Identification of the C/EBPα C-terminal tail residues involved in the protein interaction with GABP and their potency in myeloid differentiation of K562 cells.
The CCAAT/enhancer-binding protein α (C/EBPα) is the member of a family of related basic leucine zipper (bZIP) transcription factors and is critical for granulopoiesis. We previously demonstrated that C/EBPα interacts with the ETS domain of widely expressed GABPα, which leads to cooperative transcriptional activation of the myeloid-specific promoter for human FCAR encoding the Fc receptor for IgA (FcαR, CD89) in part by facilitating recruitment of C/EBPα to the promoter. The C/EBPα molecule contains transactivation domains (TADs) at its N-terminus and a DNA-binding and dimerization bZIP structure at its C-terminus. We demonstrate here that GABPα interacts with the last 18 residues of the C/EBPα C-terminus beyond the bZIP DNA-binding and dimerizing region. Deletion of this C-terminus resulted in loss of GABPα interaction but not affecting its DNA binding ability, indicating that it is not required for homodimer formation. Moreover, the C-terminus confers the ability to functionally synergize with GABP on a heterologous TAD when fused to the C-terminus of the VP16 TAD. We identified a three-amino acid stretch (amino acids 341-343) that is important for both functional and protein interactions with GABP. Ectopic expression in K562 cells of C/EBPα mutant incapable of interacting with GABPα does not induce expression of granulocytic differentiation markers including CD15, CD11b, GCSF-R and C/EBPε, and does not inhibit proliferation, whereas wild type does. These results demonstrate the functional importance of the C/EBPα C-terminus beyond the bZIP DNA-binding and dimerization region, which may mediate cooperative activation by C/EBPα and GABP of myeloid-specific genes involved in C/EBPα-dependent granulopoiesis.
Division of Molecular Cell Immunology and Allergology, Department of Biomedical Sciences, Nihon University School of Medicine, Itabashi-ku, Tokyo 173-8610, Japan. Electronic address: shimokawa.toshibumi@nihon-u.ac.jp.
Toshibumi
T
Shimokawa
Satoshi
S
Nunomura
Daisuke
D
Fujisawa
Chisei
C
Ra
D001483:Base Sequence; D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D002454:Cell Differentiation / Q000502:physiology*; D017931:DNA Primers; D024202:Electrophoretic Mobility Shift Assay; D051268:GA-Binding Protein Transcription Factor / Q000378:metabolism*; D006801:Humans; D020014:K562 Cells; D011485:Protein Binding; D020133:Reverse Transcriptase Polymerase Chain Reaction
D022763:CCAAT-Enhancer-Binding Protein-alpha; D017931:DNA Primers; D051268:GA-Binding Protein Transcription Factor
AML; BR; C/EBPα; CCAAT/enhancer-binding protein α; ChIP; DBD; DNA-binding domain; EGFP; EMSA; FcαR promoter; GABP; GST; Granulopoiesis; HSV-1; IRES; LZ; Leucine zipper; ODN; PCR; RT-PCR; TAD; WT; acute myeloid leukemia; bZIP; basic leucine zipper; basic region; chromatin immunoprecipitation; electrophoretic mobility shift assay; enhanced green fluorescent protein; glutathione S-transferase; herpes simplex virus type 1; internal ribosome entry site; leucine zipper; mtFKBP; mutated FK506-binding protein-12; oligodeoxyribonucleotides; polymerase chain reaction; reverse transcription PCR; transactivation domain; wild type