RSK-Mediated Phosphorylation in the C/EBPβ Leucine Zipper Regulates DNA Binding, Dimerization, and Growth Arrest Activity

Abstract

The bZIP transcription factor C/EBP␤ 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/EBP␤ signaling, we investigated C/EBP␤ activation by oncogenic Ras. We show that C/EBP␤ 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/EBP␤ activation and cytostatic activity. Apart from its role in DNA binding, Ser273 phosphorylation also creates an interhelical g7e 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/EBP␤ homodimer formation, whereas heterodimerization with C/EBP␥ was relatively unaffected. The S273A substitution also reduced the antiproliferative activity of C/EBP␤ 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/EBP␤-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.

C/EBPγ Suppresses Senescence and Inflammatory Gene Expression by Heterodimerizing with C/EBPβ

“…C/EBP␤ homodimers promote cell cycle arrest. Ras V12 -induced senescence in MEFs is correlated with increased C/EBP␤ DNA binding and elevated levels of C/EBP␤ homodimers (15), indicating that C/EBP␤ homodimers regulate cell cycle arrest. Hence, the proliferation defects seen in Cebpg Ϫ/Ϫ MEFs could result from increased C/EBP␤ homodimers.…”

“…C/EBP␥ lacks a transactivation domain but contains a C/EBP-like bZIP region and can inhibit transcription by C/EBP activator proteins in reporter assays through heterodimerization (12)(13)(14). Moreover, the formation of C/EBP␤ homodimers is stimulated by oncogenic Ras signaling through p90 RSK -mediated phosphorylation of a conserved leucine zipper residue, Ser273, which controls self-dimerization by increasing the electrostatic interactions between paired leucine zipper helices (15). Based on these and other considerations, we have proposed that Ras-induced C/EBP␤ homodimers are cytostatic and promote cell cycle arrest and senescence in response to oncogenic stress (8,15).…”

RSK3/4 mediate resistance to PI3K pathway inhibitors in breast cancer

“…RSK1 and RSK2 have been reported as overexpressed in breast and prostate cancer, while RSK3 has been proposed to be a tumor suppressor in ovarian cancer (55)(56)(57). RSK4 has previously been characterized as essential for p53-dependent proliferation arrest as well as stress-and oncogene-induced senescence (28,58,59). Interestingly, the RSK4 isoform exhibits constitutively high activity, is upregulated in MMTV-Myc mouse breast tumors, is aberrantly expressed in breast cancer, and has been implicated in sunitinib resistance (58,(60)(61)(62)(63).…”

RNA interference screening identifies lenalidomide sensitizers in multiple myeloma, including RSK2

“…All of these proteins are critical for the survival of MM cells. 18,36,37 RSK2 was demonstrated to phosphorylate and regulate C/EBPb, 38 which was reported to control transcription factors critical for survival of MM cells, including IRF4. 39 RSK2 has also been shown to directly regulate proteins involved in apoptosis, such as BAD 40,41 and BIM.…”