Ras Signaling Enhances the Activity of C/EBPα to Induce Granulocytic Differentiation by Phosphorylation of Serine 248

Abstract

The transcription factor C/EBPα regulates early steps of normal granulocyte differentiation since mice with a disruption of the C/EBPα gene do not express detectable levels of the granulocyte colony-stimulating factor receptor and produce no neutrophils. We have recently shown that C/EBPα function is also impaired in acute myeloid leukemias. However, how the transcriptional activity of C/EBPα is regulated both in myelopoiesis and leukemogenesis is not fully understood. The current study demonstrates that activated Ras enhances the ability of C/EBPα to transactivate the granulocyte colony-stimulating factor receptor promoter and a minimal promoter containing only C/EBP DNA binding sites. Ras signaling activates C/EBPα via the transactivation domain because it enhances the transactivation function of a fusion protein containing a Gal4 DNA binding domain and the C/EBPα transactivation domain and does not change C/EBPα DNA binding. Ras acts on serine 248 of the C/EBPα transactivation domain, because it does not enhance the transactivation function of a C/EBPα serine 248 to alanine point mutant. Interestingly, serine 248 of C/EBPα is a protein kinase C (PKC) consensus site, and a PKC inhibitor blocks the activation of C/EBPα by Ras. Ras signaling leads to phosphorylation of C/EBPα<i>in vivo</i>. Finally, mutation of serine 248 to alanine obviates the ability of C/EBPα to induce granulocytic differentiation. These data suggest a model where Ras signaling enhances the activity of C/EBPα to induce granulocytic differentiation by phosphorylation of serine 248.