Ras signaling enhances the activity of C/EBPα to induce granulocytic differentiation by phosphorylation of serine 248

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