The effect of GSK-3β phosphorylation site mutation on the stability of TSG101 protein

• Main Authors: Jung-ru He, 何忠儒
• Other Authors: Jinn-tsuey Cheng
• Format: Others
• Language: en_US
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/03461446366591875376

碩士 === 國立中山大學 === 生物科學系研究所 === 93 === Abstract： Tumor susceptibility gene 101, TSG101, is a protein exhibits multiple biological functions. For most protein, its specific function or structure stability can be regulated through protein phosphorylation or modification. The analysis of the amino acid sequence of TSG101 revealed that it has two GSK-3β phosphorylation concensus sequences. Our previous data of in vitro kinase assay have demonstrated that TSG101 can be phosphorylated by GSK-3β, a wellknown protein kinase that regulates the stability and function of it’s target protein. To investigate the effect of GSK-3β phosphorylation on the stability and the function of TSG101 protein, we first exploited the effect of GSK-3βinhibitor, LiCl, on endogenous TSG101 protein in COS1 cells. The results suggested that inhibition of GSK-3β phosphorylation could impact on the stability of TSG101 protein. Upon the transfection of an active form GSK-3β expression plasmid GSK-3β/pEGFP, additional protein products of 40, 50-80 kD were detected, suggesting that GSK-3β phosphorylation might induce modification or degradation of TSG101 protein. GSK-3β phosphorylation site mutant TSG101 protein expression plasmids were constructed using site-directed mutagenesis, and were transfected into COS1 cells to evaluate the effect of GSK-3β on TSG101 level. The results showed that GSK-3β phosphorylation site mutant TSG101 protein is more stable then wild type TSG101 due to the lack of GSK-3β phosphorylation site. The inhibition of GSK-3β activity by LiCl treatment resulted in the increase of wildtype as well as the S172AS176 and S172AS176AS202AS206A mutant TSG101 proteins, whereas the S202AS206A mutant TSG101 protein level was not affected by LiCl treatment. The above data indicated that GSK-3β might regulate the stability and biological activity of TSG101 protein through phosphorylation of serine residue at position 202, which is worthy of further investigation.