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Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein
Published on: June 30, 2019
Detecting phosphorylated MLKL in murine tissues
Riley M Williams1, Bikash Thapa1, Siddharth Balachandran1
1Center for Immunology, Fox Chase Cancer Center, Philadelphia, PA, United States.
Abstract:
The programmed cell death process of necroptosis is activated when Receptor Interacting protein Kinase (RIPK)3 phosphorylates its substrate Mixed Lineage Kinase Like protein (MLKL). Phosphorylated MLKL oligomers then traffic to cellular membranes, including the plasma membrane, and perforate these membranes. MLKL-driven pore formation in the plasma membrane alters the cell osmolarity, causing it to swell and undergo necrotic death. Phosphorylation of MLKL by RIPK3 is essential for the execution of necroptosis, and is thus a widely accepted biomarker for this mode of cell death. In this chapter, we describe our procedure for detecting phosphorylated murine MLKL in influenza A virus (IAV) infected lung tissues, and within tumors following treatment with the compound CBL0137.
Insights
Necroptosis, a programmed cell death, is triggered by RIPK3 phosphorylating MLKL. This study details detecting phosphorylated MLKL in influenza A virus-infected lungs and tumors treated with CBL0137.
Area of Science:
- Biochemistry
- Cell Biology
- Immunology
Background:
- Necroptosis is a programmed form of necrosis.
- Receptor Interacting protein Kinase 3 (RIPK3) phosphorylates Mixed Lineage Kinase Like protein (MLKL) to activate necroptosis.
- Phosphorylated MLKL forms pores in cellular membranes, leading to cell death.
Purpose of the Study:
- To describe a procedure for detecting phosphorylated MLKL.
- To assess phosphorylated MLKL in influenza A virus (IAV)-infected lung tissues.
- To evaluate phosphorylated MLKL in tumors treated with CBL0137.
Main Methods:
- Detection of phosphorylated murine MLKL.
- Analysis of lung tissues from IAV-infected mice.
- Analysis of tumor tissues from mice treated with CBL0137.
Main Results:
- The described procedure allows for the detection of phosphorylated MLKL.
- Phosphorylated MLKL was detected in IAV-infected lung tissues.
- Phosphorylated MLKL was detected in tumors treated with CBL0137.
Conclusions:
- Phosphorylation of MLKL by RIPK3 is a key event in necroptosis.
- Detecting phosphorylated MLKL can serve as a biomarker for necroptosis.
- This method can be applied to study necroptosis in various disease models.
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