Editors' ChoiceGSK-3β Signaling

Citrulline and Epithelial Identity

Science Signaling  23 Jul 2013:
Vol. 6, Issue 285, pp. ec168
DOI: 10.1126/scisignal.2004537

Citrullination is a posttranslational modification by which arginine is converted to citrulline by members of the family of peptidylarginine deiminases (PADs). PAD4 is the only PAD enzyme with a nuclear localization sequence (NLS), and it is implicated in the citrullination of various histone proteins. Noting that PAD4 is found in breast cancer cells, Stadler et al. investigated the effect of using short hairpin RNA (shRNA) to knock down PAD4 in a human noninvasive breast cancer cell line, MCF7 cells. Knockdown of PAD4 resulted in loss of epithelial markers and epithelial morphology, and an increase in the abundance of the protein vimentin, evidence of an epithelial-to-mesenchymal transition (EMT). A search of the proteome for motifs similar to those of histone proteins that undergo citrullination identified glycogen synthase kinase-3β (GSK-3β) as a potential target of PAD4, and experiments with recombinant proteins and transfected cells showed that PAD4 citrullinated GSK-3β at N-terminal arginines. Blocking citrullination of GSK-3β, by expressing a catalytically inactive PAD4 in PAD4-knockdown cells or by mutating the N-terminal arginines of GSK-3β, decreased the nuclear abundance of GSK-3β. Expression of a GSK-3β–NLS construct in PAD4-knockdown cells partially restored their epithelial characteristics. Transforming growth factor–β (TGF-β) signaling, a mediator of EMT, was enhanced in PAD4-knockdown cells compared with that in controls cells, which was partially reversed by expression of GSK-3β–NLS. Injection of PAD4-knockdown cells into nude mice resulted in the formation of tumors that were more invasive than those formed by injection of control MCF7 cells, and the tumors from PAD4-knockdown cells had decreased GSK-3β abundance and increased TGF-β signaling. Histological analysis showed that human invasive breast cancer tissue had decreased PAD4 staining (and decreased GSK-3β abundance) compared with that of normal human breast tissue or tissue from ductal carcinoma in situ (DCIS). Together, these data suggest that citrullination of GSK-3β is required to maintain its nuclear abundance and prevent EMT.

S. C. Stadler, C. T. Vincent, V. D. Fedorov, A. Patsialou, B. D. Cherrington, J. J. Wakshlag, S. Mohanan, B. M. Zee, X. Zhang, B. A. Garcia, J. S. Condeelis, A. M. C. Brown, S. A. Coonrod, C. D. Allis, Dysregulation of PAD4-mediated citrullination of nuclear GSK3β activates TGF-β signaling and induces epithelial-to-mesenchymal transition in breast cancer cells. Proc. Natl. Acad. Sci. U.S.A. 110, 11851–11856 (2013). [Abstract] [Full Text]