The Role Of Beclin 1 Phosphorylated By Checkpoint Kinase 2 In Autophagy

Objective:Autophagy is a highly conserved process in the course of biological evolution,in which cells degrade and eliminate harmful and senescent components,playing an important role in maintaining cell homeostasis and body balance.Under metabolic stress,cells can promptly remove harmful components by autophagy and be provided with necessary substances and energy for survival from this process to maintain their homeostasis.Checkpoint Kinase2(Chk2),an important member of DNA damage response pathway,plays an important role in DNA damage and repair.But our recent study found that Chk2 is also involved in the process of autophagy induced by metabolic stress and revealed the vital role of Chk2 in autophagy regulationfor the first time.By further research,we also found that among a large amount of autophagy-related proteins,Beclin 1,a homolog of yeast autophagy-related gene 6(Atg6),can be strongly bonded with Chk2 under metabolic stress and Chk2,as a serine kinase and threonine kinase,can regulate autophagy by phosphorylating Beclin 1.Therefore,in this essay,we will devote ourselves to further exploring the signaling pathway that Chk2 participates in autophagy induced by metabolic stress and clarifying its molecular mechanism of autophagy regulation.This result can provide theoretical basis for studying the crosstalk between DNA damage response and autophagy.Methods:1.With the use of H1299,human lung adenocarcinoma cell line and mouse embryonic fibroblasts,comparing the level of autophagy with and without Chk2 under the stress of metabolism,the role of Chk2 in the process of autophagy was clarified.2.With the same cell line,the physiological substrate by which Chk2 regulates autophagy was found.Among a lot of autophagy related proteins,Beclin 1 was found strongly bonded with Chk2 via immunoprecipitation experiments and kinase experiments in vitro.Besides,in the condition of metabolic stress,activation of Chk2 played an important role in the regulation of Beclin 1 phosphorylation and thus,it was clear that,autophagy-related gene Beclin 1was a significant physiological substrate for the regulation of autophagy of Chk2.3.The specific phosphorylation site of Chk2 phosphorylation Beclin 1 was determined by the kinase assayin vitro and site-specific phosphorylation antibody was produced.It was confirmed that in the condition of metabolic stress,Beclin 1 S90 / S93 site could be phosphorylated by activated Chk2,according to thephosphorylation of Beclin 1 with Chk2 overexpressed or silenced in cells.4.It was found that the binding between Beclin 1 complex and Bcl-2 could be inhibited by theactivation of Chk2-Beclin 1 signaling pathway and autophagy was promoted.5.It was confirmed that autophagy could be promoted by Chk2 phosphorylating Becliln1 by the means of western blot experiment and electron microscopy observation.Results:1.Chk2 was involved in the regulation of autophagy induced by metabolic stress.2.The physiological substrate for Chk2 regulating autophagy was Beclin 1.3.With metabolic stress,Beclin 1 S90 / S93 was activated by Chk2.4.In the condition of metabolic stress,autophagy could be promoted by a declined binding between Beclin 1 and Chk2,which was caused by activated Chk2 phosphorylating Beclin.Conclusion:Checkpointkinase2isinvolvedinautophagyregulationby phosphorylating Beclin 1.