Screening Of P70S6K Interacting Proteins By Yeast Two-hybrid System And Priliminary Study Of RPS5 Functions

p70S6K1(p70 Robosomal Protein S6 Kinase 1,p70S6K,S6K1)is a Ser/Thr kinase and one of the downstream effectors of mTORC1(Mechanistic Target of Rapamycin Complexl).p70S6K can phosphorylate the ribosomal protein S6(ribosomal protein S6,rpS6,S6)so that promote protein synthesis.The changes including nutrients or energy both extracellular and intracellular environment can regulate p70S6K1 activation and then to regulate cell metabolism and biological conditions by controlling the protein synthesis.RPS5 gene product,RPS5 protein(40S Ribosomal Protein S5)is one of the subunits of eukaryotic 40S ribosomal small subunit,and plays an important role on accuracy of protein translation.RPS5 can bind to IRES(Internal Ribosome Entry Site)of virus and has key role in initiation of protein translation related with IRES.It had yet reported about the relationship between RPS 5 and som kinase and its function is unclear by now.We used previously produced vector pGBKT7-S6K1 to express the bait protein S6K1(p70S6K1),screening the pGADT7-cDNA library and finding the interacting proteins with S6K1(p70S6Kl),and then the genes encoding the prey proteins were sequenced.The interaction between p70S6Kl and RPS5 was verified by co-immunoprecipitation assay.RPS5 gene was cloned by RT-PCR and it's over expression vector pIRES2-EGFP-RPS5 and targrting RPS5 gene's pRNAT-U6.1/Neo-RPS5-shRNA were constructed and transferred into Cashmere goat fetal fibroblatsts(GFbs)with Lipo 2000,respectively.MTT assay was used to measure cell proliferation and flow cytometry was used to analyze cell cycle,respectively.The results showed:1)27 positive clones were obtained after the Y2H yeast strain with pGBKT7-S6K1 and Y187 yeast strain with pGADT7-cDNA were mated and selected by four rounds of deficiency medium and corresponding antibiotics screening;2)the 27 Y2H yeast clones which contained the pGADT7-cDNA unknown prey proteins were managed to co-transformation with S6K1 by one-to-one pattern to obtain positive clones.The pGADT7-cDNAs harbored in positive clones were sequenced and verified by BLAST and 6 genes were confirmed.The genes are Ribosomal protein S5(RPS5),Musimon Chromosome approved 3 Open Reading Frame(C3H12orf75),Ornithine Decarboxylase antizyme1(OAZ1),Secreted Protein Acidic and Cysteine-Rich(SPARC),and Eukaryotic Translation Initiation Factor 1-Like Pseudogene(LOCI 00300942)and ER Membrane Protein Complex Subunit 8(EMC8).3).We believe that RPS5 is important for selection since its function is not clear and may has significant function in protein synthesis.As a result,the interaction between RPS5 and S6K1(p70S6K1)was verified with co-immunoprecipitation assay.It may be a potential target of S6K1(p70S6Kl)and it is possible that protein synthesis was regulated by p70S6K(S6K1)via RPS5.4)RPS5 gene was cloned by RT-PCR and the nucleotide sequence of the gene and its pridicted product RPS5 was analyzed by bioinformatics.CDS of RPS5 in Inner Mongolia Cashmere goat was 615 bp,which coding 204 amino acid residues.Overexpression of RPS5 gene can promote cell proliferation(p<0.05)and cell cycle progression;5)Knock down of RPS5 gene resulted in inhibition of cell proliferation(p<0.05)and G1/S arrest.In this study,we obtained 6 S6K1 interacting proteins using the yeast two-hybrid technology and further studies showed that RPS5 play important roles in GFb cell proliferation and cell cycle.The studies of interaction of p70S6K(S6K1)and RPS5 promote us to further explore the molecular mechanisms of amino acid signal transduction in the future.