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First published online May 16, 2007
doi: 10.1242/10.1242/dev.02853
Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, V5A 1S6, Canada.
Author for correspondence (e-mail:
everheye{at}sfu.ca)
Accepted 19 March 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Nemo, Nlk, BMP, Dpp, Mad, MH1, Smad, Drosophila
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Subsequent analyses have implicated nmo in patterning events during
embryogenesis and imaginal disc development as well as in controlling
apoptosis (Mirkovic et al.,
2002; Verheyen et al.,
2001). Nemo is the founding member of the evolutionarily conserved
Nemo-like kinase (Nlk) family of proline-directed serine/threonine (S/T)
kinases closely related to mitogen-activated protein kinases (MAPK)
(Choi and Benzer, 1994).
Biochemical and genetic studies implicate Nlk in several pathways (reviewed
by Behrens, 2000;
Martinez Arias et al., 1999).
The best-characterized role for Nlk is in Wnt/Wg signaling in numerous species
(Golan et al., 2004;
Ishitani et al., 2003a;
Ishitani et al., 1999;
Kanei-Ishii et al., 2004;
Meneghini et al., 1999;
Rocheleau et al., 1999;
Shin et al., 1999;
Smit et al., 2004;
Thorpe and Moon, 2004;
Zeng and Verheyen, 2004). Nlk
phosphorylates Tcf/Lef transcription factors and inhibits their activity.
Depending on the cellular context, Nlk either inhibits Wnt-dependent gene
expression (Ishitani et al.,
2003b; Ishitani et al.,
1999; Zeng and Verheyen,
2004) or promotes it
(Meneghini et al., 1999;
Rocheleau et al., 1999;
Thorpe and Moon, 2004). There
is increasing evidence that Nlk regulates additional HMG-domain-containing
proteins, such as Xenopus Sox11 and HMG2L1
(Hyodo-Miura et al., 2002;
Yamada et al., 2003), as well
as other transcriptional regulators such as CBP/p300, Stat3 and Myb
(Kanei-Ishii et al., 2004;
Ohkawara et al., 2004;
Yasuda et al., 2004).
Nlk can be activated by the MAPK kinase kinase Tak1 (TGF-ß activated
kinase 1) in mammals (also known as Map3k7 - Mouse Genome Informatics) and in
C. elegans (also known as MOM-4 - Wormbase) in certain contexts
(Ishitani et al., 1999;
Meneghini et al., 1999).
However, in this study we describe an inhibitory relationship between Nemo and
Drosophila TGF-ß signaling. TGF-ß signaling is initiated
when a secreted ligand of the TGF-ß, bone morphogenic protein (BMP) or
Activin family binds to a type II S/T kinase receptor (reviewed by
Attisano and Wrana, 2002;
von Bubnoff and Cho, 2001).
This receptor then recruits and phosphorylates a type I S/T kinase receptor,
which in turn phosphorylates a member of the R-Smad family of proteins on an
SSxS motif at its C-terminus. The phosphorylated R-Smad is released from the
receptor and binds the Co-Smad. In the nucleus, the Smad complex forms
complexes with transcription factors on the promoters of target genes. Nuclear
signaling is abrogated when the R-Smad is dephosphorylated at its C-terminus
(Chen et al., 2006;
Duan et al., 2006;
Knockaert et al., 2006).
During Drosophila wing patterning, BMP signaling is carried out by
two BMPs, Decapentaplegic (Dpp) and Glass bottom boat (Gbb)
(Padgett et al., 1987;
Wharton et al., 1991). Dpp
acts as a morphogen during the patterning of multiple tissues during embryonic
and imaginal disc development (reviewed by
Raftery and Sutherland, 1999).
Dpp activates the Punt receptor, which in turn phosphorylates Thickveins
(Tkv), leading to the activation of the Smads. The Smad1 ortholog, Mothers
against dpp (Mad), is phosphorylated by activated Tkv and together with the
Co-Smad Medea (Med) accumulates in the nucleus and regulates transcription of
target genes (reviewed by Moustakas et
al., 2001; Shi and Massague,
2003; ten Dijke and Hill,
2004). In the wing imaginal disc, BMP signaling regulates the
expression of several genes, including optomoter blind (omb;
also known as bifid - Flybase), spalt major (salm)
and vestigial quadrant (vgQ) enhancer
(Burke and Basler, 1996;
Grimm and Pflugfelder, 1996;
Kim et al., 1997;
Lecuit et al., 1996;
Lecuit and Cohen, 1998;
Nellen et al., 1996). The
inhibitory Smad homolog Daughters against dpp (Dad) is also
a BMP target gene that acts in a negative-feedback loop to inhibit BMP
signaling (Tsuneizumi et al.,
1997).
Dpp plays several distinct roles during larval and pupal wing development
(Segal and Gelbart, 1985;
Spencer et al., 1982). During
larval disc development, Dpp is expressed along the anterior/posterior (A/P)
boundary of the disc in response to Hedgehog signaling
(Tanimoto et al., 2000).
