Post-translational control of RIPK3 and MLKL mediated necroptotic cell death

Abstract

Several programmed lytic and necrotic-like cell death mechanisms have now been uncovered, including the recently described receptor interacting protein kinase-3 (RIPK3)-mixed lineage kinase domain-like (MLKL)-dependent necroptosis pathway. Genetic experiments have shown that programmed necrosis, including necroptosis, can play a pivotal role in regulating host-resistance against microbial infections. Alternatively, excess or unwarranted necroptosis may be pathological in autoimmune and autoinflammatory diseases. This review highlights the recent advances in our understanding of the post-translational control of RIPK3-MLKL necroptotic signaling. We discuss the critical function of phosphorylation in the execution of necroptosis, and highlight the emerging regulatory roles for several ubiquitin ligases and deubiquitinating enzymes. Finally, based on current evidence, we discuss the potential mechanisms by which the essential, and possibly terminal, necroptotic effector, MLKL, triggers the disruption of cellular membranes to cause cell lysis.

Keywords: MLKL; RIPK3; mixed lineage kinase domain-like; necroptosis; necroptotic cell death; receptor interacting protein kinase-3.

Figure 1.. Positive regulators of necroptosis.

The core necroptosis machinery, comprising RIPK3 and MLKL, are activated following RIPK3 interaction with RIPK1, TRIF or DAI via their RHIMs. CYLD-mediated deubiquitylation of RIPK1 is necessary for its participation in cell death pathways, while ubiquitylation of RIPK3 (at Lys5) and MLKL by as-yet-unidentified E3 ligases may promote necroptosis ^, . HSP90 is known to augment the necroptotic functions of RIPK1 and RIPK3. MLKL is believed to induce cell death via a membrane-directed process , perhaps by directly permeabilizing membranes ^, , with some debate over whether channel activation might be involved ^{, ,} , or, as one report suggests, there may be a role for Src in promoting MLKL-mediated death downstream of Gβγ. Abbreviations: 4HB, four-helix bundle; CYLD, cylindromatosis; DAI, DNA-dependent activator of interferon [IFN]-regulatory factors; HSP90, heat shock protein 90; IAP, inhibitor of apoptosis proteins; MLKL, mixed lineage kinase domain-like; RHIM, RIP homotypic interaction motif; RIPK, receptor interacting protein kinase; TLR, toll-like receptor; TNF, tumour necrosis factor; TNFR1, TNF receptor 1; TRADD, TNFR1-associated death domain; TRAF2, TNF receptor associated factor 2; TRPM7, transient receptor potential cation channel, subfamily M, member 7.

Figure 2.. Negative regulators of necroptosis.

Negative regulation at the levels of RIPK1, RIPK3 and MLKL have been reported to attenuate necroptotic signalling. Not only is necroptosis by definition a caspase-independent process, but caspase-8 negates cell death, potentially by proteolytically-cleaving CYLD, RIPK1 and RIPK3. Reversible tuning of the pathway is accomplished by introduction or removal of post-translational modifications, including: ubiquitylation of RIPK1 by IAPs; deubiquitylation of RIPK3 on Lys5 by A20; and dephosphorylation of mouse RIPK3 at S231/T232 by Ppm1b. Whether an analogous phosphatase exists to dephosphorylate activated MLKL and inhibit necroptosis is not clear. However, other regulators of MLKL have been proposed, such as TRAF2. It is currently unknown what factors might govern phospho-MLKL assembly into higher order oligomers on the membrane, although a role for additional factors in mediating death is suggested by the lag between MLKL membrane translocation/oligomerisation and cell death . Abbreviations: 4HB, four-helix bundle; CYLD, cylindromatosis; IAP, inhibitor of apoptosis proteins; MLKL, mixed lineage kinase domain-like; RHIM, RIP homotypic interaction motif; RIPK, receptor interacting protein kinase; TNF, tumour necrosis factor; TRADD, TNFR1-associated death domain; TRAF2, TNF receptor associated factor 2.