Abstract
BackgroundNecroptosis/programmed necrosis is initiated by a macro-molecular protein complex termed the necrosome. Receptor interacting protein kinase 1 (RIPK1/RIP1) and RIP3 are key components of the necrosome. TNFα is a prototypic inducer of necrosome activation, and it is widely believed that deubiquitination of RIP1 at the TNFR-1 signaling complex precedes transition of RIP1 into the cytosol where it forms the RIP1-RIP3 necrosome. Cylindromatosis (CYLD) is believed to promote programmed necrosis by facilitating RIP1 deubiquitination at this membrane receptor complex.Methodology/Principal FindingsWe demonstrate that RIP1 is indeed the primary target of CYLD in TNFα-induced programmed necrosis. We observed that CYLD does not regulate RIP1 ubiquitination at the TNF receptor. TNF and zVAD-induced programmed necrosis was highly attenuated in CYLD-/- cells. However, in the presence of cycloheximide or SMAC mimetics, programmed necrosis was only moderately reduced in CYLD-/- cells. Under the latter conditions, RIP1-RIP3 necrosome formation is only delayed, but not abolished in CYLD-/- cells. We further demonstrate that RIP1 within the NP-40 insoluble necrosome is ubiquitinated and that CYLD regulates RIP1 ubiquitination in this compartment. Hence, RIP1 ubiquitination in this late-forming complex is greatly increased in CYLD-/- cells. Increased RIP1 ubiquitination impairs RIP1 and RIP3 phosphorylation, a signature of kinase activation.Conclusions/SignificanceOur results show that CYLD regulates RIP1 ubiquitination in the TNFα-induced necrosome, but not in the TNFR-1 signaling complex. In cells sensitized to programmed necrosis with SMAC mimetics, CYLD is not essential for necrosome assembly. Since SMAC mimetics induces the loss of the E3 ligases cIAP1 and cIAP2, reduced RIP1 ubiquitination could lead to reduced requirement for CYLD to remove ubiquitin chains from RIP1 in the TNFR-1 complex. As increased RIP1 ubiquitination in the necrosome correlates with impaired RIP1 and RIP3 phosphorylation and function, these results suggest that CYLD controls RIP1 kinase activity during necrosome assembly.
Highlights
Programmed necrosis or necroptosis is a non-apoptotic form of cell death with important functions in pathogen infections, trauma-induced tissue injury, embryonic development and lymphocyte homeostasis [1]
Polyubiquitinated RIP1 is a known substrate of CYLD [37], a deubiquitinase that has been shown to participate in necrosis signaling [26]
We consistently observed weaker protection by siRNA against CYLD compared with those against RIP1 or RIP3 (Figure 1A). This is in contrast to the expectation that CYLD is an essential regulator of necrosis upstream of RIP1 and RIP3 activation [29,40]
Summary
Programmed necrosis or necroptosis is a non-apoptotic form of cell death with important functions in pathogen infections, trauma-induced tissue injury, embryonic development and lymphocyte homeostasis [1]. Cleavage of RIP1 and RIP3 inactivates their pro-necrotic kinase activity [5,6] This inhibitory mechanism is critical to prevent extensive necrosis during embryonic development [7,8,9], to enforce lymphocyte homeostasis [10,11], and to dampen extensive necrosis-induced inflammation in different tissues [12,13]. It is rarely observed, inhibition of programmed necrosis can result in a “switch” to apoptosis in certain cell types [14,15]. Conclusions/Significance: Our results show that CYLD regulates RIP1 ubiquitination in the TNFα-induced necrosome, but not in the TNFR-1 signaling complex. As increased RIP1 ubiquitination in the necrosome correlates with impaired RIP1 and RIP3 phosphorylation and function, these results suggest that CYLD controls RIP1 kinase activity during necrosome assembly
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