胞功能，并对体内对感染的反应具有深远的影响。在过去的一年中，我们集中在生化缺陷与突变的Tec激酶在T淋巴细胞。先前的研究已经证明Tec激酶对于抗原受体诱导的磷脂酶-c γ的活化是关键的，磷脂酶-c γ是Ca++动员所需的关键酶。我们现在已经发现Tec激酶的突变也损害肌动蛋白细胞骨架重组和WASP的激活，Wiskott-Aldrich综合征中突变的蛋白质，一种与细胞骨架组织缺陷相关的综合征。我们进一步表明，WASP激活的缺陷是次要的Cdc 42，一个Rho家族GTdR和改变鸟嘌呤核苷酸交换因子Vav的亚细胞定位的激活改变。此外，我们发现缺乏Tec激酶的细胞也对趋化因子（在免疫系统中指导细胞极化和细胞运输的小分子）显示出异常反应。我们的研究结果将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。 在继续的工作中，我们也在研究Tec激酶突变对体内T细胞应答的影响。我们以前发现Tec激酶的突变改变了T辅助细胞分化和细胞因子产生的平衡。作为这些研究的延伸，我们开始研究可能参与T辅助细胞分化的其他信号分子，包括SAP，它在遗传性疾病X连锁增殖综合征（XLP）中突变。我们以前已经产生了SAP缺陷小鼠，并发现在感染因子的攻击下，这些小鼠重现了XLP的特征，包括T细胞活化和IFN-g产生增加，Th 1应答和抗体产生减少。去年，我们发现这些小鼠中受损的抗体应答继发于CD 4 + T细胞的缺陷，即SAP缺陷型T细胞无法向B细胞提供产生长期抗体应答的必要信号，这是成功免疫和免疫应答发展的关键步骤。产生持续抗体应答的能力是生产性免疫的标志，也是成功开发疫苗的标准。因此，理解导致生产性免疫的细胞相互作用和信号是非常重要的。为了了解SAP缺陷小鼠T细胞的缺陷，我们检查了SAP缺陷小鼠细胞中T细胞功能、细胞因子产生和T细胞活化的生物化学。我们已经发现，来自Sap缺陷小鼠的T细胞响应于TCR刺激显示出Th 1细胞因子产生（IFN-g）增加和Th 2细胞因子产生（IL-4、5和10）的显著缺陷。鉴于Th 2细胞因子在促进B细胞帮助产生抗体中的作用，Th 2细胞因子产生的缺陷为XLP疾病机制提供了新的见解。我们进一步研究了T细胞活化，发现SAP缺陷型细胞显示NF κ B转录因子活化缺陷。这些研究揭示了通过NF κ B调节T辅助细胞分化的新途径。此外，我们的研究结果表明，辅助性T细胞失调可能有助于与XLP相关的表型。