Genetic and Biochemical Approaches to Tyrosine Kinase Fu

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，这些分子是参与正常细胞生长和分化以及参与癌症形成和发展的异常生长和发展的细胞内信号传导途径所需的分子。使用小鼠遗传学，细胞生物学和蛋白质生物化学的组合，我们的工作集中在这些分子如何有助于免疫系统和骨骼系统细胞的正常功能。通过这些研究，我们希望了解如何操纵这些途径可以利用开发治疗影响这些系统的疾病。近年来，我们的工作集中在Tec家族的酪氨酸激酶的研究，其中的原型成员，Btk，所需的B细胞的正常功能。Btk的突变是导致人类遗传性疾病X连锁无丙种球蛋白血症的原因。我们以前已经表明，突变的Tec家族激酶表达的T细胞可以严重损害小鼠T淋巴细胞的功能，从而首次建立了这些激酶在T细胞介导的免疫应答中的作用。我们进一步表明，这些激酶参与参与辅助性T细胞分化的信号传导途径，这是免疫应答的关键调节组分，有助于确定个体是否产生基于细胞（Th 1）或抗体（Th 2）的免疫应答。我们已经发现Tec激酶的突变可以改变参与Th 2 CD 4 + 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，而且还可能改变所产生的免疫反应的类型。在过去的一年里，我们集中在T淋巴细胞中Tec激酶突变相关的生化缺陷。我们已经发现，这些激酶的突变损害肌动蛋白细胞骨架重组和激活WASP，蛋白质突变的Wiskott-Aldrich综合征，一个综合征与缺陷的细胞骨架组织。我们的研究结果将Tec激酶作为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，我们开始研究可能参与T辅助细胞分化的其他信号分子，包括SAP，它在遗传性疾病X连锁增殖综合征（XLP）中突变。我们已经产生了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，包括增加的T细胞活化和IFN-g产生，以及减少的抗体产生。来自未感染的SAP-小鼠的脾细胞产生增加的IFN-g和减少的IL-4，表明T辅助细胞失调可能有助于与（XLP）相关的表型。在过去的一年里，我们一直专注于这些小鼠中受损的抗体反应。产生持续抗体应答的能力是生产性免疫的标志，也是成功开发疫苗的标准。因此，理解导致生产性免疫的细胞相互作用和信号是非常重要的。与R. Ahmed（Emory University Vaccine Research Center），我们发现SAP缺陷小鼠的初始B细胞应答仅受到轻度影响，但LCMV感染后，生发中心形成以及记忆和长寿命血浆（抗体分泌）B细胞的产生严重受损。使用过继性细胞转移实验，我们发现抗体产生的缺陷是CD 4 + T细胞固有的，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。

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.