Genetic and Biochemical Approaches to Tyrosine Kinase Fu

摘要：我们的实验室研究免疫系统中的信号转导，涉及酪氨酸磷酸化和非受体酪氨酸激酶，这是参与正常细胞生长和分化以及与癌症相关的异常生长和发育的细胞内信号通路所需的分子。影响这些信号通路的突变已被发现会引起多种人类疾病，包括几种原发免疫缺陷。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中细胞的正常功能。我们的目标包括了解这些通路的操纵如何有助于理解免疫系统疾病以及疾病疗法的发展。 我们的大部分工作都集中在对酪氨酸激酶Tec家族的研究上。影响该家族典型成员BTK的突变是人类遗传性疾病X连锁无丙种球蛋白血症的原因。BTK是B细胞正常发育和功能所必需的。我们已经证明，在T细胞中表达的Tec家族激酶的突变也可以严重损害小鼠的T淋巴细胞功能，并影响体内感染的反应。以前的研究表明，Tec激酶在抗原受体诱导磷脂酶-c-γ激活的过程中起关键作用，磷脂酶-c-γ是钙动员所需的关键酶。在过去的两年里，我们描述并研究了Fortec激酶在调节T淋巴细胞肌动蛋白细胞骨架中的新发现的作用。我们最近报道了在缺乏Tec激酶ITK的细胞中，Rho家族GTP酶CDc42的激活改变，它是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。影响黄蜂和Vav的突变被认为与原发免疫缺陷有关。利用遗传学和生物化学方法，我们最近扩展了我们的发现，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黏附中的关键作用。我们的结果表明Tec激酶是肌动蛋白细胞骨架、细胞黏附和迁移的关键调节因子，并提示细胞骨架缺陷可能与Tec激酶缺乏相关的表型有关，包括X连锁无丙种球蛋白血症的表型。在继续的工作中，我们还在体内检测Tec激酶突变对T细胞反应和T辅助细胞分化的影响。 作为这些研究的延伸，我们正在检测参与T辅助细胞分化的其他信号分子，包括SAP，一种含有接头蛋白的小SH2，其突变与遗传性疾病X-连锁增殖综合征(XLP)有关。SAP结合并帮助将酪氨酸激酶Fyn招募到SLAM和相关的共刺激受体的细胞内尾部。我们之前已经产生了SAP缺陷小鼠，并发现在感染病原体攻击时，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。在过去的一年里，我们扩大了这些发现，以证明SAP在免疫反应中的关键作用。我们进一步证明，这些小鼠的抗体反应受损是由于CD4+T细胞的缺陷所致，即SAP缺陷的T细胞无法向B细胞提供产生长期抗体反应的必要信号，这是成功免疫和免疫反应发展的关键步骤，而免疫反应是成功疫苗开发的标志。因此，了解这些小鼠的细胞相互作用和有缺陷的信号是非常重要的，或者理解成功开发疫苗的要求。为了了解SAP缺陷小鼠的T细胞缺陷，我们继续检测SAP缺陷小鼠的T细胞功能和T细胞激活的生化。我们发现SAP缺陷小鼠的T细胞在TCR刺激下产生的Th2细胞因子(IL-4、IL-5和IL-10)有显著缺陷。然而，我们进一步发现，他们在T细胞帮助产生抗体方面的缺陷可以从他们的Th2缺陷中分离出来。此外，虽然修复SAP缺陷细胞中产生IL-4的缺陷需要SAP-Fyn的招募，但修复体液缺陷不需要SAP-Fyn的相互作用，可能涉及不同的信号通路。这些发现为探索这种复杂疾病的T细胞功能和免疫细胞失调提供了一个分子框架。

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 and T helper cell differentiation in vivo. 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 dramatic defects in Th2 cytokine production (IL-4, 5, and 10) in response to TCR stimulation. However, we have further found that their defects in T cell help for antibody production can be separated from their Th2 defects. Moreover, while rescue of the defects in IL-4 production in SAP-deficient cells requires SAP-Fyn recruitment, rescue of the humoral defects does not require a SAP-Fyn interaction and may involve distinct signaling pathways. These findings provide a molecular framework for probing T cell function and immune cell dysregulation in this complex disorder.