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Regulation of the Erythroid Transcription Factor NF-E2 by Cyclic
Adenosine Monophosphate-Dependent Protein Kinase
Darren Casteel,
Modem Suhasini,
Tanima Gudi,
Reza Naima, and
Renate B. Pilz
From the Department of Medicine and Cancer Center, University of
California, San Diego, CA.
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 Ser169; Ala substitution for
Ser169 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 -globin gene
expression in a p45-deficient MEL cell line. Transactivation properties
of the (Ser169 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.
Blood, Vol. 91 No. 9 (May 1), 1998:
pp. 3193-3201
© 1998 by The American Society of Hematology.

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