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. 2013 Jul 16;110(29):11851-6. doi: 10.1073/pnas.1308362110. Epub 2013 Jul 1.


Peptidylarginine deiminase 4 (PAD4) is a Ca(2+)-dependent enzyme that converts arginine and methylarginine residues to citrulline, with histone proteins being among its best-described substrates to date. However, the biological function of this posttranslational modification, either in histones or in nonhistone proteins, is poorly understood. Here, we show that PAD4 recognizes, binds, and citrullinates glycogen synthase kinase-3β (GSK3β), both in vitro and in vivo. Among other functions, GSK3β is a key regulator of transcription factors involved in tumor progression, and its dysregulation has been associated with progression of human cancers. We demonstrate that silencing of PAD4 in breast cancer cells leads to a striking reduction of nuclear GSK3β protein levels, increased TGF-β signaling, induction of epithelial-to-mesenchymal transition, and production of more invasive tumors in xenograft assays. Moreover, in breast cancer patients, reduction of PAD4 and nuclear GSK3β is associated with increased tumor invasiveness. We propose that PAD4-mediated citrullination of GSK3β is a unique posttranslational modification that regulates its nuclear localization and thereby plays a critical role in maintaining an epithelial phenotype. We demonstrate a dynamic and previously unappreciated interplay between histone-modifying enzymes, citrullination of nonhistone proteins, and epithelial-to-mesenchymal transition.

Publication types

  • Research Support, American Recovery and Reinvestment Act
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Calcium Ionophores
  • Citrulline / metabolism*
  • Epithelial-Mesenchymal Transition / physiology*
  • Fluorescent Antibody Technique
  • Gene Knockdown Techniques
  • Glycogen Synthase Kinase 3 / metabolism*
  • Glycogen Synthase Kinase 3 beta
  • Humans
  • Hydrolases / metabolism*
  • Immunoblotting
  • Immunohistochemistry
  • Immunoprecipitation
  • MCF-7 Cells
  • Mass Spectrometry
  • Microscopy, Interference
  • Mutagenesis, Site-Directed
  • Protein-Arginine Deiminase Type 4
  • Protein-Arginine Deiminases
  • Real-Time Polymerase Chain Reaction
  • Signal Transduction / physiology*
  • Statistics, Nonparametric
  • Transforming Growth Factor beta / metabolism*


  • Calcium Ionophores
  • Transforming Growth Factor beta
  • Citrulline
  • GSK3B protein, human
  • Glycogen Synthase Kinase 3 beta
  • Glycogen Synthase Kinase 3
  • Hydrolases
  • PADI4 protein, human
  • Protein-Arginine Deiminase Type 4
  • Protein-Arginine Deiminases