Localized phosphorylation and activation of Mad (pMad) results in a Mad
activity gradient that drives characteristic patterns of reporter gene
expression across the wing disc, providing positional information to guide
wing vein organization. In addition to a patterning function, BMP signaling is
required for proliferation of the disc, as clones of cells lacking
tkv or Mad are smaller than sister clones and are eliminated
from the wing disc, whereas ectopic BMP signaling results in outgrowths
(Martin-Castellanos and Edgar,
2002; Rogulja and Irvine,
2005). It is speculated that the slope and extent of the pMad
gradient is important for both the proliferative and patterning functions of
Dpp, but the temporal and spatial characteristics for each are distinct
(Rogulja and Irvine,
2005).
In this study we describe a detailed analysis of a novel interaction between nmo and BMP signaling mediated by Mad. Genetic studies in the wing suggest a role for nmo as an antagonist of BMP signaling. These genetic interactions are supported by the finding that elevated Nemo levels can attenuate BMP target gene expression, whereas loss of nmo results in elevated target gene expression. Biochemical and cell culture studies show that Nemo can bind to and phosphorylate Mad and promote its nuclear export. Nemo phosphorylates the MH1 domain of Mad at Ser25 and mutation of this site to alanine causes ligand-independent nuclear localization, whereas substitution with the phosphomimetic aspartic acid results in cytoplasmic localization of Mad. This is the first example of the inhibition of Drosophila BMP signaling by a MAPK and represents a novel mechanism of Smad inhibition by a Nemo-like kinase family member.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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),
nmoadk1 and UAS-nmoC5-1e
(Verheyen et al., 2001),
UAS-nmob27, nmoP also referred to as
nmo-lacZ (Choi and Benzer,
1994; Zeng and Verheyen,
2004), AyGal4.25-UAS-GFP.S65T
(Ito et al., 1997;
Zecca et al., 1996),
Ubi-GFP FRT 79D, ap-Gal4 (expressed in the dorsal wing disc
compartment), dpp-Gal4 (expressed along the A/P boundary),
ptc-Gal4 (expressed along the A/P boundary), vg-Gal4
(expressed along the D/V boundary), prd-Gal4 (expressed in
alternating stripes in the embryo), 69B-Gal4 (expressed ubiquitously
in the wing pouch), omb-Gal4 (expressed in a wide domain along the
A/P axis in the wing pouch), dppd5, dpphr56,
dpphr4, UAS-Mad, UAS-MadS25A, UAS-tkvQD
(Nellen et al., 1996),
UAS-tkvwt, UAS-sog (Yu
et al., 2000), P{lacW}Dadj1E4
(Tsuneizumi et al., 1997),
vgQ-lacZ, salm-lacZ, rlSem/CyO,
UAS-Sem3-1 (Rintelen et
al., 2003) and UAS-GFP.
Clonal analysis
nmoDB24 somatic clones were induced using the FLP/FRT
method (Xu and Rubin, 1993).
To induce nmo loss-of-function clones, embryos from the appropriate
crosses were collected for 24 hours and the hatched larvae were heat shocked
at 38°C for 90 minutes at 48 hours of development. More than 30 clones
were examined in each experiment.
Immunostaining and wing handling
Dissection of imaginal discs, X-Gal staining and antibody staining were
performed following standard protocols. The antibodies used were: rabbit
anti-pMad (1:1000) (Persson et al.,
1998), anti-Delta 9B ascites (1:5000; DSHB), mouse
anti-ß-galactosidase (1:500; Promega) and rabbit
anti-ß-galactosidase (1:2000; Cappel). Secondary antibodies used were:
donkey anti-mouse FITC, donkey anti-rabbit CY3, donkey anti-rabbit FITC and
biotinylated goat anti-rabbit (all from Jackson ImmunoResearch), donkey
anti-mouse Alexa Fluor 594 (Molecular Probes). All secondary antibodies were
used at a 1:200 dilution.
Adult wings were dissected and rinsed in 100% ethanol followed by mounting in Aquatex (EM Science).
Nemo expression vectors
Full-length nmo coding sequences were cloned into the pXJ-Flag
expression vector. The kinase-dead Nemo construct encodes a substitution of a
lysine residue at position 69 for a methionine (K69M). This was modeled on the
kinase-dead form of Nlk described by Brott et al.
(Brott et al., 1998).
Mutagenesis was performed using the QuickChange Site-Directed Mutagenesis Kit
according to the manufacturer's instructions (Stratagene).
