Gajula, Rajendra P.Chettiar, Sivarajan T.Williams, Russell D.Nugent, KatrianaKato, YoshinoriWang, HailunMalek, ReemTaparra, KekoaCades, JessicaAnnadanam, AnveshYoon, A.-RumFertig, ElanaFirulli, Beth A.Mazzacurati, LuciaBurns, Timothy F.Firulli, Anthony B.An, Steven S.Tran, Phuoc T.2016-04-112016-04-112015-01Gajula, R. P., Chettiar, S. T., Williams, R. D., Nugent, K., Kato, Y., Wang, H., … Tran, P. T. (2015). Structure-Function Studies of the bHLH Phosphorylation Domain of TWIST1 in Prostate Cancer Cells. Neoplasia (New York, N.Y.), 17(1), 16–31. http://doi.org/10.1016/j.neo.2014.10.0091476-5586https://hdl.handle.net/1805/9244The TWIST1 gene has diverse roles in development and pathologic diseases such as cancer. TWIST1 is a dimeric basic helix-loop-helix (bHLH) transcription factor existing as TWIST1-TWIST1 or TWIST1-E12/47. TWIST1 partner choice and DNA binding can be influenced during development by phosphorylation of Thr125 and Ser127 of the Thr-Gln-Ser (TQS) motif within the bHLH of TWIST1. The significance of these TWIST1 phosphorylation sites for metastasis is unknown. We created stable isogenic prostate cancer cell lines overexpressing TWIST1 wild-type, phospho-mutants, and tethered versions. We assessed these isogenic lines using assays that mimic stages of cancer metastasis. In vitro assays suggested the phospho-mimetic Twist1-DQD mutation could confer cellular properties associated with pro-metastatic behavior. The hypo-phosphorylation mimic Twist1-AQA mutation displayed reduced pro-metastatic activity compared to wild-type TWIST1 in vitro, suggesting that phosphorylation of the TWIST1 TQS motif was necessary for pro-metastatic functions. In vivo analysis demonstrates that the Twist1-AQA mutation exhibits reduced capacity to contribute to metastasis, whereas the expression of the Twist1-DQD mutation exhibits proficient metastatic potential. Tethered TWIST1-E12 heterodimers phenocopied the Twist1-DQD mutation for many in vitro assays, suggesting that TWIST1 phosphorylation may result in heterodimerization in prostate cancer cells. Lastly, the dual phosphatidylinositide 3-kinase (PI3K)-mammalian target of rapamycin (mTOR) inhibitor BEZ235 strongly attenuated TWIST1-induced migration that was dependent on the TQS motif. TWIST1 TQS phosphorylation state determines the intensity of TWIST1-induced pro-metastatic ability in prostate cancer cells, which may be partly explained mechanistically by TWIST1 dimeric partner choice.en-USAttribution-NonCommercial-NoDerivs 3.0 UnportedBasic Helix-Loop-Helix Transcription FactorsmetabolismNuclear ProteinsProstatic NeoplasmsProtein Interaction Domains and MotifsTwist Transcription FactorArticle