Genetic and Biochemical Approaches to Tyrosine Kinase Fu

我们的实验室研究酪氨酸磷酸化和非受体酪氨酸激酶的信号转导，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进展的异常生长和发育。结合小鼠遗传学、细胞生物学和蛋白质生物化学，我们的工作集中在这些分子如何促进免疫系统和骨骼系统细胞的正常功能。通过这些研究，我们希望了解如何利用这些途径来开发治疗影响这些系统的疾病的方法。近年来，我们的工作主要集中在Tec酪氨酸激酶家族的研究上，该家族的典型成员Btk是B细胞正常功能所必需的。Btk的突变是人类遗传疾病x连锁无核球蛋白症的原因。我们之前已经证明，在T细胞中表达的Tec家族激酶的突变可以严重损害小鼠T淋巴细胞的功能，从而首次确立了这些激酶在T细胞介导的免疫应答中的作用。我们进一步表明，这些激酶参与辅助性T细胞分化的信号通路，T细胞分化是免疫反应的关键调节成分，有助于确定个体是否产生细胞（Th1）或基于抗体（Th2）的免疫反应。我们发现Tec激酶的突变可以改变参与Th2 CD4+ T辅助细胞分化的转录因子的激活和抑制。我们的研究结果表明，TCR反应的损伤不仅可能降低动物对抗原的反应能力，还可能改变所产生的免疫反应的类型。在过去的一年里，我们集中研究了与T淋巴细胞Tec激酶突变相关的生化缺陷。我们发现这些激酶的突变会损害肌动蛋白细胞骨架重组和WASP的激活，WASP在Wiskott-Aldrich综合征（一种与细胞骨架组织缺陷相关的综合征）中发生突变。我们的研究结果表明Tec激酶是WASP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。作为这些研究的延伸，我们开始研究其他可能参与T辅助细胞分化的信号分子，包括SAP，它在遗传疾病x连锁增殖综合征（XLP）中发生突变。我们培养了缺乏SAP的小鼠，并发现在感染因子的攻击下，这些小鼠再现了XLP的特征，包括T细胞活化和IFN-g产生增加，抗体产生减少。未感染SAP-小鼠的脾细胞产生增加的IFN-g和减少的IL-4，表明T辅助细胞失调可能导致与（XLP）相关的表型。在过去的一年里，我们专注于这些小鼠的受损抗体反应。产生持续抗体反应的能力是产生性免疫的标志，也是疫苗研制成功的标准。因此，了解细胞相互作用和信号导致生产免疫是非常重要的。我们与R. Ahmed （Emory大学疫苗研究中心）合作发现，在sap缺陷小鼠中，初始B细胞反应仅受到轻微影响，但在感染LCMV后，生发中心的形成、记忆和长寿命血浆（抗体分泌）B细胞的产生严重受损。通过过继细胞转移实验，我们发现抗体产生的缺陷是CD4+ T细胞固有的，即SAP缺陷T细胞不能向B细胞提供产生长期抗体反应的必要信号，而长期抗体反应是成功免疫和免疫反应发展的关键步骤。

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 mouse genetics, cell biology and protein biochemistry, our work has concentrated on how these molecules contribute to normal function of cells of the immune system and the skeletal system. Through these studies we hope to understand how manipulation of these pathways can be utlilized to develop therapeutics for diseases affecting these systems. 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 previously shown that mutation of Tec family kinases expressed in T cells can severely impair T lymphocyte function in mice, thereby establishing for the first time a role for these kinases in T cell mediated immune responses. We have further shown that these kinases participate in the signaling pathways involved in T helper cell differentiation, a critical regulatory component of immune responses that helps determine whether an individual mounts a cellular (Th1) or antibody based, (Th2) immune response. We have found that mutation of Tec kinases can alter activation and repression of transcription factors involved in Th2 CD4+ T helper cell differentiation. Our results suggest that impairment of TCR responses may not only reduce the ability of an animal to respond to antigen, but may also alter the type of immune response generated. In the last year, we have concentrated on the biochemical defects associated with mutation of the Tec kinases in T lymphocytes. We have found that mutation of these kinases impairs actin cytoskeletal reorganization and activation of WASP, the protein mutated in Wiskott-Aldrich Syndrome, a syndrome associated with defective cytoskeleton organization. Our results place the Tec kinases as critical regulators of WASP and suggest that cytoskeletal defects may contribute to the phenotypes associated with Tec kinase deficiency. 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 have 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, and decreased antibody production. Splenocytes from uninfected SAP- mice produced increased IFN-g and decreased IL-4, suggesting that T helper cell misregulation may contribute to phenotypes associated with (XLP). In the last year, we have focused on the impaired antibody response in these mice. 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. In collaboration with R. Ahmed (Emory University Vaccine Research Center), we have found that initial B cell responses are only mildly affected in SAP-deficient mice but germinal center formation and the generation of memory and long-lived plasma (antibody secreting) B cells is severely impaired post-infection with LCMV. Using adoptive cell transfer experiments we found that the defect in antibody production is intrinsic to 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.