HPK1 MEDIATED T CELL SIGNAL TRANSDUCTION MECHANISMS

JNK激酶级联在T细胞增殖中起关键作用， 凋亡 HPK 1是一种PAK/STE 20样激酶，是一种新的造血- JNK信号通路的特异性激活剂。 我们的工作假设 关于HPK 1信号通路如下：TCR，CD 28产量 衔接子产生HPK 1产生MEKK 1，TAK 1产生MKK 4，MKK 7产生JNK 产生T细胞活化/凋亡。 这项拟议的研究将侧重于 在T细胞活化中HPK 1上游的信号传导事件 和凋亡。 该方法旨在（i）检验我们的假设， Crk（或其他衔接子）作为HPK 1的上游调节子，（ii） 了解HPK 1在T细胞中的调节、功能和修饰。 细胞激活，和（iii）测试我们的假设，即HPK 1参与 T细胞凋亡。 我们对宿主因素的未来理解 HPK 1介导的T细胞信号通路将提供信息， 发现、设计和评估有效的 用于AIDS、免疫性疾病和 癌的 具体目标是： 1. T细胞增殖过程中衔接子对HPK 1功能调控的研究 Activation. 这一目标将检验我们的假设，即Crk（或其他 adaptors）是HPK 1的上游调节因子。 我们将研究物理 HPK 1和衔接子之间的相互作用以及 HPK 1-接头复合物在T细胞活化中的调节。 我们将 进一步研究磷酸化在HPK 1-接头相互作用中的作用， 检查HPK 1通过适配器的潜在调节，映射适配器- 结合域的HPK 1，并评估个人的作用， 富含脯氨酸的结构域在T细胞中的HPK 1信号转导中的作用。 2. HPK 1在T细胞中的磷酸化及其调控研究 共刺激。 我们将检验HPK 1激酶活性 受磷酸化和T细胞共刺激信号调节。 我们 将研究HPK 1与TCR或TCR之间潜在的复合物形成， CD28。 我们还将研究HPK 1的潜在磷酸化， TCR/CD 28相关酪氨酸激酶。我们将绘制磷酸化 通过磷酸肽定位/测序，确定HPK 1的位点，然后确定 它们在T细胞活化、激酶活性、亚细胞 定位和蛋白质-蛋白质相互作用。 3. HPK 1在T细胞凋亡中的调控及功能研究 Fas信号传导导致HPK 1的裂解，这可能导致细胞凋亡。 HPK 1的不可逆激活。 我们将检验HPK 1 与T细胞凋亡有关。 HPK 1在T细胞中的生物学作用 将使用野生型、激酶结构域或 HPK 1的显性失活突变体。 我们将鉴定Fas诱导的裂解 HPK 1上的位点和负责HPK 1切割的蛋白酶活性。 这种蛋白水解切割的功能将通过产生 HPK 1的显性阴性不可裂解突变体。

The JNK kinase cascade plays a pivotal role in T-cell proliferation and apoptosis. HPK1, a PAK/STE20-like kinase, is a novel hematopoietic- specific activator of the JNK signaling pathway. Our working hypothesis about the HPK1 signaling pathway is as follows: TCR, CD28 yields Adaptors yields HPK1 yields MEKK1, TAK1 yields MKK4, MKK7 yields JNK yields T-Cell Activation/Apoptosis. This proposed study will focus on the signaling events immediately upstream of HPK1 in T-cell activation and apoptosis. The approach is designed to (i) test our hypothesis of Crk (or other adaptors) as the upstream regulator of HPK1, (ii) understand the regulation, functions, and modifications of HPK1 in T- cell activation, and (iii) test our hypothesis that HPK1 is involved in T-cell apoptosis. Our future understanding of host factors involved in the HPK1-mediated T-cell signaling pathways will provide information fundamental to the discovery, design, and evaluation of effective intracellular therapeutic agents for AIDS, immunological disorders, and cancers. The specific aims are: 1. Study of the Regulation of HPK1 Function by Adaptors during T-Cell Activation. This aim will test our hypothesis that Crk (or other adaptors) is an upstream regulator of HPK1. We will study the physical interaction between HPK1 and adaptors as well as the potential regulation of the HPK1-adaptor complex in T-cell activation. We will further study the role of phosphorylation in HPK1-adaptor interactions, examine the potential regulation of HPK1 by adaptors, map the adaptor- binding domains of HPK1, and evaluate the role of the individual proline-rich domains in HPK1 signal transduction in T cells. 2. Study of the Regulation and Phosphorylation of HPK1 in T-Cell Costimulation. We will test the hypothesis that HPK1 kinase activity is regulated by phosphorylation and T-cell costimulatory signals. We will study potential complex formation between HPK1 and either TCR or CD28. We will also study the potential phosphorylation of HPK1 by TCR/CD28-associated tyrosine kinases. We will map the phosphorylation sites of HPK1 by phosphopeptide mapping/sequencing, and then determine their role in T-cell activation, kinase activity, subcellular localization, and protein-protein interactions. 3. Study of the Regulation and Function of HPK1 in T-Cell Apoptosis. Fas signaling causes the cleavage of HPK1 which may lead to the irreversible activation of HPK1. We will test the hypothesis that HPK1 is involved in T-cell apoptosis. The biological role of HPK1 in T-cell apoptosis will be studied using the wild-type, the kinase domain, or a dominant-negative mutant of HPK1. We will identify Fas-induced cleavage sites on HPK1 and the protease activities responsible for HPK1 cleavage. The function of this proteolytic cleavage will be studied by generating dominant-negative, uncleavable mutants of HPK1.