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Assaying Protein Kinase Activity with Radiolabeled ATP
Published on: May 26, 2017
GW806742X can induce mouse MLKL activation by directly promoting MLKL kinase like domain dimerization
Feiyang Yuan1, Yu Zhang1, Xinxin Zhu1
1School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China; State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, 200031, China; University of Chinese Academy of Sciences, Beijing, 100049, China.
Abstract:
The dimerization of the MLKL kinase-like domain (KLD) is a crucial step for MLKL activation in necroptosis. In 2014, it was discovered that GW806742X can directly bind to the mouse MLKL KLD via surface plasmon resonance (SPR) (Kd = 9.3 μM), inhibiting TNF-induced membrane translocation of MLKL and necroptosis. Consequently, GW806742X is considered a mouse MLKL inhibitor. In this study, we found that GW806742X blocks TNF-induced RIP1-dependent necroptosis but promotes necroptosis triggered by either RIP3 or MLKL self-oligomerization in the FKBPv chemical dimerizer system. In addition, higher doses of GW806742X can directly induce MLKL-dependent necroptosis and promote MLKL oligomerization, as detected by non-reducing Western blot. Through chemical cross-linking assays, we observed that GW806742X induces dimerization of recombinant mouse MLKL KLD proteins. The dimerization of the MLKL KLD is a direct consequence of RIP3 phosphorylation, a crucial step in RIP3-induced MLKL activation and necroptosis. Therefore, GW806742X exerts a dual effect on necroptosis: it inhibits necroptosis, likely by interfering with RIP1 function, while promoting necroptosis by facilitating MLKL activation.
Insights
GW806742X, a known inhibitor of mixed lineage kinase domain-like protein (MLKL), paradoxically promotes necroptosis by directly inducing MLKL dimerization and activation, revealing a dual role in cell death pathways.
Area of Science:
- Cellular Biology
- Molecular Mechanisms of Cell Death
- Signal Transduction Pathways
Background:
- Mixed lineage kinase domain-like protein (MLKL) activation, involving kinase-like domain (KLD) dimerization, is essential for necroptosis execution.
- The compound GW806742X was previously identified as a direct binder to mouse MLKL KLD, inhibiting TNF-induced necroptosis.
Purpose of the Study:
- To investigate the complex role of GW806742X in necroptosis.
- To elucidate the mechanism by which GW806742X influences MLKL activation and dimerization.
Main Methods:
- Surface Plasmon Resonance (SPR) for binding affinity.
- Chemical cross-linking assays to detect protein dimerization.
- Non-reducing Western blot to assess MLKL oligomerization.
- FKBPv chemical dimerizer system to trigger necroptosis.
Main Results:
- GW806742X inhibits TNF-induced RIP1-dependent necroptosis but promotes necroptosis induced by RIP3 or MLKL self-oligomerization.
- Higher doses of GW806742X directly induce MLKL-dependent necroptosis and promote MLKL oligomerization.
- GW806742X was shown to directly induce dimerization of recombinant mouse MLKL KLD proteins.
- This GW806742X-induced MLKL KLD dimerization is linked to RIP3 phosphorylation.
Conclusions:
- GW806742X exhibits a dual role in necroptosis, inhibiting it via potential interference with RIP1, while promoting it by facilitating MLKL KLD dimerization and activation.
- The findings highlight a novel mechanism of MLKL activation and suggest GW806742X as a valuable tool for dissecting necroptosis pathways.
