Grass Carp (Ctenopharyngodon Idella) DYRK2 (Dual Specificity Tyrosine Phosphorylation Regulated Kinase 2) Modulates Cell Apoptosis Through Phosphorylating P53

Apoptosis is the autonomous and orderly death of cells controlled by genes,which involves the activation,expression and regulation of a series of genes.DYRK2 is a serine/threonine kinase that can regulate various pathways through modification of phosphorylation.In mammals,DYRK2 interacts with p53 to increase p53 phosphorylation,thereby promoting apoptosis.However,whether DYRK2 promotes apoptosis through p53 in fish has not been elucidated.In this study,we use grass carp as a model to explore this problem.First,we homologously cloned the CDS of grass carp DYRK2(Ci DYRK2)and found that the CDS is 1773 bp in length and encodes 590 amino acids.SMART prediction analysis showed that Ci DYRK2 possesses a serine/threonine kinase domain.Through amino acid sequence analysis,we found that the DYRK2 of grass carp is similar to the amino acid sequence of lilang whitefish,human and zebrafish,which indicates that it is relatively conservative.Subsequently,we stimulated grass carp with ds RNA analogs poly(I:C)and GCRV,extracted different tissues,and performed QPCR experiments.The results showed that the expression level of Ci DYRK2 m RNA was significantly up-regulated after poly(I:C)stimulation for 24 h and 12 h after GCRV stimulation,and the expression levels in brain and liver were higher than those in other tissues.In order to investigate the relationship between grass carp DYRK2 and the proapoptotic protein p53,we used co-immunoprecipitation experiment in grass carp ovary cells to observe the combination of them.The result showed that Ci DYRK2 can bind to p53,that is,there is an interaction.At the same time,we found that the phosphorylation level of p53 was enhanced in the case of overexpression of DYRK2.In order to explore the function of Ci DYRK2,we carried out overexpression and interference experiments in grass carp kidney cells,and used Q-PCR,TUNEL staining and flow cytometry to evaluate the ratio of Bax/Bcl-2 m RNA,the number of TUNEL positive cells,and FITC and PI,respectively.The number of positive cells was detected to evaluate the effect of Ci DYRK2 on apoptosis.The results showed that overexpression of Ci DYRK2 could significantly up-regulate the Bax/Bcl-2 m RNA ratio,as well as increase the number of TUNEL-positive cells and the proportion of Annexin V-positive cells;the Bax/Bcl-2 m RNA ratio was significantly down-regulated after Ci DYRK2 interference.Therefore,Ci DYRK2 can promote apoptosis.In conclusion,grass carp DYRK2 can promote apoptosis by enhancing p53 phosphorylation.This study also demonstrated that the mechanism of DYRK2-promoting apoptosis is highly conserved in mammals and fish.