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。我们最近报告了Cdc 42的激活改变，Cdc 42是Rho家族GTPase，是Tec激酶Itk缺陷细胞中Wiskott奥尔德里奇综合征蛋白（WASp）的上游激活剂。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。我们现在正在扩展这些研究，使用转基因和基因靶向小鼠的组合来改变T淋巴细胞中Tec激酶的表达。此外，我们正在研究相关的信号分子，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SH 2，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CD 4 + 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。我们已经进一步证明SAP缺陷型T细胞中IL-4分泌的缺陷与IFN-γ产生的增加无关，但继发于TCR诱导的PKC-θ募集、Bcl-10磷酸化、IkappaB-α降解和核NF-κ B1/p50水平的降低。野生型（WT）而非SAP的Fyn结合突变体的再表达挽救了SAP缺陷细胞中PKC-θ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但非SAP缺陷型T细胞中TCR介导的PKC-θ募集、核p50水平和IL-4产生，表明SLAM在T细胞信号传导中的潜在新作用。我们的数据表明，SAP/Fyn通路是PKC-θ/Bcl-10的有效募集以及NF-κ B激活的适当模式所必需的，并表明T辅助细胞2（IL-4）细胞因子调节的潜在新途径。然而，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 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.