Hematopoietic cells from patients with Fanconi anemia (FA) exhibit both hypersensitive to cross-linking agents such as mitomycin C and exaggerated apoptotic responses to a variety of extracellular cues for mitogenic inhibition and apoptosis. One of the FA proteins, FANCC, functions to protect cells from cytotoxicity induced by combinations of either interferon gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) or double-stranded RNA (dsRNA) and IFN-gamma. Our studies have demonstrated that the pro-apoptotic protein kinase PKR is required for cytokine hypersensitivity of FA cells. More recently, we showed that FANCC forms a ternary complex with the PKR kinase and molecular chaperone Hsp70, and mutations in 3 major FA genes, FANCA, FANCC and FANCG, affect the structural integrity of the complex. This suggests that 1 or more FA proteins participate in the formation of a multi-subunit protein complex, which we term the PKR-FA protein complex. We hypothesize that the FA proteins modulate stress responses by forming the PKR-FA protein complex and that a proper control of the activity of the stress kinase PKR is one consequence of structural and functional integrity of the complex. The long-term goals of our study are to characterize the functional assembly of the PKR-FA protein complex and to ultimately define the role of this newly identified FA protein-containing complex in hematopoiesis. We plan to build on our preliminary structural and biochemical characterizations of the PKR-FANCC-Hsp70 ternary complex to explore in molecular details the roles that FA proteins and other components play in the formation and function of the PKR-FA protein complex, interactions between components of the complex and functional significance of these interactions, and the roles of individual components in the structural and functional integrity of the complex. We will also investigate the in vivo role of the PKR-FA complex in proliferation and apoptotic response (to inhibitory cytokines IFN-gamma and TNF-alpha or DNA damaging agents such as mitomycin C) of hematopoietic progenitor cells using bone marrow cells from FA patients and FA knockout mice. Finally, we will examine the assembly of the FA protein-containing complexes in a living cell exposed to specified cellular stresses. The expectation is that the proposed study will provide insight into the biological function of the PKR-FA protein complex with regard to hematopoiesis and the relationship between the PKR-FA protein complex and the FA nuclear complex identified for DNA damage response.
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