Phosphorylation of serine 21 modulates the proliferation inhibitory more than the differentiation inducing effects of C/EBPα in K562 cells

The CCAAT/enhancer binding protein α (C/EBPα) is a transcription factor required for differentiation of myeloid progenitors. In acute myeloid leukemia (AML) cells expressing the constitutively active FLT3‐ITD receptor tyrosine kinase, MAP kinase‐dependent phosphorylation of serine 21 (S21) inhibits the ability of C/EBPα to induce granulocytic differentiation. To assess whether this post‐translational modification also modulates the activity of C/EBPα in BCR/ABL‐expressing cells, we tested the biological effects of wild‐type and mutant C/EBPα mimicking phosphorylated or non‐phosphorylatable serine 21 (S21D and S21A, respectively) in K562 cells ectopically expressing tamoxifen‐regulated C/EBPα‐ER chimeric proteins. We show here that S21D C/EBPα‐ER induced terminal granulocytic differentiation of K562 cells almost as well as wild‐type C/EBPα‐ER, while S21A C/EBPα‐ER was less efficient. Furthermore, wild‐type C/EBPα suppressed the proliferation and colony formation of K562 cells vigorously, while S21D and S21A C/EBPα mutants had more modest anti‐proliferative effects. Both mutants were less effective than wild‐type C/EBPα in suppressing endogenous E2F‐dependent transactivation and bound less E2F‐2 and/or E2F‐3 proteins in anti‐C/EBPα immunoprecipitates. Together, these findings suggest that mutation of S21 more than its phosphorylation inhibits the anti‐proliferative effects of C/EBPα due to reduced interaction with or impaired regulation of the activity of E2F proteins. By contrast, phosphorylation of serine 21 appears to have a modest role in modulating the differentiation‐inducing effects of C/EBPα in K562 cells. J. Cell. Biochem. 113: 1704–1713, 2012. © 2011 Wiley Periodicals, Inc.