Co-immunoprecipitations
HEK293 cells were cultured in Dulbecco's modified Eagle's medium (DMEM;
Gibco) supplemented with 10% fetal bovine serum (Gibco). Cells at 70-80%
confluency were subjected to transient transfection with 8 µg total DNA
using Polyfect transfection reagent (Qiagen) following the manufacturer's
instruction. Cells were lysed 24-48 hours after transfection in lysis buffer
[10% glycerol, 1% Triton X-100, 50 mM Tris pH 7.5, 5 mM EDTA, 150 mM NaCl, 4%
protease inhibitors (Roche), 100 mM ß-glycerol phosphate, 1 mM sodium
vanadate, 5 mM NaF]. Mouse anti-Flag (Sigma) or mouse anti-T7 (Novagen)
coupled to protein G-sepharose beads (Sigma) were used for immunoprecipitation
for 1 hour at 4°C. The immunocomplexes were washed three times with lysis
buffer and boiled in Laemmli buffer, then subjected to SDS-PAGE and western
analysis according to standard protocols. Primary antibodies used were mouse
anti-Flag (1:1000) or mouse anti-T7 (1:5000), and the secondary antibody was
goat anti-mouse HRP light chain-specific (1:5000; Jackson ImmunoResearch). The
western blot was visualized using the Enhanced Chemiluminescence (ECL) Western
Blotting System (Amersham).
Kinase assays
Cell lysates were precleared with protein G-sepharose beads and incubated
with appropriate antibodies. Antibody-protein complexes were precipitated with
protein G-sepharose beads, then washed three times with lysis buffer and once
with kinase assay buffer (25 mM HEPES pH 7.2, 25 mM MgCl2, 50 mM
ß-glycerol phosphate, 2 mM dithiothreitol, 0.5 mM sodium vanadate, 0.1 mM
ribo-ATP). Kinase reactions were initiated by the addition of kinase assay
buffer containing 10 µCi of [
-32P]ATP at room temperature
and stopped after 20 minutes by the addition of Laemmli buffer. Samples were
boiled and subjected to SDS-PAGE and transferred to nitrocellulose membrane
(Perkin Elmer Life Sciences) according to standard protocols and visualized by
autoradiography.
Immunostaining of cultured cells and nuclear export assays
COS-7 and HeLa cells were grown on glass coverslips in 6-well plates 24
hours prior to transfection. Cells at 50-70% confluency were transiently
transfected with various combinations of vectors: pCMV-T7-mad; pCMV-T7-mad and
pCDNA-HA-tkvQD (Inoue et al.,
1998); pCMV-T7-mad, pCDNA-HA-tkvQD and pXJ-Flag-nmo;
pCMV-T7-mad, pCDNA-HA-tkvQD and pXJ-Flag-nmoK69M;
pCMV-T7-mad-S25A; pCMV-T7-mad-S25D. Sixteen hours post-transfection, the cells
were fixed in 4% paraformaldehyde for 15 minutes, followed by permeabilization
with 0.25% Triton X-100. Following two washes in PBS, immunostaining was
performed using mouse anti-T7 antibody (1:2000; Novagen) and rabbit anti-HA
(1:1000; Sigma). Secondary staining was performed using donkey anti-mouse FITC
and goat anti-rabbit CY3 (1:200). Coverslips were mounted cell-side down with
Prolong Gold Antifade Reagent with DAPI (Molecular Probes). For Crm1-dependent
nuclear export assays, leptomycin B (Sigma) was added to a final concentration
of 5.53 ng/ml for 2 hours prior to fixation.
Site-directed mutagenesis of Mad and generation of the Mad MH1 deletion construct
Mutagenesis was performed on the pCMV-T7-mad plasmid, using the QuickChange
Site-Directed Mutagenesis Kit according to the manufacturer's instructions
(Stratagene). Forward and reverse PCR primers were designed to harbor several
nucleotide changes, with the rest of the sequence corresponding to the
template. Serines 25, 146, 202, 212 and 226 were respectively substituted with
alanines as indicated in Fig.
6. In addition, S25 was replaced with aspartic acid (S25D) to
introduce a phosphomimetic residue.
The Mad-
MH1 construct was made by excision of an EcoRI
fragment from the 5' coding region of the pCMV-T7-mad plasmid.
pCMV-T7-mad contains two EcoRI sites: one is located in the 5'
multiple cloning site, the other is at the boundary of the MH1 domain and the
linker domain. Mad-MH1 was obtained by EcoRI digestion, gel
purification of the vector plus 3' sequences and religation resulting in
an in-frame fusion of T7 with the remainder of the Mad coding region, thereby
deleting the MH1 domain.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Mirkovic et al.,
2002; Verheyen et al.,
2001). Notably, the wing phenotypes are indicative of altered BMP
signaling. The adult wing blade consists of two epithelial sheets of intervein
cells intersected at regular intervals by an invariant pattern of longitudinal
veins (numbered L2-L5), the anterior crossvein (ACV) and posterior crossvein
(PCV) (Fig. 1A)
(Bier, 2000). Mutations that
target the early role of Dpp result in vein loss, vein fusions and narrowing
of wing tissue (Fig. 1B,C)
(Cook et al., 2004;
Haerry et al., 1998;
Segal and Gelbart, 1985;
Spencer et al., 1982). Later,
during pupal wing development, dpp expression in vein primordia
functions to maintain and refine the veins
(de Celis, 1997;
Yu et al., 1996).
