CCAAT/enhancer binding protein epsilon: changes in function upon phosphorylation by p38 MAP kinase.

C/EBPepsilon, a member of the CCAAT/enhancer binding protein family, is a transcription factor important in neutrophil differentiation. We have determined that it is phosphorylated on multiple serine and threonine residues and can be a target for phosphorylation by a number of kinases. We identified a threonine at amino acid 75, part of a consensus mitogen-activated protein (MAP) kinase site within the transactivation domain of C/EBPepsilon, as being phosphorylated only by p38 MAP kinase. Phosphorylation of this residue resulted in enhanced transcriptional activity on a myeloid-specific promoter in in vitro transient transfection reporter assays. We also determined that phosphorylation at Thr75 yielded a protein that was more effective at binding its cognate DNA sequence compared with the wild-type nonphosphorylated C/EBPepsilon. Stable expression of C/EBPepsilonT75A in interleukin 3 (IL-3)-dependent 32Dcl3 did not result in the up-regulation of expression of secondary granule genes compared with wild-type C/EBPepsilon or C/EBPepsilonT75D. Therefore we suggest that C/EBPepsilon is a target for p38 MAP kinase activity.