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.
Department of Biomedical Sciences, University of Modena and Reggio Emilia, Modena, Italy.
Valentina
V
Fragliasso
Yuri
Y
Chiodo
Giovanna
G
Ferrari-Amorotti
Angela Rachele
AR
Soliera
Gloria
G
Manzotti
Sara
S
Cattelani
Olivia
O
Candini
Giulia
G
Grisendi
Jenny
J
Vergalli
Samanta Antonella
SA
Mariani
Clara
C
Guerzoni
Bruno
B
Calabretta
D019943:Amino Acid Substitution; D001483:Base Sequence; D001549:Benzamides / Q000494:pharmacology; D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002454:Cell Differentiation / Q000235:genetics / Q000502:physiology; D049109:Cell Proliferation; D017931:DNA Primers / Q000235:genetics; D050684:E2F Transcription Factors / Q000378:metabolism; D006098:Granulocytes / Q000378:metabolism / Q000473:pathology; D006801:Humans; D020014:K562 Cells; D015470:Leukemia, Myeloid, Acute / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D020935:MAP Kinase Signaling System / Q000187:drug effects; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D016188:Receptors, Granulocyte Colony-Stimulating Factor / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D014410:Tumor Stem Cell Assay
C120227:2-(2-chloro-4-iodophenylamino)-N-cyclopropylmethoxy-3,4-difluorobenzamide; D001549:Benzamides; D022763:CCAAT-Enhancer-Binding Protein-alpha; D017931:DNA Primers; D050684:E2F Transcription Factors; D016188:Receptors, Granulocyte Colony-Stimulating Factor; D011993:Recombinant Fusion Proteins; D012694:Serine