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

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- p90^RSK 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 Ras^V12-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.