MORT1 in Apoptosis of Malignant and Primary T cells

描述(申请人提供)：最初分离的Morti或FADD(Fas相关死亡结构域)是作为传递Fas凋亡信号的适配分子而分离的。随后，FADD被证明是被Fas和属于肿瘤坏死因子受体超家族的其他含有死亡结构域的受体所缓解的细胞凋亡所必需的。然而，在研究FADD在体内的作用的过程中，人们发现FADD在小鼠胚胎发育和T细胞增殖中也起着重要的作用。FADD缺陷小鼠在怀孕第11天左右在子宫中死亡，FADD-/-RAG-1-/-嵌合体中的成熟T细胞功能缺陷。这些T细胞表现出细胞周期异常和细胞周期机制的异常调节。同样，对T细胞特异性FADD缺陷小鼠的特征表明，FADD缺陷导致T细胞发育停滞在未成熟T细胞增殖阶段。有趣的是，FADD在细胞周期的GJM期被G2IM特异性的激酶磷酸化，这表明FADD的磷酸化可能在细胞周期的进展中很重要。在本申请中，我们提出了三个特定的目标，旨在了解FADD如何在增殖中发挥作用。在目标1中，将产生并分析磷酸化缺陷和结构性磷酸化的FADD小鼠。FADD磷酸化在增殖和凋亡以及小鼠胚胎发育中的作用将被研究。在目标2中，将通过产生突变的FADD小鼠来评估含有死亡结构域的受体在增殖和小鼠发育中的作用，这些突变的FADD小鼠在FADD死亡结构域点突变以削弱其适配功能。这应该可以在体内对缺乏任何死亡区域受体功能的小鼠进行研究。在目标3中，将研究FADD在增殖过程中可能发挥作用的分子机制。DNA微阵列将用于评估表达磷酸化缺陷和死亡结构域缺陷FADD的FADD/-I细胞和I细胞的基因表达谱。酵母双杂交系统和生化分析将被用来分离和鉴定新的FADD相互作用蛋白。这些目标的成功实现将使人们对癌细胞中的细胞凋亡和增殖如何协调，以及增殖如何主导细胞的凋亡途径有一个重要的理解。

DESCRIPTION (provided by applicant): Morti or FADD (Fas associated death domain) was initially isolated as an adapter molecule that transmits the Fas apoptotic signals. FADD was subsequently shown to be required for apoptosis mitigated by Fas as well as other death-domain containing receptors that belong to the tumor necrosis factor receptor superfamily. However, during the course of studying the role of FADD in vivo, it was discovered that FADD also plays an essential function in mouse embryogenesis and in T cell proliferation. FADD-deficient mice die in utero around day 11 of gestation and mature T cells in FADD-/- RAG-1-/- chimeras are functionally defective. These T cells exhibit cell cycle abnormalities and aberrant regulation of the cell cycle machinery. Similarly, characterization of T-cell specific FADD-deficient mice showed that FADD-deficiency leads to an arrest of T cell development at the stage of immature T cell proliferation. Interestingly, FADD is phosphorylated during GJM phase of the cell cycle by a G2IM-specific kinase, suggesting that FADD phosphorylation may be important during cell cycle progression. In this application, we propose three specific aims that are designed to understand how FADD functions in proliferation. In aim 1, phosphorylation-defective and constitutive phosphorylated FADD mice will be generated and analyzed. The role of FADD phosphorylation in proliferation and apoptosis as well as mouse embryogenesis will be studied. In aim 2, the role of death-domain containing receptors in proliferation and mouse development will be assessed by generating mutant FADD mice that contain a point mutation at the FADD death domain to cripple its adapter function. This should allow studies of mice devoid of any death-domain receptor function in vivo. In aim 3, the molecular mechanisms of how FADD might function during proliferation will be studied. DNA microarrays will be used to assess gene expression profile of FADD/- I cells and I cells expressing phosphorylation-defective and death-domain defective FADD. The yeast-two hybrid system and biochemical analysis will be used to isolate and characterize novel FADD-interacting proteins. Successful completion of these aims should lead to a significant understanding of how apoptosis and proliferation are coordinated and how proliferation might dominate over apoptotic pathway in cancer cells.