Molecular Analysis of TTP, a Key Regulator of TNF Activity
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RESULTS. We have determined that various cell types undergo programmed cell death in response to constitutive low-level TTP/TIS11 protein expression. This cell death involves DNA cleavage, caspase activation, and stimulation of the mitochondrial death machinery, indicating that it proceeds by apoptosis. Mapping experiments revealed that both the zinc finger region and C-terminal domain of TTP are necessary to induce significant apoptosis. Surprisingly, expression of TTP but not TIS11b or TIS11d sensitizes cells to the apoptotic effects of TNFα, 4. Upon conducting a yeast-2-hybrid analysis to screen for interacting proteins, we identified 143-3 as a potential interactor with the C-terminal domain of TTP. We verified that this interaction could also occur in a mammalian system via co-immunoprecipitation of overexpressed TTP and endogenous 14-3-3. All three TTP/TIS11 proteins can bind to GST-143-3. Binding is phosphorylation dependent, and different 14-3-3 isoforms appear to bind to a varying pattern of phosphorylated forms of TTP. We have also identified a TTP point mutant with diminished binding to 14-3-3. DISCUSSION. Our findings that modest levels of TTP/TIS11 expression induce apoptosis suggest that all three of these proteins may influence growth or survival pathways. TTP alone however is able to sensitize cells to TNFα mediated cell death, leading us to postulate that