D. Casteel et al., REGULATION OF THE ERYTHROID TRANSCRIPTION FACTOR NF-E2 BY CYCLIC ADENOSINE MONOPHOSPHATE-DEPENDENT PROTEIN-KINASE, Blood, 91(9), 1998, pp. 3193-3201
Activation of cyclic adenosine monophosphate (cAMP)-dependent protein
kinase (A-kinase) promotes hemoglobin synthesis in several erythropoie
tin-dependent cell lines, whereas A-kinase-deficient murine erythroleu
kemia (MEL) cells show impaired hemoglobin production; A-kinase may re
gulate the erythroid transcription factor NF-E2 by directly phosphoryl
ating its p45 subunit or by changing p45 interactions with other prote
ins. 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 affinit
y as wild-type p45 and functioned normally to restore beta-globin gene
expression in a p45-deficient MEL cell line. Transactivation properti
es of the (Ser(169) --> Ala) mutant p45 were also indistinguishable fr
om wild-type p45 when Gal4-p45 fusion constructs were tested with a Ga
l4-dependent reporter gene. Transactivation of the reporter by both mu
tant and wild-type p45 was significantly enhanced when A-kinase was ac
tivated by membrane-permeable cAMP analogs or when cells were cotransf
ected with the catalytic subunit of A-kinase, Stimulation of p45 trans
activation by A-kinase required only the N-terminal transactivation do
main of p45, suggesting that A-kinase regulates the interaction of p45
with downstream effecters, (C) 1998 by The American Society of Hemato
logy.