RSK-mediated phosphorylation in the C/EBP{beta} leucine zipper regulates DNA binding, dimerization, and growth arrest activity.
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.
Lee
Sook
S
Laboratory of Cancer Prevention, Bldg. 539 Room 122, NCI-Frederick, Frederick, MD 21702-1201, USA.
Shuman
Jon D
JD
Guszczynski
Tad
T
Sakchaisri
Krisada
K
Sebastian
Thomas
T
Copeland
Terry D
TD
Miller
Maria
M
Cohen
Michael S
MS
Taunton
Jack
J
Smart
Robert C
RC
Xiao
Zhen
Z
Yu
Li-Rong
LR
Veenstra
Timothy D
TD
Johnson
Peter F
PF
D000595:Amino Acid Sequence; D000818:Animals; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002453:Cell Cycle / Q000502:physiology*; D002460:Cell Line; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D006133:Growth Substances / Q000378:metabolism; D006801:Humans; D016350:Leucine Zippers; D020935:MAP Kinase Signaling System / Q000502:physiology; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010766:Phosphorylation; D055503:Protein Multimerization; D011499:Protein Processing, Post-Translational; D051381:Rats; D019893:Ribosomal Protein S6 Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D015533:Transcriptional Activation; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism
D022782:CCAAT-Enhancer-Binding Protein-beta; D006133:Growth Substances; D004247:DNA; D019893:Ribosomal Protein S6 Kinases; D020928:Mitogen-Activated Protein Kinases; D018631:ras Proteins