RSK-mediated phosphorylation in the C/EBPβ leucine zipper regulates DNA binding, dimerization, and growth arrest activity

Sook Lee, Jon D. Shuman, Tad Guszczynski, Krisada Sakchaisri, Thomas Sebastian, Terry D. Copeland, Maria Miller, Michael S. Cohen, Jack Taunton, Robert C. Smart, Zhen Xiao, Li Rong Yu, Timothy D. Veenstra, Peter F. Johnson

Research output: Contribution to journalArticle

44 Scopus citations

Abstract

The bZIP transcription factor C/EBPβ is a target of Ras signaling that has been implicated in Ras-induced transformation and oncogene-induced senescence (OIS). To gain insights into Ras-C/EBPβ signaling, we investigated C/EBPβ activation by oncogenic Ras. We show that C/EBPβ DNA binding is autorepressed and becomes activated by the Ras-Raf-MEK-ERK- p90RSK cascade. Inducible phosphorylation by RSK on Ser273 in the leucine zipper was required for DNA binding. In addition, three other modifications (phosphorylation on Tyr109 [p-Tyr109], p-Ser111, and monomethylation of Arg114 [me-Arg114]) within an N-terminal autoinhibitory domain were important for Ras-induced C/EBPβ activation and cytostatic activity. Apart from its role in DNA binding, Ser273 phosphorylation also creates an interhelical g↔e′ salt bridge with Lys268 that increases attractive electrostatic interactions between paired leucine zippers and promotes homodimerization. Mutating Ser273 to Ala or Lys268 to Glu decreased C/EBPβ homodimer formation, whereas heterodimerization with C/EBPγ was relatively unaffected. The S273A substitution also reduced the antiproliferative activity of C/EBPβ in RasV12-expressing fibroblasts and decreased binding to target cell cycle genes, while a phosphomimetic substitution (S273D) maintained growth arrest function. Our findings identify four novel C/EBPβ-activating modifications, including RSK-mediated phosphorylation of a bifunctional residue in the leucine zipper that regulates DNA binding and homodimerization and thereby promotes cell cycle arrest.

Original languageEnglish (US)
Pages (from-to)2621-2635
Number of pages15
JournalMolecular and cellular biology
Volume30
Issue number11
DOIs
StatePublished - Jun 2010

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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    Lee, S., Shuman, J. D., Guszczynski, T., Sakchaisri, K., Sebastian, T., Copeland, T. D., Miller, M., Cohen, M. S., Taunton, J., Smart, R. C., Xiao, Z., Yu, L. R., Veenstra, T. D., & Johnson, P. F. (2010). RSK-mediated phosphorylation in the C/EBPβ leucine zipper regulates DNA binding, dimerization, and growth arrest activity. Molecular and cellular biology, 30(11), 2621-2635. https://doi.org/10.1128/MCB.00782-09