| Summary: | 碩士 === 國立陽明大學 === 生命科學暨基因體科學研究所 === 98 === The members of signal transducer and activator of transcription (STAT) family are a group of latent transcription factors. In response to cytokines and growth factors, they are post-translationally modified which allows them to form dimmers and translocate to the nucleus to regulate gene expression. Stat3 is a multifaceted oncogene, which is frequently activated in many human cancers. Stat3 regulates the expression of a variety of genes in response to cell stimuli, and plays a key role in a plethora of cellular processes such as cell growth and apoptosis. It is well established that the dimerization of Stat3 is mediated through the phosphorylation of Y705. Acetylation of K685 has also been reported to promote Stat3 dimerization. In our previous study, we have demonstrated that ligation of CD44, a type I transmembrane protein known to be involved in cell/cell and cell/matrix interactions, promotes acetylation of Stat3. The acetylated Stat3 forms homodimer and translocates into the nucleus, where it promotes cell proliferation through transactivation of cyclin D1. It is noted that CD44-mediated Stat3 activation is independent of cytokine- as well as growth factor-mediated phosphorylation process. To gain further understanding of the molecular mechanisms involving Stat3 activation, in this study, I further characterize the activation and transcriptional activity of phosphorylated and acetylated Stat3. In HeLa cells, I showed that IL-6 treatment leads to phosphorylation and acetylation of the endogenous Stat3, whereas ligation of CD44 by anti-CD44 monoclonal antibody Hermes-3 induces Stat3 acetylation. Both IL-6- and CD44 ligation-induced Stat3 modification promotes nuclear localization of Stat3. In addition, I have overexpressed the wild-type, phosphorylation-deficient (K685R), acetylation-deficient (Y705F), and double mutants in the Stat3-null prostate cancer PC-3 cells and the Stat3-knockout mouse embryonic fibroblasts (MEF). Similarly, the ectopically expressed Stat3(WT) can be phosphorylated and acetylated under IL-6 treatment, while Stat3(Y705F) is acetylated, Stat3(K685R) is phosphorylated, and Stat3(Y705F/K685R) is not modified. To further examine the transcriptional activity, vector encoding the individual Stat3 was co-transfected into PC-3 cells with an sis-inducible element (SIE)-directly luciferase reporter plasmid. My data showed that Stat3(WT) and Stat3(K685R) can transactivate the expression of luciferase to the same extent in response to Il-6 treatment, but Stat3(Y705F) failed to do so. Furthermore, chromatin immunoprecipitation (ChIP) assay revealed that Stat3(Y705F) and Stat3(K685R) have distinct binding preference towards the Stat consensus sequences on cyclin D1 promoter. These data clearly demonstrate that phosphorylated and acetylated Stat3, although both are capable of regulating gene expression, display varying DNA binding activity and transcriptional activity. This study thus paths the groundwork for further characterization of the function of Stat3 upon differential modification.
|
|---|
