Abstract
Sphingomyelinase (SMase) activation and ceramide generation have emerged as an important signaling pathway transducing diverse biological effects of cytokine receptors like p55 tumor necrosis factor (TNF) receptor or Fas. Here we describe the TNF-dependent activation of acid SMase (A-SMase) through the p55 TNF receptor-associated proteins TRADD and FADD. Overexpression of TRADD and FADD in 293 cells did not change basal activity of A-SMase but enhanced TNF-induced stimulation of A-SMase. Other TNF R55-associated proteins like TRAF2 and RIP, which were reported to mediate TNF R55-mediated activation of nuclear factor kappaB, did not affect activation of A-SMase. Caspase inhibitors markedly reduced A-SMase activity, suggesting the involvement of an ICE-like protease in TRADD/FADD-mediated activation of A-SMase. Overexpression of caspase-8/a (FLICE/MACH) or caspase-10/b (FLICE2) did not change A-SMase activity, suggesting that TRADD/FADD-mediated activation of A-SMase involves a yet to be defined caspase-like protease distinct from caspase-8/a or -10/b.
Highlights
Protein kinase C (PKC), JNK, and caspases (6 – 8)
To examine whether one of the so far identified tumor necrosis factor (TNF)-R55 death domain-associated proteins mediates the activation of acid SMase (A-SMase), 293 cells were first transfected with an expression vector coding for FLAG-tagged TRADD
We show that acid sphingomyelinase is activated through p55 TNF receptor-associated proteins TRADD and FADD
Summary
N-SMase and A-SMase are activated independently by distinct cytoplasmic domains of TNF-R55 [1]. The NSD binds a WD-repeat protein, FAN, that mediates activation of N-SMase. The cytoplasmic protein TRADD was recently identified to associate with the death domain of TNF-R55 in a TNF-dependent process [12, 13]. FADD, which directly binds to TRADD, has been shown to mediate activation of a pro-apoptotic protease, caspase-8/a (FLICE/ MACH), eventually leading to apoptosis [16, 17]. We show that TNF-induced activation of A-SMase is mediated through TRADD and FADD. Several tetrapeptide caspase inhibitors as well as cytokine response modifier A (crmA) attenuated TNF-induced activation of A-SMase activity, suggesting that FADD mediates enhancement of A-SMase activity through activation of a protease distinct from FLICE
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