Ectopic expression of Nemo using the Gal4-UAS system causes a number of
different wing phenotypes (Brand and
Perrimon, 1993; Mirkovic et
al., 2002; Verheyen et al.,
2001; Zeng and Verheyen,
2004). Expression of nmo with omb-Gal4 resulted
in a narrowing of the regions between longitudinal veins, notably L2 and L3
(Fig. 1D), a phenotype seen
with certain dpp alleles (Brummel
et al., 1994; Segal and
Gelbart, 1985). Expression of two copies of UAS-nmo with
omb-Gal4 (omb>2x nmo) resulted in loss of wing tissue,
narrowing of the interval between veins, loss of the PCV and loss of some
longitudinal veins (Fig. 1E,F).
This phenotype is reminiscent of BMP inhibition caused by brinker
(Cook et al., 2004), and
phenocopies that seen with expression of dominant-negative versions of the Dpp
receptors tkv and punt
(Haerry et al., 1998) and in
certain dpp mutants (Bangi and
Wharton, 2006). 69B-Gal4>nmo results in
varied loss of the PCV and a narrower wing blade
(Fig. 1J)
(Verheyen et al., 2001). This
phenotype resembles loss-of-function mutations in the gbb, Medea and
crossveinless genes (Conley et
al., 2000; Hudson et al.,
1998; Khalsa et al.,
1998; Segal and Gelbart,
1985). Similarly, ectopic expression of the BMP antagonist
sog also leads to loss of PCV tissue
(Fig. 1I)
(Yu et al., 1996).
By contrast, nmo loss-of-function alleles displayed a broader wing
blade and ectopic veins emanating from the PCV, posterior to L5 and between L2
and L3 (Fig. 1H). The distance
between the longitudinal veins was also expanded
(Fig. 2H; see below). The
nmo phenotype is similar to those found in flies ectopically
expressing Dpp, Mad or Gbb (Haerry et al.,
1998; Yu et al.,
2000; Yu et al.,
1996). Using vestigial-Gal4 (vg-Gal4) to express
UAS-Mad along the dorsal/ventral (D/V) boundary also resulted in a
broader wing and ectopic veins along L2 and L5 and emanating from the PCV
(Fig. 1G) (see also
Tsuneizumi et al., 1997). This
affect on wing shape, size and vein position in loss-of-function and ectopic
nmo flies suggests that Nemo might negatively influence BMP
signaling.
Modulation of Nemo affects wing disc proliferation
To quantitate the effect of Nemo on the width of the wing blade and the
spacing of veins as processes directly regulated by BMP signaling, we measured
wing blades of different genotypes. Superimposition of wild-type and
nmo wings (Fig. 2A-C)
showed that the positions of L2 and L5 are shifted from the central A/P
boundary towards the margins in nmo wings. The abnormal vein
positions in both genotypes were statistically significant
(Fig. 2H) and highly
reproducible; namely, nmo mutant wings showed an almost identical
pattern of vein spacing. Conversely, ectopic Nemo in omb>nmo
caused a shift of L2 and L5 towards the A/P boundary
(Fig. 2D-F).
To address whether the abnormal wing size in nmo mutants is a result of changes in cell proliferation, we determined cell density within a given region in the wing blade (Fig. 2I-L, Table 1) and also measured overall wing area. Each wing blade cell possesses a single hair (trichome) and counting trichomes thus reflects cell number. nmo wings possessed more cells per given area, and this difference was statistically significant (Table 1, P<0.01). This suggests that nmo mutant cells are slightly smaller than wild type cells. Area measurements determined that nmo wings were consistently larger than wild type wings (Table 1, P<0.0001). This indicates that there is more proliferation in a nmo wing. Since BMP signaling is required for proliferation, it follows that a putative antagonist of the pathway would normally act to inhibit growth, and its mutation would result in increased growth.
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) resulted
in a 20.8% penetrant bifurcated wing blade phenotype (n=53), which
was completely suppressed by co-expression of UAS-nmo
(Fig. 3A-C; n=49).
Although the bifurcation was suppressed, ectopic nmo was unable to
fully restore the wing to wild-type morphology. Use of patched
(ptc-Gal4) to drive expression of wild-type UAS-tkv caused a
vein defect along the A/P boundary (Fig.
3D) that was suppressed by UAS-nmo
(Fig. 3F). Marquez et al.
(Marquez et al., 2001) also
observed ectopic vein phenotypes upon ectopic expression of Mad.
vg>Mad caused a broader wing shape and an abnormal wing vein
phenotype (Fig. 1G,
Fig. 3G). Whereas
vg>nmo caused no discernable phenotype
(Fig. 3H), co-expression of
UAS-nmo and UAS-Mad led to dose-sensitive suppression of the
phenotype induced by UAS-Mad (Fig.
3I), as two copies of Nemo almost completely suppressed the
vg>Mad phenotype (Fig.
5D).
