TRAF1 Is a Substrate of Caspases Activated during Tumor Necrosis Factor Receptor-α-induced Apoptosis

Abstract

TRAF family proteins are signal-transducing adapter proteins that interact with the cytosolic domains of tumor necrosis factor (TNF) family receptors. Here we show that TRAF1 (but not TRAF2-6) is cleaved by certain caspases in vitro and during TNF-␣-and Fas-induced apoptosis in vivo.160 LEVD 163 was identified as the caspase cleavage site within TRAF1, generating two distinct fragments. Significant enhancement of TNF receptor-1 (CD120a)-and, to a lesser extent, Fas (CD95)-mediated apoptosis was observed when overexpressing the C-terminal TRAF1 fragment in HEK293T and HT1080 cells. The same fragment was capable of potently suppressing TNF receptor-1-and TRAF2-mediated nuclear factor-B activation in reporter gene assays, providing a potential mechanism for the enhancement of TNF-mediated apoptosis. Cell death induced by other death receptor-independent stimuli such as cisplatin, staurosporine, and UV irradiation did not result in cleavage of TRAF1, and overexpression of the C-terminal TRAF1 fragment did not enhance cell death in these cases. TRAF1 cleavage was markedly reduced in cells that contain little procaspase-8 protein, suggesting that this apical protease in the TNF/Fas death receptor pathway is largely responsible. These data identify TRAF1 as a specific target of caspases activated during TNF-and Fasinduced apoptosis and illustrate differences in the repertoire of protease substrates cleaved during activation of different apoptotic pathways.

Caspase-mediated Cleavage Converts the Tumor Necrosis Factor (TNF) Receptor-associated Factor (TRAF)-1 from a Selective Modulator of TNF Receptor Signaling to a General Inhibitor of NF-κB Activation

“…1A). Although it has already been shown elsewhere that the caspase-generated cleavage product of TRAF1 inhibits TNF-mediated NF-B activation (18,19), our data show further that this TRAF domain com-…”

“…TRAF1 was demonstrated to inhibit apoptosis (11,12), but it is also a substrate of caspase-8 and the respective carboxylterminal fragment that is generated during apoptosis was previously shown to inhibit TNF mediated NF-B activation (18,19). Our functional analysis of this TRAF1 fragment discussed above revealed a remarkable difference to full-length TRAF1.…”

“…We have addressed this question by analyzing the impact of TRAF1 on a range of NF-B-inducing receptors of the TNF receptor family in transient reporter gene assays. As a caspasegenerated, truncated form of TRAF1 occurs naturally during apoptosis (18,19), we included an expression plasmid encoding this fragment (TRAF1 amino acids 164 -416, designated as TRAF1-TD) in our analysis. With exception of a significant inhibitory effect of full-length TRAF1 on CD40-mediated NF-B activation, we found no evidence for a major inhibitory effect of transient TRAF1 expression on the following NF-B stimulators: CD30, TNF-R2, LT␤R, EDAR, DR3, and TNF-R1 (Fig.…”

“…Remarkably, TRAF1 is transcriptionally up-regulated by NF-B and could have a role in receptor cross-talk and/or feedback regulation of activated receptor signaling complexes. Moreover, TRAF1 can be cleaved during apoptosis by caspase-8 (18,19). The carboxyl-terminal cleavage product of TRAF1 produced thereby comprises only the TRAF domain of the molecule, is still able to interact with TRAF2 and interferes with TNF-induced NF-B activation (18,19).…”

Tumor necrosis factor receptor-associated factors (TRAFs)

“…An explanation for these apparently divergent roles of TRAF1 may in part be provided by the observation that TRAF1, but not other members of the TRAF family, is a substrate for caspases that are activated by TNF family death receptors (Leo et al, 2001;Jang et al, 2001). TRAF1 is cleaved at a 160 LVED 163 motif to yield two fragments of approximately 28 and 22 kd.…”