Abstract
Receptor-interacting protein kinase 1 (RIPK1) is an important component of the tumor necrosis factor receptor 1 (TNFR1) signaling pathway. Depending on the cell type and conditions, RIPK1 mediates MAPK and NF-κB activation as well as cell death. Using a mutant form of RIPK1 (RIPK1ΔID) lacking the intermediate domain (ID), we confirm the requirement of this domain for activation of these signaling events. Moreover, expression of RIPK1ΔID resulted in enhanced recruitment of caspase-8 to the TNFR1 complex II component Fas-associated death domain (FADD), which allowed a shift from TNF-induced necroptosis to apoptosis in L929 cells. Addition of the RIPK1 kinase inhibitor necrostatin-1 strongly reduced recruitment of RIPK1 and caspase-8 to FADD and subsequent apoptosis, indicating a role for RIPK1 kinase activity in apoptotic complex formation. Our study shows that RIPK1 has an anti-apoptotic function residing in its ID and demonstrates a cellular system as an elegant genetic model for RIPK1 kinase-dependent apoptosis that, in contrast to the Smac mimetic model, does not rely on depletion of cellular inhibitor of apoptosis protein 1 and 2 (cIAP1/2).
Highlights
Receptor-interacting protein kinase 1 (RIPK1) is a central kinase in tumor necrosis factor receptor 1 (TNFR1) signaling participating in NF-B activation, necroptosis, and apoptosis
Our results further indicate that intermediate domain (ID) is involved in induction of necrotic cell death, as ectopic expression of RIPK1⌬ID did not induce cell death
RIPK1 is dispensable for the classical apoptotic pathway induced by TNF, in the presence of Smac mimetics, RIPK1 is converted into a prodeath molecule that is required for the formation of a caspase-8 activating complex [7, 9, 19, 21,22,23]
Summary
RIPK1 is a central kinase in TNFR1 signaling participating in NF-B activation, necroptosis, and apoptosis. Upon TNFR1 stimulation, a prosurvival complex I is formed by recruitment of, among others, TNF receptor-associated death domain (TRADD), receptor-interacting protein kinase 1 (RIPK1), TRAF2 (TNF receptor-associated factor 2), cellular inhibitor of apoptosis protein 1 and 2 (cIAP1 and cIAP2) and the linear ubiquitin chain assembly complex [1, 2, 4]. RIPK1 Kinase-dependent Apoptosis the presence of Smac mimetics, which induce proteasomal degradation of cIAP1/2 and inhibit RIPK1 ubiquitylation in complex I, cells switch to a RIPK1-dependent apoptotic pathway in response to TNF [7, 9, 19, 21,22,23] In these conditions, kinase-active RIPK1 was crucial for the formation of a caspase-8 activating complex, whereas in the absence of Smac mimetics, RIPK1 had no role in apoptosis induction upon TNF stimulation [23]. Our model resembles the model of RIPK1-dependent apoptosis induced by TNF plus Smac mimetics but has the advantage that no additional factors are required so that this cellular model is very useful to study the downstream targets of RIPK1 kinase leading to apoptosis
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