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The observation of a synergistic interaction between nmo and
Dad provided further support for the proposal that Nemo antagonizes
BMP signaling. Dad is an antagonist that is also a transcriptional target of
the pathway (Tsuneizumi et al.,
1997). A P-element enhancer trap insertion into the Dad
gene caused no discernible wing phenotype in homozygous flies
(Fig. 3J), yet in the
Dadj1E4; nmoadk1 double-mutant fly we observed
ectopic vein phenotypes much more severe than nmoadk1
normally displayed (Fig. 3, compare L with
K). This suggests that both genes contribute to the inhibition of
the pathway and that this Dad allele might have partially reduced
function, but not below the threshold needed to see a defect on its own.
Nemo can modulate BMP-dependent gene expression
To further characterize the inhibitory effect of nmo, the
expression of BMP-target genes was monitored in third instar larval wing discs
bearing either nmo mutant clones or ectopic expression of
nmo. The vestigial quadrant (vgQ)
enhancer is expressed in domains flanking the D/V and A/P boundaries
(Fig. 4A). Mad has been shown
to bind directly to the Dpp-responsive element within the
vgQ enhancer (Kim et
al., 1997); thus, this gene serves well as a readout of
Mad-mediated gene expression. UAS-nmo driven by the dorsally
expressed apterous-Gal4 severely reduced vgQ-lacZ
staining in the dorsal wing pouch (Fig.
4B). To further characterize this effect, vgQ
expression was monitored in wing discs containing nmo
loss-of-function somatic clones (Fig.
4C-E). nmoDB24 clones in the central region of
the wing where Dpp signaling is most active (and nmo is normally
enriched, see Fig. 4I) showed
elevated vgQ expression
(Fig. 4E, arrow), whereas
clones outside of this region showed no change in reporter gene expression
(Fig. 4E, arrowhead).
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;
Lecuit and Cohen, 1998;
Sturtevant et al., 1997).
Measurements of the width of salm expression at the D/V boundary
(Fig. 4, white lines) were
normalized against wild type (taken as 100%). In nmo mutants, the
width of the salm domain was consistently wider than in the wild type
(113.92%, n=20, Fig.
4G), whereas in omb>2x nmo the width was dramatically
reduced to just 56.62% (n=20, Fig.
4H).
nmo has a dynamic expression pattern in wing discs
(Verheyen et al., 2001;
Zeng and Verheyen, 2004). In
addition to expression along the D/V boundary, in late third instar wing discs
nmo is enriched in two stripes flanking the A/P boundary of the wing
and is expressed ubiquitously throughout the disc at lower levels
(Fig. 4I). This expression
overlaps with the peaks of pMad staining and corresponds to the site of the
future longitudinal veins L3 and L4 (Fig.
4I-K) (Tanimoto et al.,
2000). During pupal wing development, nmo is expressed in
intervein regions and is enriched in the cells flanking the presumptive veins
(Verheyen et al., 2001). This
pattern of expression together with phenotypic observations suggest a role for
nmo during BMP function in vein patterning and refinement
(Conley et al., 2000).
To determine if nmo can affect levels of pMad, we examined pMad antibody staining in nmo mutant clones. In nmoDB24 mutant clones (Fig. 4L, marked by the absence of GFP fluorescence) there was no detectable change in the levels of pMad (Fig. 4N and merged image in Fig. 4M). In omb>1x nmo discs where the width of the salm expression domain was altered (data not shown), we observed a slight narrowing of the interval between pMad stripes (Fig. 4Q), whereas in homozygous nmo mutant discs the domain was subtly wider (Fig. 4P). Although the mechanism responsible for this observation is not yet known, it is possible that the early role of Nemo in regulating proliferation affects cell numbers in the disc and wing (Fig. 2, Table 1).
Inhibition of Mad is specific to Nemo and not a general feature of MAPK in Drosophila wings
There is a precedent for inhibition of Smad signaling by MAPK proteins from
a number of studies using mammalian cell culture
(Aubin et al., 2004;
Grimm and Gurdon, 2002;
Kretzschmar et al., 1997;
Kretzschmar et al., 1999;
Pera et al., 2003). We sought
to examine whether Drosophila Erk MAPK, encoded by the
rolled (rl) locus, could play a similar role. In flies, both
Epidermal growth factor receptor (Egfr) and BMP signaling are required for
vein specification (Bier,
2000). Hyperactivity of Erk, as found in the
rlSem allele, results in ectopic veins
(Fig. 5C)
(Brunner et al., 1994),
similar to those seen upon loss of nmo
(Fig. 1H). Whereas
co-expression of Nmo and Mad suppressed the ectopic veins induced by
Mad (Fig. 3I,
Fig. 5D), the combination of
ectopic Mad and rlSem (either through ectopic
expression of a UAS-rlSem transgene or introduction of the
rlSem hypermorphic mutation) resulted in an extreme
synergistic vein promotion and excess proliferation
(Fig. 5E,F). We conclude that
in this context, Erk MAPK does not inhibit Mad signaling.
