Spi-1/PU-1 and Spi-B are hematopoietic transcription factors which, in
vitro, display similar affinities for DNA target sequences containing
the consensus binding site 5'GGAA-3'. While the role of Spi-1 in the
transcriptional regulation of B cell and myeloid specific genes has be
en largely demonstrated, the biological function of Spi-B still remain
s to be elucidated. Since Spi-B and Spi-1 are very divergent in their
transactivator domain, these domains might acquire functional specific
ity in vivo by interacting with different co-factors and/or by undergo
ing different phosphorylations. First, we observed that casein kinase
II phosphorylates Spi-B as well as Spi-1, in vitro, Then. by affinity
chromatographies and in vitro kinase assays with fusion proteins betwe
en glutathione-S-transferase and the transactivator domain of Spi-B, t
wo kinases were identified on their ability to interact and phosphoryl
ate this domain; the MAP kinase ERK1 and the stress activated protein
kinase JNK1. The Threonine 56 was defined as the ERK1 phosphorylation
site by using phosphoamino-acid analyses and a Spi-B mutant version wi
th the substitution T56 to A56. Strikingly, ERK1 failed to phosphoryla
te Spi-1, in vitro, whereas JNK1, like CK II, phosphorylated Spi-B and
Spi-1. In addition, other purified Spi-B-kinase activities, unidentif
ied as yet, display similar specificity than ERK1 for Spi-B versus Spi
-1. Furthermore, the evident interaction of pRb protein with the trans
activator domain of Spi-B in an unphosphorylated state disappeared whe
n this domain was first phosphorylated in vitro either by ERK1 or by t
he purified Spi-B-kinase activities. Our data revealed multiple phosph
orylation sites within Spi-B whose some of them appeared specific for
Spi-B versus Spi-1 and which may account for differential regulation o
f their activities.