| Abstract |
Erf is an ETS-domain protein with strong transcriptional repressor
activity that represses transcription through a distinct C- terminus located
repressor domain. The 2.8 kb Erf mRNA encodes for an 80 kDa phosphoprotein
that is ubiquitously expressed in the developing mouse embryo and
adult tissues as well as in all cell lines tested, except for the placenta. Erf is an
effector of the Ras/MAPK signaling pathway that is regulated through direct
Erk phosphorylation. Erf-Erk physical interaction is mediated through two FXF
motifs, lying at the central part of the Erf protein. Both are required for the
interaction with the active form of Erk, while only one is sufficient for the
binding to the non-phosphorylated form of Erk, although with less affinity. Erf
is phosphorylated by Erk in multiple serine and threonine residues within the
nucleus. Concomitantly, this phosphorylation determines its subcellular
localization and thus its function as a transcriptional repressor. After mitogenic
stimulation Erf is phosphorylated by Erk and exported from the nucleus to the
cytoplasm, while in the absence of mitogenic stimulation, Erf is accumulating
in the nucleus in a non-phosphorylated state. Phosphorylation-deficient and
Erk-binding-deficient Erf mutants are primarily nuclear, irrespective of the
growth conditions and Erk activity and can arrest cell cycle at the G0/G1
phase, further confirming that Erf constitutes a bona-fide Erk substrate and
Ras/Erk pathway effector. It has also been suggested that the Erf mediated
cell cycle arrest is Rb-dependent and is abolished in the overexpression of
cyclins D1 and E. Erf can also act as a tumor suppressor since it has been
shown that can suppress ets- and ras-induced tumorigenicity as well as
Ewing’s sarcoma in cellular and murine systems.
Recent findings from the analysis of the Erf knockout mouse suggest
that Erf plays significant role in the placenta development. Erf KO mice die at
10.5dpc, due to severe placenta malformation. During placenta development,
Erf is expressed in the trophoblast stem cells of the extraembryonic ectoderm
and in later stages in the chorion diploid cells and the labyrinthine
trophoblasts. Erf -/- placentas exhibit compact chorion layer, absence of
labyrinth, expanded giant cell layer and diminished spongiotrophoblast layer.
Marker analysis for different cell types of the trophoblast lineage by in situ
hybridization, indicated that Erf -/- placentas lack post- mitotic chorion cells as
well as the terminal differentiated labyrinthine cell type, the
syncytiotrophoblasts, while they show prolonged expression of trophoblast
stem cell (TSC) markers, like Errb. These data suggest that loss of Erf may
block terminal differentiation of the chorion diploid cells.
Although much is known about Erf regulation through the Ras/Erk
pathway and its physiological role in vivo, there is no evidence for cellurar
direct Erf transcriptional targets. In this study we used microarrays to identify
genes that are directly regulated by Erf. Fourteen genes were characterised
as potential direct Erf targets, with Olig1 and c-Myc genes being the most
prominent. These genes were shown to be upregulated in the absence of Erf
and serum in primary fibroblasts, while c-Myc is increased in Erf KO
empbryos and placentas. Conversely, Erf overexpression could downregulate
c-Myc gene, in conditions that Erf is nuclear and inhibits the transformation of
the MCF-7 adenocarcinoma cell line. Furthermore, endogenous Erf could bind
the 5’ upstream regions of c-Myc in serum-starved fibroblasts and regulate its
promoter activity, suggesting that c-Myc is a direct Erf target gene. Erf is
totally unable to inhibit cell proliferation in the absence of c-Myc, showing that
c-Myc is downstream of Erf in the regulation of the cell cycle. Finally, in Rb-/-
fibroblasts, c-Myc is marginally expressed and cannot be regulated by Erf.
Together these data show that Erf functions are mediated through the direct
regulation of the c-Myc gene.
In an effort to characterise proteins that regulate Erf activity, a yeast
two-hybrid screen was performed in the lab. Hipk1 nuclear kinase emerged as
one potential interactant protein and this interactio was further analysed. Erf
wt, as well as the nuclear mutant form of Erf, interact preferentially with Hipk1
in mammalian cells, through its aminoterminal region. Both Hipk1 and and
Hipk2 phosphorylate this part of Erf, but not a portion of the Erf protein
consisting of the Erk-interaction domain and the the C-terminus. However, we
failed to identify specific residues that are phosphorylated by the Hipks.
Functionally, Hipk-mediated phosphorylation leads to partial Erf export to the
cytoplasm of fibroblasts and concomitantly to loss of Erf transcriptional
repression activity at both artificial and native promoters. The biological
significance of the Erf-Hipk interaction is still unclear, although it is speculated
that it may play a role in the development of the neural tube by regulating
Olig1 expression.
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