Nemo binds to and phosphorylates Mad
Since Nemo can genetically inhibit BMP signaling, we sought to address the
underlying biochemical mechanism. Nlk can target a number of transcriptional
regulators and affect their function both positively and negatively. Since
Nemo can antagonize Mad-dependent target gene expression in vivo,
co-immunoprecipitation studies were carried out. HEK293 cells were transfected
with T7-tagged Mad and Flag-tagged Nemo and immunoprecipitations revealed
binding of Mad and Nemo (Fig.
6A).
Next we addressed whether Nemo could phosphorylate Mad. In vitro kinase assays were performed on cell lysates and Nemo was found to phosphorylate Mad, as well as to autophosphorylate (Fig. 6B). This was dependent on the kinase activity of Nemo as a dominant-negative Nemo (K69M) construct, in which the lysine residue in the ATP-binding domain was changed to methionine, did not show phosphorylation of Mad, nor did it show Nemo autophosphorylation (Fig. 6B).
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). Since Nemo is a proline-directed S/T kinase, we sought to
identify Nemo target residues in Mad. We identified all S/T residues followed
directly by prolines (S/TP). Based on the precedent seen with Erk-mediated
inhibition of Smads, we first targeted residues within the linker region of
Mad. Site-directed mutagenesis was employed to alter serine 212 (S212) to
alanine in the single consensus Erk phosphorylation site (PNSP) in the linker
domain. In addition, two putative phosphorylation sites (S202 and S226) in the
linker and one in the C-terminus of the MH1 domain (S146) were mutated to
alanine (Fig. 6C).
Surprisingly, a construct expressing Mad in which these four sites were
altered to alanine residues (Mad-4SA) was still phosphorylated by Nemo
(Fig. 6D).
BMP receptor activation leads to phosphorylation of serines (SSVS) at the
C-terminus of Mad (reviewed by ten Dijke
and Hill, 2004). A Mad construct in which these sites were altered
(Mad-AAVA; Fig. 6C) was also
still phosphorylated by Nemo (Fig.
6D), ruling out these residues as possible Nemo target sites.
To map the domain in which the target residue was located, a truncated Mad
protein was generated from which the MH1 domain was deleted (Mad-MH1;
Fig. 6C). This protein was no
longer phosphorylated by Nemo (Fig.
6D), indicating that the target site was contained within the
deleted fragment. Within the deleted MH1 fragment there are two putative Nemo
target sites, S25 and S146. Since the S146 residue had been altered in the
Mad-4SA construct that was still phosphorylated by Nemo, we focused on S25.
Site-directed mutagenesis of S25A was performed and in vitro kinase assays
from transfected cells revealed that Nemo was unable to phosphorylate MadS25A
(Fig. 6D). Thus, we determined
that Nemo can phosphorylate the single serine 25 residue in the MH1 domain of
Mad. This residue has not previously been shown to be targeted by any MAPK
proteins and has not previously been implicated in regulation of Mad function.
The serine found in Mad at position 25 is conserved in the mammalian ortholog
Smad1, but not in the related Smads 2 and 3.
Nemo blocks Tkv-dependent nuclear accumulation of Mad
Activation of BMP signaling leads to nuclear accumulation of
receptor-phosphorylated Smads (reviewed by
ten Dijke and Hill, 2004). In
vertebrate cell culture experiments, Erk MAPK can inhibit this nuclear
localization through its phosphorylation of Smads in the linker domain
(reviewed by Massague, 2003).
Since we have shown that Nemo can also phosphorylate Mad, we examined whether
this affected the nuclear localization of Mad in transfected cells.
Transfection of COS-7 cells with T7-Mad resulted in a uniform subcellular
distribution of Mad (Fig. 7A).
Quantitation showed that Mad expression is nuclear in 11.9% of transfected
COS-7 cells (n=388), and cytoplasmic in the remaining cells.
Co-transfection of an activated Tkv receptor (tkvQD) led to the
dramatic nuclear accumulation of Mad (91.2% of cells; n=457;
Fig. 7B). This nuclear
localization was inhibited by co-transfection of wild-type Nemo with Mad and
Tkv (Fig. 7C). Quantitation
showed that Mad is nuclear in 40.1% (n=424) of transfected cells.
This effect is kinase-dependent, as transfection with kinase-dead Nemo (K69M)
was unable to inhibit nuclear accumulation of Mad
(Fig. 7D), with 87.1% of cells
(n=417) showing nuclear Mad.
Nemo phosphorylation of Mad promotes nuclear export
Examination of the subcellular localization of the MadS25A protein in COS-7
and HeLa cells revealed a primarily nuclear localization as compared with
wild-type Mad (compare Fig. 7E
and Fig. 8A with
Fig. 7A). Significantly, the
nuclear localization was found to be constitutive and unaffected by either
expression of activated receptor or the presence of Nemo (data not shown).
