Genetic/Biochemical Approaches-Tyrosine Kinase Function

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中细胞的正常功能。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。我们已经证明，在T细胞中表达的Tec家族激酶突变也会严重损害小鼠T淋巴细胞的功能，并对体内感染的反应产生深远的影响。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。先前的研究表明，Tec激酶对于抗原受体诱导的磷脂酶-c γ激活至关重要，磷脂酶-c γ是钙离子动员所需的关键酶。我们现在发现Tec激酶的突变也会损害肌动蛋白细胞骨架重组和WASP的激活，WASP是Wiskott-Aldrich综合征（一种与细胞骨架组织缺陷相关的综合征）中发生突变的蛋白质。我们进一步表明，WASP激活缺陷是继发于Rho家族GTPase Cdc42的激活改变和鸟嘌呤核苷酸交换因子Vav的亚细胞定位改变。此外，我们发现缺乏Tec激酶的细胞也对趋化因子（在免疫系统中指导细胞极化和细胞运输的小分子）表现出异常反应。我们的研究结果表明Tec激酶是肌动蛋白细胞骨架、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，包括在x连锁无球蛋白血症中看到的表型。

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 genetics, cell biology and protein biochemistry, our work has concentrated on how these molecules contribute to normal function of cells in the immune system. Through these studies we hope to understand how manipulation of these pathways can be utlilized to develop therapeutics for disease. 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 shown that mutation of Tec family kinases expressed in T cells can also severely impair T lymphocyte function in mice and has profound effects on responses to infections in vivo. In the last year, we have concentrated on biochemical defects associated with mutation of the Tec kinases in T lymphocytes. 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. We have now found that mutation of Tec kinases also impairs actin cytoskeletal reorganization and activation of WASP, the protein mutated in Wiskott-Aldrich Syndrome, a syndrome associated with defective cytoskeleton organization. We have further shown that the defect in WASP activation is secondary to altered activation of Cdc42, a Rho family GTPase and altered subcellular localization of the guanine nucleotide exchange factor Vav. Moreover, we have found 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. 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 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 IFN-g production, a Th1 response, and decreased antibody production. Last year, we showed that the impaired antibody response 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 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. To understand the defect in T cells in the Sap-deficient mice, we have examined T cell function, cytokine production 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 new insights into the mechanism of disease in XLP. We have further examined T cell activation and have found that SAP-deficient cells show defective activation of the NFKB transcription factor. These studies are revealing a new pathway for the regulation of T helper cell differentiation via NFKB. Furthermore, our results suggest that T helper cell misregulation may contribute to phenotypes associated with XLP.