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.
