Regulation of the erythroid transcription factor NF-E2 by cyclic adenosine monophosphate-dependent protein kinase.
Activation of cyclic adenosine monophosphate (cAMP)-dependent protein kinase (A-kinase) promotes hemoglobin synthesis in several erythropoietin-dependent cell lines, whereas A-kinase-deficient murine erythroleukemia (MEL) cells show impaired hemoglobin production; A-kinase may regulate the erythroid transcription factor NF-E2 by directly phosphorylating its p45 subunit or by changing p45 interactions with other proteins. We have mapped the major A-kinase phosphorylation site of p45 to Ser(169); Ala substitution for Ser(169) resulted in a protein that was no longer phosphorylated by A-kinase in vitro or in vivo. The mutant protein formed NF-E2 complexes that bound to DNA with the same affinity as wild-type p45 and functioned normally to restore beta-globin gene expression in a p45-deficient MEL cell line. Transactivation properties of the (Ser (169)--> Ala) mutant p45 were also indistinguishable from wild-type p45 when Gal4-p45 fusion constructs were tested with a Gal4-dependent reporter gene. Transactivation of the reporter by both mutant and wild-type p45 was significantly enhanced when A-kinase was activated by membrane-permeable cAMP analogs or when cells were cotransfected with the catalytic subunit of A-kinase. Stimulation of p45 transactivation by A-kinase required only the N-terminal transactivation domain of p45, suggesting that A-kinase regulates the interaction of p45 with downstream effectors.
Casteel
D
D
Department of Medicine and Cancer Center, University of California, San Diego, CA, USA.
Suhasini
M
M
Gudi
T
T
Naima
R
R
Pilz
R B
RB
D000818:Animals; D000242:Cyclic AMP / Q000502:physiology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D050981:Erythroid-Specific DNA-Binding Factors; D004920:Erythropoiesis; D004921:Erythropoietin / Q000502:physiology; D051548:Histone Acetyltransferases; D051379:Mice; D016297:Mutagenesis, Site-Directed; D050983:NF-E2 Transcription Factor; D050984:NF-E2 Transcription Factor, p45 Subunit; D056921:Nuclear Receptor Coactivator 3; D010768:Phosphoserine / Q000378:metabolism; D013329:Structure-Activity Relationship; D015534:Trans-Activators / Q000502:physiology; D014157:Transcription Factors / Q000378:metabolism*; D015533:Transcriptional Activation
D004268:DNA-Binding Proteins; D050981:Erythroid-Specific DNA-Binding Factors; D050983:NF-E2 Transcription Factor; D050984:NF-E2 Transcription Factor, p45 Subunit; C494693:Nfe2 protein, mouse; D015534:Trans-Activators; D014157:Transcription Factors; D004921:Erythropoietin; D010768:Phosphoserine; D000242:Cyclic AMP; D051548:Histone Acetyltransferases; C538867:Ncoa3 protein, mouse; D056921:Nuclear Receptor Coactivator 3; D017868:Cyclic AMP-Dependent Protein Kinases