Abstract
Receptor-interacting protein (RIP) kinase 3 (RIPK3)-dependent necroptosis directs inflammation and tissue injury, as well as anti-viral host defense. In human cells, herpes simplex virus 1 (HSV1) UL39-encoded ICP6 blocks RIP homotypic interacting motif (RHIM) signal transduction, preventing this leakage form of cell death and sustaining viral infection. TNF receptor 1 (TNFR1)-induced necroptosis is known to require the formation of a RIPK1-RIPK3-mixed lineage kinase domain-like pseudokinase (MLKL) signaling complex (necrosome) that we find compartmentalizes exclusively to caveolin-1-associated detergent-resistant membrane (DRM) vesicles in HT-29 cells. Translocation proceeds in the presence of RIPK3 kinase inhibitor GSK'840 or MLKL inhibitor necrosulfonomide but requires the kinase activity, as well as RHIM signaling of RIPK1. ICP6 impedes the translocation of RIPK1, RIPK3, and MLKL to caveolin-1-containing DRM vesicles without fully blocking the activation of RIPK3 or phosphorylation of MLKL. Consistent with the important contribution of RIPK1 RHIM-dependent recruitment of RIPK3, overexpression of RHIM-deficient RIPK3 results in phosphorylation of MLKL, but this does not lead to either translocation or necroptosis. Combined, these data reveal a critical role of RHIM signaling in the recruitment of the MLKL-containing necrosome to membrane vesicle-associated sites of aggregation. A similar mechanism is predicted for other RHIM-containing signaling adaptors, Z-nucleic acid-binding protein 1 (ZBP1) (also called DAI and DLM1), and TIR domain-containing adapter-inducing interferon-β (TRIF).
Highlights
Receptor-interacting protein (RIP) kinase 3 (RIPK3)– dependent necroptosis directs inflammation and tissue injury, as well as anti-viral host defense
There was modest recruitment of P42-caspase 8 (Casp8) (P42-C8) and receptor interacting protein kinase 1 (RIPK1) following TNF ϩ SMAC mimetic (T/S) treatment but robust recruitment of mixed lineage kinase domain–like pseudokinase (MLKL), pMLKL, P42-C8, RIPK1, RIPK3, and -actin to Cav1-containing membrane fractions following T/S/V treatment, indicating that apoptotic complexes form independently of detergent-resistant membrane (DRM), whereas necroptotic complexes associate with DRM
This is consistent with the recruitment of the necroptotic machinery (RIPK1, RIPK3, and MLKL) to detergent-insoluble fractions previously identified in high-speed centrifugation pellets [13, 14, 46]
Summary
The TNFR1-induced necrosome associates with Cav1-containing intracellular vesicles. Many receptors recruit downstream signaling machinery components into specialized membrane sites, exhibiting reduced density caused by the association with lipids. Treatment of HSV1 ⌬ICP6-infected HT29 –FLAG–mutR3 cells with T/S/ V/NSA resulted in P42-C8 in both soluble and pellet fractions without appearing in the FLAG– co-IP fraction (Fig. 6A), which indicates that the detected interaction of RIPK3 with P42-C8 is RHIM-dependent. RIPK3 co-localization with Cav was lost in HT29 –FLAG–mutR3 infected with HSV1 ⌬ICP6, indicating the role of RHIM signaling in necrosome aggregation under viral infection conditions (Fig. 6B) These results align with previous reports showing RIPK3 RHIM-dependent interaction with the ripoptosome. Lar puncta following T/S/V/NSA treatment (Fig. 7D, lower panels), structures that were not detected in HT29 –FLAG–mutR3 (Fig. 7D, upper panels) These results suggest that RHIM interactions induce RIPK3 oligomers as a requisite step leading to pMLKL oligomerization and translocation to the plasma membrane
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