Research On Serine Kinase TBK1 Interact With ERα And Regulate Its Transcriptional Acitivity

Estrogen receptors act as ligand-activated transcription factors, which are members of the steroid hormone superfamily of nuclear receptors. Estrogen receptors can regulate transcription level by binding to estrogen-responsive elements or interacting with other transcription factors. ERαis important for many biological processes, such as differentiation and growth, morphogenesis and programmed cell death. In addition, ERαplays an important role in development and progression of breast cancer by regulating genes and signaling pathways which involved in cellular proliferation. Regulation ERαactivity requires the coordinate ligand binding or phosphorylation. ERαconsists of 6 regions (A-F), which contains two transactivation function domains: AF1 domain which has ligand-independent transactivation function and AF2 domain which has ligand-dependent transactivation function. In addition, ERαhas a hinge region and a DNA binding domain (DBD).ERαcan be phosphorylated by many kinases in order to regulate its activities. Phosphorylations of ERαon serines 104, 106, 118,167 and 236, and tyrosine 537 have been demonstrated. Mutation of serine 118 to alanine reduces ERαtranscriptional activity, whereas mutation of tyrosine 537 leads to increased ligand-independent ERαt?ranscriptional activation by affecting its DNA binding ability and dimerization. However, how the signaling pathways regulating ERαtranscriptional activity by phosphorylation are not fully understood.TBK1 is a serine kinase that plays important roles in innate immunity,and it is a member of IKKs that has serine phosphorylation activity. Recent studies suggest that TBK1 kinases are constitutively activated in some breast cancer cells and lung cancer. Although the mechanism by which TBK1 drives lung cancer development is well understood, little is known regarding how TBK1 promotes progression of breast cancer tumors. Here we show:Firstly, we show that TBK1 interacts with ERαin vivo. We also show that DBD domain of ERαinteracts with 292-510 domain and kinase domain of TBK1.Secondly,we show that ERαcan be phosphorylated by TBK1 but cannot be phosphorylated by TBK1 kinasedead mutant. Point mutation analysis revealed that the phosphorylation sites of ERαat S305, S309, S468 and S578 may be mediated by TBK1. The phosphorylation sites of ERαS468 and S578 were not reported elsewhere. Our results also showed that TBK1 can enhance the ability of ERαbinding to the transcription factor ERE, but TBK1 cannot improve the stability of ERα.Thirdly, we study the effect of TBK1 on the transcription activity of ERα. We show that TBK1 can enhance the transcription activity of ERαby Luciferase assay. We show that the transcription activity of ERαwas enhanced by TBK1 in estrogen independence and dependence manner. We further find that TBK1 kinase-dead mutation(K38A)or ERα(S305A, S309A, S468A, S578A)mutation can completely abrogates TBK1-dependent ERαtranscriptional activation without changing its basic transcription activity level. This finding indicates that TBK1 enhances the transcription activity of ERαthrough ERαphosphorylation. TBK1 ULD domain mutation led to enhanced transcription activity of ERαwithout changing the transcription activity of IFN-β, indicating an independent role of TBK1 invovling in ERαsignalling pathway.Finally, to further test the function of endogenous TBK1 in breast cancer cells, specific suppression of TBK1 by using RNA interference technology was monitored. Growth curve showed that TBK1 knockdown cells decreased the growth rate of MCF7 cells in monolayer culture. Reconstitution of MCF7 TBK1 knockdown cells with an interfering RNA-resistant version of WT TBK1 or ULD mutant TBK1 to endogenous levels resumed the growth of MCF7 cells, while reconsitution of MCF7 TBK1 knockdown cells with TBK1 kinase dead mutant did not. In wound healing assays, TBK1 knockdown cells exhibited decreased migratory capacity to the wounding areas, as compared with the vector control cells, suggesting that TBK1 expression has a positive effect on breast cancer cell motility.Taken together, we show here that ERαassociates with TBK1 serine kinase. Mutational analysis and in vitro kinase assays show that ERα?c?an be phosphorylated on four sites, resulting in enhanced ERα?t?ranscriptional activity and increased expression of endogenous ERαtarget genes. Moreover, the TBK1 deficiency mutant displays reduced breast cancer cell growth and cell mobility. Taken together, these results demonstrates that TBK1 is a novel kinase that upregulates ERαtranscription acitivity and promotes breast cancer cell proliferation and migration, suggesting a great potential for this kinase as a therapeutic target for breast cancer...