Studies On The Functions Of TH1

The human Trihydrophobin 1 (TH1) gene is the homolog of Drosophila TH1, which was originally identified during the positional cloning of mei-41. The TH1 gene lies adjacent to mei-41 and was characterized further by the D. T. Bonthron group in 2000. According to their studies, the TH1 gene was highly conserved from Drosophila to human by sequence comparison. The human TH1 gene was located in chromosome 20ql3, which had a transcript product of 2. 4 kb. Multiple tissues detected by Northern blots showed that TH1 was widely expressed. The human TH1 protein has been predicted having a molecular mass of 65.8 kDa and displays high levels of expression in cardiac and skeletal muscle, kidney, adrenal, and thyroid. Although ubiquitously expressed, its function is not clear at present.Our previous work indicated that instead of binding to B-Raf or C-Raf, TH1 (trihydrophobin 1) specifically binds to A-Raf kinase both in vitro and in vivo. In this work, we investigated its function further. Using confocal microscopy, we found that TH1 colocalizes with A-Raf, which confirms our former results. The region of TH1 responsible for the interaction with A-Raf is mapped to amino acids 1 - 372. Coimmunoprecipitation experiments demonstrate that TH1 is associated with A-Raf in both quiescent and serum-stimulated cells. Wild type A-Raf binds increasingly to TH1 when it is activated by serum and/or upstream oncogenic Ras/Src compared with that of "kinase- dead" A-Raf. The latter can still bind to TH1 under the same experimental condition. The binding pattern of A-Raf implies that this interaction is mediated in part by the A-Raf kinase activity. As indicated by Raf protein kinase assays, TH1 inhibits A-Raf kinase, whereas neither B-Raf nor C-Raf kinase activity is influenced. Furthermore, we observed that TH1 inhibited cell cycle progression in TH1 stably transfected 7721 cells compared with mock cells, and flow cell cytometry analysis suggested that the TH1 stably transfected 7721 cells were G0/G1 phase-arrested. Taken together, our data providea clue to understanding the cellular function of TH1 on Raf isoform-specific regulation.In following studies on TH1, we find that TH1 could interact with other components (MEK1.ERK1) involved in ERK MAPK pathway and we further investigated the mechanism that TH1 interfered with the activation of ERK pathway. TH1 can form ternary complexes with A-Raf, MEK1 and ERK. TH1 interferes with A-Raf/MEK1 interaction, behaving as a competitive inhibitor of MEK1 phosphorylation. From our observation, TH1 itself can be phosphorylated by A-Raf kinase, thus it seems to be a new substrate of A-Raf kinase. Interestingly, TH1 can interfere with the interaction between MEK1 and ERK1, either. It has not effect on phosphorylation of ERK1 by MEK1 kinase and on ERK1's kinase ability. TH1 overexpression could inhibit the downstream transcriptional factor Elk—1 activtiy, which indicated by reporter gene luciferase assay. In this study, we identified TH1 as a new scaffolding protein in the ERK MAPK pathway.In order to searching for potential interaction partner of TH1, we used the yeast two-hybrid system to screen human fetal brain cDNA library, and found two candidates proteins: E6-associated protein(E6AP/UBE3A), nuclear protein 220(NP220). Interaction between TH1 and E6AP were proved by coimmunoprecipitation assays. E6AP functions as a ubiquitin-protein ligase (E3) in ubiquitin-dependend proteolytic pathway and we found that TH1 is a new target protein for degradation by E6AP. This probably provides us a new clue in understanding TH1's function and study on the mechanism that TH1 paticipates in Raf/MEK/ERK signaling pathway need to be furthered.