Regulation of erythroid Krüppel-like factor (EKLF) transcriptional activity by phosphorylation of a protein kinase casein kinase II site within its interaction domain.
Erythroid Krüppel-like factor (EKLF) is a red cell-specific activator whose presence is crucial for establishing high levels of adult beta-globin expression in definitive cells during erythroid ontogeny. However, its simple presence within the erythroid lineage is not sufficient to activate the beta-globin promoter. One explanation that may account for this is that post-translational modification of EKLF differs within erythroid cell populations and regulates its activity. We have therefore addressed whether phosphorylation plays a role in modulating EKLF action. First, in vivo analyses implicate serine/threonine kinases as important players in the terminal differentiation of MEL cells, and demonstrate that EKLF is phosphorylated at serine and threonine residues within its transactivation region. Second, directed disruption of a protein kinase casein kinase (CK) II site, located within the EKLF interaction domain, abolishes EKLF transactivation and in vivo competition activity. Third, in vitro assays demonstrate that CKIIalpha interacts with EKLF, and that the EKLF interaction domain is phosphorylated by CKII only at Thr-41; however, the CKII-site mutant is not phosphorylated. Finally, the transactivation capability of EKLF is augmented by co-transfection of CKIIalpha. We conclude that EKLF is a phosphoprotein whose ability to transcriptionally activate an adjacent promoter is critically dependent on the phosphorylation status of a specific site located within the EKLF interaction domain, and that serine/threonine kinases play an important role in this process.
Brookdale Center for Molecular Biology, Mount Sinai School of Medicine, New York, New York 10029, USA.
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Ouyang
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Chen
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Bieker
D000595:Amino Acid Sequence; D000818:Animals; D047390:Casein Kinase II; D016384:Consensus Sequence; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005914:Globins / Q000096:biosynthesis*; D051741:Kruppel-Like Transcription Factors; D004915:Leukemia, Erythroblastic, Acute; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D013912:Threonine / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D051741:Kruppel-Like Transcription Factors; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C080774:erythroid Kruppel-like factor; D013912:Threonine; D012694:Serine; D005914:Globins; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases