Interaction Between AES, TLE1 And Gp130 Molecules And Investigation On Their Functions

As the common signal transducer for IL-6, IL-11, LIF(leukemia inhibitory factor), CT-1 (cardiotrophin-1), CNTF(ciliary neurotrophic factor) and OSM(oncostain M) receptors, gp!30 plays important roles in various aspects. It has been proved that gplSO mediated signal transduction by both Ras-MAPK pathway and JAK-STAT pathway. But there are proofs indicating that other signal transducing molecules or pathways might be involved in gp!30 signaling. AES(amino-terminal enhancer of split), also named GAM(gpl30 associated molecule), had been proved to be associated with gp!30 at its cytoplasmic membrane proximal part, and co-expression of AES. gp!30 and JAK kinase in COS cells inhibited the association of gp!30 and JAK kinase. AES contains a glutamine-rich (Q) domain in its N-terminal, which sharing high homology with the amino terminal region of Groucho/TLE(transducin like enhancer of split). The function of AES is not clear yet, while Groucho/TLE were found to be a family of transcription co-repressor and play important roles in Notch signaling, Wnt-0 catenin signaling and other transcription regulation events.By yeast two-hybrid assay, AES was found to interact with gp130 intracellular region through its conserved Q domain. Results from the yeast two-hybrid assay, gluthione S-transferase fusion protein pull-down assayand immuno-co-precipitation assay indicated that the Q domain of TLE1 is capable of binding gp130 intracellular domain, and the intracellular membrane proximal region of gp130 containing conserved Boxl and Box2 motifs seemed essential for this interaction. To investigate the consequence of this interaction, TLE1-GFP fusion protein expression vector was constructed and co-transfected into NIH 3T3 cells with gp130 expression vector. Confocal microscopy observation followed immune-fluorescence staining with anti-gp130 showed that gp130 could interact with TLE1 at the cytomembrane region. Moreover, this interaction inhibited the concentration of TLE1 into nucleus. These results indicated that molecules of TLE family might play a role in gp130 signaling.The amino-terminal regions of AES and TLE are highly homologous, both contains Q(glutamine) domain. To investigate the possible interaction between AES and the ubiquitously expressed TLE molecule, TLE1, we constructed a serial of expression vectors containing various domains of AES and TLE1. Yeast two hybrid system assay indicated that AES and TLE1 interact by their homologous Q domain. To investigate the consequence of this interaction, AES-RFP fusion protein expression vector was constructed and co-transfected into NIH 3T3 cells with TLE1-GFP fusion protein expression vector. Confocal microscopy observation showed that AES could interact with TLE1 at the cytomembrane region. Moreover, this interaction inhibited the concentration of TLE1 into nucleus. These results indicated that AES might function as an antagonist of TLE by binding with TLE and inhibit the translocation of TLE into nucleus.It has been reported that TLE1 expression was up-regulated in cervix cancer cells and peptide derived from AES could be recognized by CTLs from ovary cancer patients. To investigate the relationship between AES/TLE1 and tumors, the expression patterns of AES/TLE1 were detected in samples of gastric cancer, cervix cancer, hepatoma and corresponding non-tumor tissues by immunohistochemistry assay. It was proved that TLE1 expression was up-regulated in cervix cancer, while AES expression was up-regulated in gastric cancer. Antisense oligonucleotide or small interfering RNA specific against AES or TLE1 could inhibit theexpression of corresponding molecule. The decrease of AES expression inhibited the proliferation of SGC7901 gastric cancer cells and induce some of the cells to die, while enhance the proliferation of HeLa cervix cancer cells. On the other hand, the decrease of TLE1 expression inhibited the proliferation of HeLa cells and induces some of the cells to die. These results indicated that AES/TLE1 might play distinct and important role...