摘要/Abstract

摘要： 目的 ·研究新型受体相互作用蛋白激酶 3（receptor interacting protein kinase 3，RIPK3）突变体的激酶活性。方法 ·分别对 RIPK3中的 4个氨基酸（ Q84WDF87）进行突变，并将这些突变体与混合谱系蛋白激酶样假激酶（ mixed lineage kinase domain like pseudokinase，MLKL）共同转染到 HEK293T细胞中。通过 Western blotting检测新型 RIPK3突变体 S232位点自磷酸化的情况及其对 MLKL S345位点磷酸化的影响；通过免疫共沉淀法观察 RIPK3与 MLKL之间的相互作用；采用非还原裂解法检测 MLKL的寡聚化情况。结果 · RIPK3ΔQ84、RIPK3ΔW85和 RIPK3ΔD86的激酶活性显著降低， RIPK3Q84A/RIPK3Q84E、RIPK3W85Y和 RIPK3D86A/RIPK3D86Y的激酶活性无明显改变； RIPK3W85A的自磷酸化减少，但不影响 MLKL的磷酸化与寡聚化。结论 · Q84、 W85与 D86是调节 RIPK3激酶活性的关键氨基酸位点，RIPK3W85A的激酶活性减弱，但其对 MLKL无影响。
关键词: 受体相互作用蛋白激酶 3, 混合谱系蛋白激酶样假激酶, 激酶活性, 程序性细胞坏死
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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