›› 2019, Vol. 39 ›› Issue (8): 856-.doi: 10.3969/j.issn.1674-8115.2019.08.008

• Original article (Basic research) • Previous Articles     Next Articles

Kinase activity of novel receptor interacting protein kinase 3 mutants

ZHANG Yue1, ZHANG Hai-wei2, ZHANG Hai-bing2, LUO Yan 1   

  1. 1. Department of Anesthesiology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China; 2. Key Laboratory of Nutrition and Metabolism, Institute of Nutrition and Health, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
  • Online:2019-08-28 Published:2019-09-23
  • Supported by:
    Foundation of Science and Technology Commission of Shanghai Municipality, 16ZR1421100

Abstract: Objective · To explore the kinase activity of novel receptor interacting protein kinase 3 (RIPK3) mutants. Methods · The four amino acids (Q84WDF87) of RIPK3 were mutated respectively and these mutants were co-transfected with mixed lineage kinase domain like pseudokinase (MLKL) into HEK293T cells. The auto-phosphorylation of these mutants at S232 and phosphorylation of MLKL at S345 were detectedWestern blotting. The interaction between RIPK3 and MLKL was testedco-immunoprecipitation. The oligomerization of MLKL was detectednon-reducing gel. Results · The kinase activities of RIPK3ΔQ84, RIPK3ΔW85 and RIPK3ΔD86 were effectively decreased. Nevertheless, the kinase activities of RIPK3Q84A/RIPK3Q84E, RIPK3W85Y and RIPK3D86A/RIPK3D86Y did not change markedly. The auto-phosphorylation of RIPK3W85A at S232 was decreased without affecting phosphorylation and oligomerization of MLKL. Conclusion · The amino acid site Q84, W85 or D86 plays a critical role in RIPK3 kinase activity. The kinase activity of RIPK3W85A is decreased, but it does not affect MLKL.

Key words: receptor interacting protein kinase 3 (RIPK3), mixed lineage kinase domain like pseudokinase (MLKL), kinase activity, necroptosis

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