This suggests that the phosphorylation of Mad by Nemo at S25 regulates its
nuclear accumulation, and this regulation is disrupted when the residue is
rendered immune to Nemo phosphorylation (MadS25A). Consistent with the
prediction that the phosphorylation status of S25 influences the localization
of Mad, we found that MadS25D was localized primarily in the cytoplasm
(Fig. 8B), even in the presence
of activated receptor (data not shown).
|
|
; Xiao et al.,
2001). If Nemo is required for cytoplasmic tethering of Mad, then
LMB treatment should not affect the cytoplasmic localization of Mad after
co-transfection with Nemo. If, however, Nemo participates in stimulating
nuclear export, then treatment with LMB should result in Mad accumulation in
the nucleus, even in the presence of Nemo. We found that the nuclear retention
of Mad increased upon treatment with LMB
(Fig. 8E,F), supporting the
second scenario, i.e. that Nemo acts to promote nuclear export of Mad, thus
reducing the effectiveness of Mad signaling.
|
;
Xiao et al., 2001), our
prediction would be that a nuclear-trapped Mad would signal weakly, at most.
Consistent with this prediction, we found that in vivo expression of MadS25A
with engrailed-Gal4 (en-Gal4) resulted in very mild
phenotypic consequences (Fig.
7G), as compared with the severe defects caused by expression of
wild-type Mad (Fig. 7F). Among
20 independently generated transgenic lines, this S25A line displayed the
strongest phenotypic consequences. In situ hybridizations performed with
several independently isolated lines confirmed that the UAS transgenes were
expressed (data not shown). |
DISCUSSION |
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|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Regulation of Mad nuclear localization by phosphorylation
The vertebrate Mad ortholog Smad1 normally shuttles between the cytoplasm
and nucleus in the absence of signal, but upon receptor activation becomes
phosphorylated at its C-terminus, binds the Co-Smad and accumulates primarily
in the nucleus (Xiao et al.,
2001). Such nucleocytoplasmic shuttling is observed with R-Smads
participating in both BMP and TGF-ß signaling (reviewed by
ten Dijke and Hill, 2004). The
shuttling provides a tightly regulated mechanism for monitoring the activation
status of the receptors (Inman et al.,
2002). Receptor-phosphorylated Smads are dephosphorylated in the
nucleus, most likely causing them to detach from Co-Smads and DNA and allowing
them to shuttle back to the cytoplasm
(Chen et al., 2006;
Duan et al., 2006;
Knockaert et al., 2006). Their
nuclear retention is aided by the formation of the R-Smad-Co-Smad complex and
DNA binding. Thus, receptor activation leads to elevated nuclear retention.
The actual rates of nuclear import are not altered by receptor-mediated
phosphorylation (Schmierer and Hill,
2005).
From our findings we conclude that under normal conditions, endogenous Nemo acts to modulate the level of active Mad that is retained in the nucleus. Since Nemo is expressed ubiquitously at low levels and is enriched in cells with elevated levels of pMad, it fulfils the requirements for such a molecule involved in fine-tuning the BMP response. The phosphorylation by Nemo might control a delicate balance between promoting cytoplasmic localization of Mad, while allowing certain levels of Mad signaling to proceed in a receptor-dependent manner.
Differential control of Mad by Nemo and Erk MAPKs
We show that Nemo can inhibit BMP signaling by antagonizing the nuclear
localization of Mad in a kinase-dependent manner. Such a mechanism has been
attributed previously to crosstalk between Erk MAPK signaling and
TGF-ß/BMP signaling (reviewed by
Massague, 2003). Our research
presents Nemo as the first MAPK-like protein to attenuate Drosophila
BMP pathway activity through phosphorylation of Mad. We have also found that
murine Nlk can bind to Mad (data not shown), raising the intriguing
possibility that this mechanism is conserved across species.
|
;
Grimm and Gurdon, 2002;
Kretzschmar et al., 1997;
Kretzschmar et al., 1999;
Pera et al., 2003). The
BMP-specific Smad1 is a target of cross-regulation by EGF signaling through
the Erk MAPK pathway. Erk phosphorylates Smad1 in the linker domain and
inhibits both the nuclear accumulation and transcriptional activity of Smad1
in cell culture and, in consequence, the in vivo function of Smad1 in neural
induction and tissue homeostasis (Aubin et
al., 2004; Kretzschmar et al.,
1997; Pera et al.,
2003). Ras-stimulated Erk also phosphorylates two R-Smads involved
in TGF-ß/Activin signaling and prevents their nuclear accumulation
(Kretzschmar et al., 1999).
The phosphorylation sites within these Smads differ, thus providing a
mechanism for preferentially selective inhibition of one subtype (reviewed by
Massague, 2003). Thus, the
distinct Nemo phosphorylation site in the MH1 domain represents an additional
level of regulation of these proteins. Interestingly, in our studies, we have found that the Drosophila Erk MAPK does not inhibit Mad during wing development. In fact, Erk and Mad appear to synergize in the wing blade, as would be predicted given that both Egfr and BMP signaling are required for vein specification.
