HPK1 MEDIATED T CELL SIGNAL TRANSDUCTION MECHANISMS

JNK激酶级联在T细胞增殖和增殖中起关键作用 细胞凋亡。HPK1是一种类似PAK/STE20的蛋白激酶，是一种新的造血因子。 JNK信号通路的特异性激活物。我们的工作假说 关于HPK1的信号通路如下：TCR、CD28产生 适配器产生HPK1产生MEKK1，TAK1产生MKK4，MKK7产生JNK 产生T细胞激活/凋亡。这项拟议的研究将重点放在 T细胞活化中HPK1上游的信号转导事件 和细胞凋亡。该方法旨在(I)检验我们的假设 CRK(或其他适配器)作为HPK1的上游调节器，(Ii) 了解HPK1在T-T细胞中的调节、功能和修饰 细胞激活，以及(Iii)检验我们的假设，即HPK1参与 T细胞凋亡。我们未来对宿主因素的理解涉及到 HPK1介导的T细胞信号通路将提供信息 有效发现、设计和评估的基础 细胞内治疗艾滋病、免疫紊乱和 癌症。具体目标是： 1.T细胞中接头对HPK1功能调节的研究 激活。这个目标将检验我们的假设，即Crk(或其他 适配器)是HPK1的上游调节因子。我们将学习体能。 HPK1与接头的相互作用及其潜能 HPK1-接头复合体在T细胞激活中的调节。我们会 进一步研究磷酸化在HPK1-适配器相互作用中的作用， 研究适配器对HPK1的潜在调控，绘制适配器- HPK1的结合域，并评估个体的作用 T细胞HPK1信号转导中的富含脯氨酸结构域。 2.HPK1在T细胞中的调节和磷酸化的研究 共刺激效应。我们将检验HPK1激酶活性的假设 受磷酸化和T细胞共刺激信号的调节。我们 将研究HPK1与TCR或TCR之间潜在的复合体形成 CD28。我们还将研究HPK1的潜在磷酸化。 TCR/CD28相关的酪氨酸激酶。我们会绘制出磷酸化的图谱 通过磷酸肽定位/测序，确定HPK1的位置 它们在T细胞激活、激酶活性、亚细胞中的作用 定位和蛋白质-蛋白质相互作用。 3.研究HPK1在T细胞凋亡中的调控和作用。 Fas信号导致HPK1的裂解，这可能导致 HPK1的不可逆激活。我们将检验HPK1的假设 参与T细胞的凋亡。HPK1在T细胞中的生物学作用 细胞凋亡的研究将使用野生型、激活域或 HPK1的显性负性突变体。我们将确定Fas诱导的卵裂 HPK1上的位点和与HPK1裂解有关的蛋白酶活性。 这种蛋白水解性切割的功能将通过生成 HPK1的显性阴性、不可切割的突变体。

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.