Necroptosis signalling is tuned by phosphorylation of MLKL residues outside the pseudokinase domain activation loop

Area of Science:

• Cell Biology
• Molecular Biology
• Immunology

Background:

• Mixed lineage kinase domain-like (MLKL) is a pseudokinase essential for necroptosis.
• Phosphorylation of MLKL's activation loop by RIPK3 is critical for necroptosis initiation.
• The role of other MLKL phosphorylation sites in regulating necroptosis remains unclear.

Purpose of the Study:

• To investigate the function of novel MLKL phosphorylation sites.
• To compare the impact of known and novel MLKL phosphorylation sites on necroptosis.
• To explore the potential role of RIPK3 as a suppressor of MLKL activation.

Main Methods:

• Utilized CRISPR-Cas9 to generate Mlkl(-/-), Ripk3(-/-), and Mlkl(-/-)Ripk3(-/-) knockout cells.
• Reconstituted knockout cells with MLKL phospho-site mutants, including phosphomimetic and alanine substitution mutants.
• Identified novel MLKL phosphorylation sites (Ser158, Ser228, Ser248) using mass spectrometry (MS).

Main Results:

• RIPK3 phosphorylation of the MLKL activation loop (S345D mutant) was sufficient to induce necroptosis independently of death stimuli.
• MLKL mutants S228A, S228E, and S158A induced cell death without stimuli, most significantly in double knockout cells.
• These findings suggest RIPK3 may act as a suppressor of MLKL activation.

Conclusions:

• Phosphorylation events beyond RIPK3 activation loop modification can fine-tune MLKL's necroptosis-inducing activity.
• Specific MLKL phosphorylation sites (e.g., S158, S228) can promote cell death even in the absence of canonical necroptotic stimuli.
• The results highlight a complex regulatory network involving MLKL phosphorylation in controlling necroptosis.