Targeting of the Mad MH1 domain by Nemo kinase
The phosphorylation of serine 25 in the MH1 domain of Mad represents a
novel site of regulation of Smads. This protein domain is involved in nuclear
localization, DNA binding and association with transcriptional regulators
(Kretzschmar and Massagué,
1998). Based on known protein structures of Smads, one can predict
that the Mad MH1 domain is composed of several elements. The most N-terminal
sequence predicts a flexible region, then a short alpha-helix followed by a
linker region and a longer, second alpha-helix
(Chai et al., 2003). The
second alpha-helix contains the predicted nuclear localization sequence (NLS)
(Xiao et al., 2001). Serine 25
is located just N-terminal to the first alpha-helix. The added negative charge
following phosphorylation by Nemo could modify the interaction between the two
alpha-helical regions by potentially neutralizing the positively charged NLS
and thereby influencing nuclear localization of Mad. Such a model is also
supported by our finding that mutation of serine to alanine renders Mad
constitutively nuclear. Interestingly, Kretzschmar et al.
(Kretzschmar et al., 1997)
observed a similar constitutively nuclear localization when they mutated the
Erk phosphorylation sites in Smad1. This suggests that both Nemo and Erk MAPK
are involved in the inhibition of BMP signaling and that their distinct sites
of action function to block the nuclear accumulation of Smads. Thus, the
cellular factors that induce either Nlk or Erk activity can oppose the
functions of BMP signaling.
In vivo inhibition of BMP signaling by Nemo during wing patterning and growth
In addition to the biochemical and cell culture evidence that Nemo targets
the MH1 domain of Mad to promote its nuclear export, we present in vivo
evidence which clearly demonstrates that the expression of Nemo or absence of
nmo has a measurable effect on the readout of the BMP pathway in
terms of Mad target gene expression, wing size, wing vein spacing and vein
patterning. Specifically, elevated Nemo can attenuate the expression of
vgQ and salm, whereas nmo somatic clones
and mutant discs show elevated or expanded target gene expression. Genetic
interaction studies confirm such an antagonistic role, as elevated Nemo can
suppress the mutant phenotypes induced by elevated BMP signaling, and
reductions in nmo enhanced the penetrance of activated BMP
phenotypes. Thus, the phenotypic analyses support and extend the biochemical
model of the inhibition of Mad and BMP signaling by Nemo.
Modulation of Nemo does not affect the levels of pMad found at the peaks of the BMP response gradients, suggesting that the effect of Nemo is at the level of the nuclear function of Mad. Our LMB studies demonstrate that Nemo can affect the nuclear localization of Mad. Thus, we propose that Nemo promotes the nuclear export of Mad and that this results in a fine-tuning of the levels of target genes in regions where nmo is expressed.
We propose that one role for nmo is in refining the level of BMP signaling regulating proliferation. This early role for BMP signaling also relies on Mad and is therefore a candidate for Nemo-mediated inhibition. The effect on proliferation may affect the spacing, but not levels, of the pMad gradient. We consistently observe that the genotypes in which wing width is affected do have a mild effect on the spacing of pMad stripes, and we suggest this might be due to actual changes in cell number in the disc. Additionally, nmo mutations manifest in alterations in wing size, wing shape and cell density.
nmo mutations also affect the later larval and pupal patterning and differentiation functions of BMP, and these can be correlated to changes in target gene expression and with vein patterning abnormalities. Thus, it appears that Nemo can modulate levels of BMP signaling at several developmental stages in wing growth and patterning.
Nlks integrate multiple signaling pathways during development
We have previously demonstrated that Nemo can antagonize
Drosophila Wg signaling during wing development
(Zeng and Verheyen, 2004). In
this study we demonstrate that Nemo also acts to attenuate BMP signaling by
targeting the activity of Mad. In both of these signaling pathways the net
outcome is the inhibition by Nemo of pathway-dependent target gene expression.
These results demonstrate that Nemo - and by extension the Nemo-like kinases -
play important roles in refining signaling pathways during development.
An intriguing but still incomplete picture is emerging regarding the
regulation of both Nlk expression and activity and it represents a potential
point of crosstalk between signaling pathways. We have shown that nmo
is transcriptionally regulated by Wg signaling
(Zeng and Verheyen, 2004).
Others have found that the kinase activity of Nlk is stimulated by Tak1 after
Wnt induction (Ishitani et al.,
2003a; Smit et al.,
2004; Kanei-Ishii et al.,
2004) and that Tak1 can be activated by BMP signaling
(Yamaguchi et al., 1995).
Activated Nlk can inhibit Tcf/Lef proteins and modulate Wnt-dependent gene
expression (Ishitani et al.,
2003b; Ishitani et al.,
1999; Zeng and Verheyen,
2004). In this study, we found that Drosophila Nlk is
playing an important role in modulating BMP signaling and Mad-dependent gene
expression, revealing an additional point of cross-regulation and refinement
between signaling molecules.
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
Present address: Department of Developmental Biology, Stanford University,
Stanford, CA 94305-5323, USA
|
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