Abstract
The bZIP transcription factor C/EBPbeta 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/EBPbeta signaling, we investigated C/EBPbeta activation by oncogenic Ras. We show that C/EBPbeta 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/EBPbeta 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/EBPbeta homodimer formation, whereas heterodimerization with C/EBPgamma was relatively unaffected. The S273A substitution also reduced the antiproliferative activity of C/EBPbeta 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/EBPbeta-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.