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

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