Ras signaling enhances the activity of C/EBP alpha to induce granulocytic differentiation by phosphorylation of serine 248.
The transcription factor C/EBP alpha regulates early steps of normal granulocyte differentiation since mice with a disruption of the C/EBP alpha gene do not express detectable levels of the granulocyte colony-stimulating factor receptor and produce no neutrophils. We have recently shown that C/EBP alpha function is also impaired in acute myeloid leukemias. However, how the transcriptional activity of C/EBP alpha is regulated both in myelopoiesis and leukemogenesis is not fully understood. The current study demonstrates that activated Ras enhances the ability of C/EBP alpha to transactivate the granulocyte colony-stimulating factor receptor promoter and a minimal promoter containing only C/EBP DNA binding sites. Ras signaling activates C/EBP alpha via the transactivation domain because it enhances the transactivation function of a fusion protein containing a Gal4 DNA binding domain and the C/EBP alpha transactivation domain and does not change C/EBP alpha DNA binding. Ras acts on serine 248 of the C/EBP alpha transactivation domain, because it does not enhance the transactivation function of a C/EBP alpha serine 248 to alanine point mutant. Interestingly, serine 248 of C/EBP alpha is a protein kinase C (PKC) consensus site, and a PKC inhibitor blocks the activation of C/EB alpha by Ras. Ras signaling leads to phosphorylation of C/EBP alpha in vivo. Finally, mutation of serine 248 to alanine obviates the ability of C/EBP alpha to induce granulocytic differentiation. These data suggest a model where Ras signaling enhances the activity of C/EBP alpha to induce granulocytic differentiation by phosphorylation of serine 248.
Behre
Gerhard
G
Department of Internal Medicine III, University Hospital Grosshadern, Ludwig-Maximilians-University Munich and GSF-National Research Center for Environment and Health, D-81377 Munich, Germany. gerdbehre@aol.com
Singh
Sheo M
SM
Liu
Huaitian
H
Bortolin
Laura T
LT
Christopeit
Max
M
Radomska
Hanna S
HS
Rangatia
Janki
J
Hiddemann
Wolfgang
W
Friedman
Alan D
AD
Tenen
Daniel G
DG
D000208:Acute Disease; D000409:Alanine / Q000378:metabolism; D019943:Amino Acid Substitution; D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000235:genetics / Q000378:metabolism*; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D006098:Granulocytes / Q000166:cytology*; D006801:Humans; D007951:Leukemia, Myeloid / Q000378:metabolism; D010766:Phosphorylation; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D011493:Protein Kinase C / Q000378:metabolism; D016188:Receptors, Granulocyte Colony-Stimulating Factor / Q000235:genetics; D012694:Serine / Q000378:metabolism*; D015398:Signal Transduction; D015533:Transcriptional Activation; D018631:ras Proteins / Q000502:physiology*
D022763:CCAAT-Enhancer-Binding Protein-alpha; D016188:Receptors, Granulocyte Colony-Stimulating Factor; D012694:Serine; D011493:Protein Kinase C; D018631:ras Proteins; D000409:Alanine