Abstract
Tumor Necrosis Factor-α canonically induces the activation of NF-κB and associated gene product cellular FLICE-like inhibitory protein (cFLIPL) to promote cell survival. Previously, we demonstrated that ectopic expression of the Fas associated death domain (FADD) diminishes the expression of cFLIPL and transduces caspases-8 mediated apoptosis, independent of FasL stimulation in HEK 293T cells. However, the underlying molecular mechanism of FADD mediated ablation of cFLIP and NF-κB signaling to determining the fate of cell death or survival remains elusive. Here, we explored a novel molecular mechanism of FADD mediated apoptotic cell death that was directed by ubiquitination of cFLIPL and inhibition of NF-κB activation, independent of TNF-α stimulation. We found that induced expression of FADD firmly interacts with procaspase-8 and precludes cFLIPL to from the death inducing signaling complex (DISC). In addition, FADD negatively regulates cellular inhibitor of apoptosis protein 2 (cIAP2) and Bcl-2. Furthermore, FADD restrains cIAP2 expression and interacts with RIP1 and procaspase-8 to accomplish apoptotic cell death signaling. Interestingly, FADD was also found to promote JNK1 mediated activation of E3 ubiquitin ligase ITCH to degrade cFLIPL that may lead to commencement of apoptosis. Thus, FADD is an important regulator for determining the fate of cell death or survival.
Highlights
Tumor Necrosis Factor-α canonically induces the activation of NF-κB and associated gene product cellular FLICE-like inhibitory protein to promote cell survival
A growing body of evidence demonstrates that elevated expression of cFLIPL competitively excludes the interaction of initiator procaspase-8/10 with adaptor protein Fas associated death domain (FADD) at death inducing signaling complex (DISC) to block death receptor signaling of apoptosis[28,29,30]
We observed that mutations in DED of the FADD at Serine 16, Serine and lysine did not affect the binding of procaspase-8, but restrict the binding of cFLIPL at the DISC (Fig 1f)
Summary
Tumor Necrosis Factor-α canonically induces the activation of NF-κB and associated gene product cellular FLICE-like inhibitory protein (cFLIPL) to promote cell survival. We demonstrated that ectopic expression of the Fas associated death domain (FADD) diminishes the expression of cFLIPL and transduces caspases-8 mediated apoptosis, independent of FasL stimulation in HEK 293T cells. We explored a novel molecular mechanism of FADD mediated apoptotic cell death that was directed by ubiquitination of cFLIPL and inhibition of NF-κB activation, independent of TNF-α stimulation. FADD was found to promote JNK1 mediated activation of E3 ubiquitin ligase ITCH to degrade cFLIPL that may lead to commencement of apoptosis. We observed that induced expression of FADD regulates NF-κ B activation and cIAP2 and cFLIPL expression accompanied with JNK1 mediated ubiquitination of cFLIPL to commence apoptotic cell death signaling
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