Characterization of RIPK3-mediated phosphorylation of the activation loop of MLKL during necroptosis

Area of Science:

• Cellular biology
• Molecular mechanisms of cell death

Background:

• Mixed lineage kinase domain-like pseudokinase (MLKL) is a key mediator of necroptosis, a programmed form of cell death.
• MLKL activation involves its translocation to the plasma membrane and subsequent rupture, a process regulated by the necrosome complex containing RIPK3.
• Previous studies indicated RIPK3 phosphorylates MLKL at Ser345, Ser347, and Thr349, with Ser345 substitution to aspartic acid conferring constitutive activity.

Purpose of the Study:

• To elucidate the specific roles of MLKL phosphorylation sites (Ser345, Ser347, Thr349) in RIPK3-mediated necroptosis.
• To investigate the necessity of MLKL Ser345 phosphorylation for MLKL translocation and plasma membrane accumulation.

Main Methods:

• Generation of a specific monoclonal antibody for detecting phospho-MLKL Ser345 in murine cells.
• Utilized a series of MLKL mutants to assess the function of individual phosphorylation sites.
• Employed a novel RIPK3 inhibitor to study the upstream regulation of MLKL phosphorylation.

Main Results:

• Phosphorylation of MLKL at Ser345 is critical for RIPK3-mediated necroptosis, while Ser347 plays a minor role and Thr349 appears irrelevant.
• MLKL Ser345 phosphorylation is not required for MLKL interaction within the necrosome complex.
• Phosphorylation of Ser345 is essential for MLKL translocation to the plasma membrane, its accumulation there, and the subsequent induction of necroptosis.

Conclusions:

• MLKL Ser345 phosphorylation is a crucial step for initiating necroptosis downstream of RIPK3.
• Targeting MLKL Ser345 phosphorylation could offer a therapeutic strategy for modulating necroptotic cell death.