Genetic /Biochemical Approaches to Tyrosine Kinase Funct

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，这是参与正常细胞生长和分化的细胞内信号通路所需的分子，以及参与癌症形成和发展的异常生长和发育。影响这种信号通路的突变已被发现会引起许多人类疾病，包括几种主要的免疫缺陷。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，该家族的原型成员BTK是人类遗传性疾病X连锁无丙球蛋白血症的罪魁祸首，是正常B细胞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。在过去的一年里，我们主要集中在新发现的Fortec激酶在调节T淋巴细胞肌动蛋白细胞骨架中的作用。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。此外，我们最近发表的文章指出，缺乏Tec激酶的细胞也对趋化因子表现出异常反应，趋化因子是一种指导细胞极化和细胞在免疫系统中运输的小分子。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，我们还在研究Tec激酶突变对体内T细胞反应的影响。我们先前发现，Tec激酶的突变改变了辅助性T细胞分化和细胞因子产生的平衡。我们现在正在利用转基因和基因靶向小鼠的组合来扩展这些研究，以改变T淋巴细胞中Tec激酶的表达。 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。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细胞活化的生化。我们发现SAP缺陷小鼠的T细胞在TCR刺激下表现出Th1细胞因子产生(干扰素-g)的增加和Th2细胞因子产生的显著缺陷(IL-4、5和10)。鉴于Th2细胞因子在促进B细胞帮助产生抗体方面的作用，Th2细胞因子产生的缺陷为XLP的疾病机制提供了潜在的新见解。在过去的一年里，我们在理解这些表型方面取得了重大进展。我们证明SAP缺陷T细胞的IL-4分泌缺陷与干扰素-γ的产生增加无关。SAP缺陷细胞对极化细胞因子的反应正常，但表现出TCR介导的GATA-3和IL-4诱导受损。TCR信号检测显示SAP缺陷细胞内钙离子动员正常，ERK、JNK和Vav激活，但PKC-theta募集、Bcl10磷酸化、IkappaB-α降解和核转录因子-kappaB1/p50水平降低。尽管如此，另一种核因子-kappaB亚单位c-rel的核水平是正常的。在Fyn缺失的细胞中也观察到了类似的缺陷。野生型(WT)的重新表达，而不是SAP的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。此外，SLAM参与增加了TCR介导的PKC-theta募集、核p50水平和WT中IL-4的产生，但不增加SAP缺陷的T细胞，这表明SLAM在T细胞信号转导中可能发挥新的作用。我们的数据表明，SAP/Fyn通路是PKC-theta/Bcl-10有效募集以及NF-kappaB激活所必需的，并提示了一条潜在的T辅助2(IL-4)细胞因子调节的新途径。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。

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. Mutations affecting such signaling pathways have been found to give rise to a number of 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 development of therapeutics for disease. A large portion of our work is focused on studies of the Tec family of tyrosine kinases, the prototypical member of which, Btk, is responsible for the human genetic disorder X-linked agammmaglobulimemia and 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. In the last year, we have concentrated on the 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 in cells deficient for the Tec kinase Itk. and altered subcellular localization of the guanine nucleotide exchange factor Vav. Moreover, we have recently published 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. 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. 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. We further showed that the impaired antibody responses 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 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 continuedd 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. Given the role of Th2 cytokines in promoting B cell help for antibody production, the defect in Th2 cytokine production provides potential new insights into the mechanism of disease in XLP. In the last year, we have made significant progress in understanding these phenotypes. We demonstrate that the defect in IL-4 secretion in SAP-deficient T cells is independent of increased IFN-gamma production. SAP-deficient cells respond normally to polarizing cytokines, yet show impaired TCR-mediated induction of GATA-3 and IL-4. Examination of TCR signaling revealed normal Ca++ mobilization as well as ERK, JNK and Vav activation in SAP-deficient cells, but decreased PKC-theta recruitment, Bcl-10 phosphorylation, IkappaB-alpha degradation and nuclear NF-kappaB1/p50 levels. Nonetheless, nuclear levels of c-Rel, another NF-kappaB subunit, are normal. Similar defects were observed in Fyn-deficient cells. 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. 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. These findings provide a molecular framework for probing T cell function and immune cell dysregulation in this complex disorder.