Genetic and Biochemical Approaches to Tyrosine Kinase Fu

总结：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展的细胞内信号传导途径所需的分子。已经发现影响这种信号传导途径的突变引起多种人类疾病，包括几种原发性免疫缺陷。使用遗传学，蛋白质生物化学和细胞生物学的组合，我们的目标是了解这些分子如何有助于免疫系统中细胞的正常功能。我们的目标包括了解这些途径的操纵如何有助于理解免疫系统疾病和开发疾病治疗方法。 我们的工作的很大一部分集中在Tec家族的酪氨酸激酶的研究。影响该家族原型成员Btk的突变导致人类遗传性疾病X连锁无丙种球蛋白血症。Btk是正常B细胞发育和功能所必需的。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的T淋巴细胞功能，并影响体内对感染的反应。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。在过去的两年中，我们描述和研究了一个新发现的作用forTec激酶在调节肌动蛋白细胞骨架的T淋巴细胞。我们最近报道了Cdc42的活化改变，Cdc42是Rho家族GTdR，是Tec激酶Itk缺陷细胞中Wiskott Aldrich综合征蛋白（WASp）的上游活化剂。和改变的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。使用遗传和生物化学方法，我们最近扩展了我们的研究结果，以帮助确定Itk和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激酶在淋巴细胞粘附激活中的关键作用。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细胞应答和T辅助细胞分化的影响。 作为这些研究的延伸，我们正在研究参与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）相关。SAP结合并帮助将酪氨酸激酶Fyn募集到SLAM和相关共刺激受体的细胞内尾部。我们之前已经产生了SAP缺陷的小鼠，并发现在用感染因子攻击时，这些小鼠重现了XLP的特征，包括T细胞活化增加和抗体产生减少。在过去的一年中，我们扩展了这些发现，以证明SAP在免疫反应中的关键作用。我们进一步表明，这些小鼠中受损的抗体应答继发于CD4 + T细胞的缺陷，即SAP缺陷型T细胞不能向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，是成功疫苗开发的标志。因此，了解这些小鼠中有缺陷的细胞相互作用和信号非常重要，或者了解成功疫苗开发的要求。为了了解SAP缺陷小鼠中T细胞的缺陷，我们继续检查了SAP缺陷小鼠细胞中T细胞功能和T细胞活化的生物化学。我们已经发现，来自SAP缺陷型小鼠的T细胞在响应TCR刺激的Th2细胞因子产生（IL-4、5和10）中显示出显著缺陷。然而，我们进一步发现，它们在T细胞中的缺陷有助于抗体产生，这可以与它们的Th2缺陷分开。此外，虽然SAP缺陷细胞中IL-4产生缺陷的拯救需要SAP-Fyn募集，但体液缺陷的拯救不需要SAP-Fyn相互作用，并且可能涉及不同的信号传导途径。这些发现为探测这种复杂疾病中的T细胞功能和免疫细胞失调提供了分子框架。

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.