Grass Carp(Ctenopharyngodon Idella)RSK2 Protects Cells Against Apoptosis By Up-regulating BCL-2

Ribosomal protein S6 Kinase α3(RSK2)is a member of the serine/threonine kinase and plays an important role in the innate immune response induced by TRL3.In mammals,an important feature of RSK2 is the presence of two functional domains,the N-terminal kinase domain(NTKD)and the C-terminal kinase domain(CTKD).CTKD is involved in the auto-phosphorylation of RSK2,while NTKD is primarily responsible for the phosphorylation of substrates.There is an ERK1-docking domain in CTKD of RSK2,which is an important site for the interaction between ERK1 and RSK2.In recent years,it has been found that RSK2 plays a very important role in cancer,cell proliferation,apoptosis and immune response.However,the function of RSK2 in innate immunity and apoptosis is still unclear.For example,ultraviolet radiation can activate RSK2 in mice and phosphorylate serine 112 of BAD,thereby regulating cell apoptosis.But the anti-apoptotic function of RSK2 has not been reported in relation to BCL-2,which is closely related to apoptosis.This study mainly explored the innate immunity and apoptosis mechanism of ERK1-RSK2 in fish MAPK pathway.We cloned the full-length cDNA sequences of ERK1(named CiERKl,MH985854)and RSK2(named CiRSK2,MH844551)from grass carp tissues by homologous cloning.The result shows that the open reading frame(ORF)of CiERK1 is 1182 bp,encoding a polypeptide of 394 amino acids.And the ORF of CiRSK2 is 2202 bp,encoding a polypeptide of 734 amino acids.Phylogenetic tree analysis showed that CiERK1 and CiRSK2 had a close relationship with most teleost fish,and the grass carp ERK1 has the greatest homology with Cyprinus carpio,while the grass carp RSK2 has the highest homology with zebrafish.Protein structure analysis shows that CiRSK2 has a typical secondary structure,and the ERK1-docking domain of CiRSK2 is conserved among humans,mice,and zebrafish.So it’s indicating that grass carp RSK2 may have similar functions as mammals.To demonstrate that ERK1-RSK2 can respond to innate immunity induced by TLR3,poly(I:C)was used to stimulate grass carp individuals at different time periods,then we used Q-PCR to analyze the expression of RSK2 in eight different tissues.The results showed that,except for the eyes,the expression of CiRSK2 was significantly up-regulated and reached the maximum expression after 48 h of stimulation.In addition,the expression of CiRSK2 is highest in gills,intestines,and kidneys,and the lowest in brain and eyes.In grass carp kidney cells(CIK),the expression of CiERKl and CiRSK2 can be significantly up-regulated by endogenous poly(I:C)stimulation.The expression of CiERKl reached its maximum at 24 h,while the expression of CiRSK2 reached its maximum at 48 h.In order to further study the function of grass carp ERK1-RSK2,we used Western blot assay by incubating anti-phospho-Ser/Thr/Tyr antibody,the result shows that poly(I:C)stimulation can make CiERK1 phosphorylated.we demonstrated that CiRSK2 interacts with CiERK1 using immunofluorescence assay and CO-IP assay.Then we performed a subcellular localization of CiRSK2 at different time intervals of CiERK1 stimulation and found that CiERX1 can induce CiRSK2 into the nucleus.In our further experiments,we used Q-PCR to analyze the expression of CiBCL-2 in the conditions of overexpression and interference with CiRSK2.It was found that CiRSK2 had a significant positive regulatory effect on CiBCL-2.Then,Dual luciferase assay and Western blot assay showed that CiRSK2 could significantly increase the transcription and protein levels of CiBCL-2.In Hoechst 33258 and TUNNEL assay,it was found that CiRSK2 exerted a significant inhibitory effect on apoptosis.Therefore,it shows that CiRSK2 plays an important role in the innate immunity of grass carp and may inhibit apoptosis by up-regulating CiBCL-2.