Mothers against dpp
MAD is required downstream of DPP receptors in eye morphogenesis. dpp expression in the morphogenic furrow is dependent on Hedgehog which diffuses anteriorly and antagonizes the repression of dpp by Patched and PKA, the catalytic subunit of Protein kinase A. The DPP response mediated by MAD plays a central role in the initiation of the morphogenetic furrow but is largely dispensable for its subsequent anterior propagation (Wiersdorff, 1996).
The furrow is generally more curved in discs of mutant Mad alleles, possible due to a delay in initiation at the lateral edges of the disc. Normally, the furrow seems to continue to initiate along the posterior margin faster than it is propagated in the center of the disc, thus ensuring that it forms a straight line across the eye disc during later stages. In wild type discs, dpp is expressed at the lateral margin until the morphogenetic furrow has passed. This expression is absent at this stage in the central part of Mad mutant discs, suggesting that Mad function is required for the expression of dpp at the margin. Once a partial initiation of the furrow has occurred, furrow progression appears unaffected across the anterior eye field. Clones of Mad mutant cells that include the posterior margin of the eye disc completely abolish initiation of the furrow within the mutant tissue; furthermore, furrow progression in neighboring wild-type tissue is unable to spread laterally into the clone, suggesting that MAD plays a minor role in the propagation of the furrow (Wiersdorff, 1996).
In cells transfected with Medea, the protein is predominantly localized to the cytoplasm; this localization was not altered by coexpression of activated TKV type I receptor. Thus, activation of DPP type I receptor alone is not sufficient to cause accumulation of Medea in the nucleus. In contrast, Mad protein is detected throughout the cell in the absence of signal, and coexpression with activated TKV receptor results in accumulation of Mad in the nucleus. Mutation of the putative phosphorylation sites in Mad, