Fas is a cell surface molecule which transduces a signal that results in apoptosis. Fas has been implicated in the removal of self reactive T and B cells and in the maintenance of self tolerance. A defect in Fas gene expression has been identified as the cause of the lymphoproliferative syndrome and autoimmune disease of MRL/pr/pr mice. Mutational analysis of the human Fas antigen has defined a 68 a.a. signal-transducing cytoplasmic domain which is required for the transduction of an apoptotic signal. However, the mechanism of signal transduction by Fas has yet to be determined. We have recently cloned, using the yeast two hybrid system, a novel intracellular Fas associated protein (FAP). FAP shows strong homology to the recently cloned ubiquitin conjugating (UBC) enzyme UBC9 which controls yeast cell cycle progression from G2 to mitosis and regulates the degradation of S and M phase cyclins in Saccharomyces cerevisiae. A single amino acid substitution 238 Val-> Asn in the death domain of the intracellular region of Fas that abolishes Fas mediated apoptosis also abolished Fas-FAP interaction suggesting that FAP plays an important role in Fas killing. Wild type FAP, but not a mutant in which Cys93 in the consensus sequence of the active site for ubiquitin conjugation was replaced by Ser, complemented UBC9 temperature sensitive mutants suggesting that FAP possesses ubiquitin conjugating activity and that it is likely to play an important role in cell cycle regulation. We propose to: l. Analyze the role of FAP in Fas mediated apoptosis by examining Fas killing in a) cells with a temperature sensitive ubiquitin activating enzyme (E1) mutant and b) in cells transfected with a dominant negative FAP mutant. 2. Analyze the structural and functional interaction of Fas and FAP by a) mapping the FAP domains and residues involved in Fas-FAP interactions using truncated and mutant proteins; b) examining the effect of Fas crosslinking on FAP phosphorylation, ubiquitination, association with Fas and intracellular localization. 3. Identify FAP targets by a) identifying the targets for FAP ubiquitin conjugating activity ; b) identifying proteins that interact with FAP in the yeast two hybrid system. 4. Examine FAP expression and association with Fas in T cell development and in autoimmune disease. The proposed studies may lead to a better understanding of the mechanisms of apoptosis and self tolerance and to novel strategies for the treatment of autoimmune diseases.
| Dunn, I F; Sannikova, T Y; Geha, R S et al. (2000) Identification and characterization of two CD40-inducible enhancers in the mouse TRAF1 gene locus. Mol Immunol 37:961-73 |
| Dunn, I F; Geha, R S; Tsitsikov, E N (1999) Structure of the murine TRAF1 gene. Mol Immunol 36:611-7 |
| Tsitsikov, E N; Wright, D A; Geha, R S (1997) CD30 induction of human immunodeficiency virus gene transcription is mediated by TRAF2. Proc Natl Acad Sci U S A 94:1390-5 |
| Wright, D A; Futcher, B; Ghosh, P et al. (1996) Association of human fas (CD95) with a ubiquitin-conjugating enzyme (UBC-FAP). J Biol Chem 271:31037-43 |
