Proteomics Study Of Interaction Between Rheb And FKBP38in Cashmere Goat Cells

mTOR signal pathway coordinates the regulation of cell growth, proliferation as well as differentiation and Rheb along with FKBP38are two important regulatory proteins in this pathway. Rheb is a member of the Ras super family and it is a upstream regulator factor in mTOR signal pathway, with the function of integrating nutrition and promoting cell division; As a member of FKBPs family, FKBP38can inhibit the activity of mTOR so as to control cell growth. Early in2007, it was first reported Rheb could regulate the activity of mTOR by FKBP38meaning that Rheb could activate mTOR by antagonizing FKBP38. But later some researchers proved that they did not detect the interaction between Rheb and FKBP38with different methods. What is worth to point out is, those different conclusions were according to human cells, but there was no such report about the research in cashmere goat fetal fibroblasts cells (GFb cells). In order to discuss the interaction between Rheb and FKBP38, we studied the relationships of them in GFb cells using proteomics technology appending previously reports, expecting it may provide new evidence by using of new technology and new material and offer data for research of mTOR signal pathway in GFb cells at the same time.Firstly, we made use of RT-PCR to clone the CDS fragments in cDNA of Rheb and FKBP38genes, analyzing their expressing pattern; Secondly, two recombinant plasmids were transfected into the GFb cells, and then co-immunopricipitation (co-IP) and Western-Blot were used to detect the co-existed of Rheb and FKBP38. Finally, we defined the interaction relationship between Rheb and FKBP38by using yeast two-hybrid and Co-IP.The results showed that, the ORF of Rheb gene in Inner Mongolia Cashmere Goat is555bp long encoding184amino acids (HM569224), this mRNA expresses comparatively higher in brain but lower in heart. The cDNA of FKBP38gene is1248bp long with1236bp ORF, encoding411amino acids (JF714970), this mRNA expressed comparatively higher level in testis but lower in kidney.According to the results of gene cloning, the recombinating eukaryotic expression vector pIRES2-EGFP-Rheb and pIRES2-DsRed2-FKBP38were got, following co-transfected GFB cells, two weeks later, we screened by G418and extracted their proteins to detect by co-IP and Western-Blot. The results showed Rheb and FKBP38were identified in Co-IP complex. Bait vectors (pGBKT7-Rheb/FKBP38) and prey vectors (pGADT7-Rheb/FKBP38) were got. The screening of yeast mating showed that there was an interaction between Rheb and FKBP38. We concluded that the two of them could interact with each other directly.We proved the direct interactions between Rheb and FKBP38in GFb cells by proteomics technology, offering a new evidence for correlation between Rheb and FKBP38. The results enriched the genetical and functional information of Rheb and FKBP38gene, and providing experimental evidence for clarifying the function and mechanism of mTOR Signal Pathway.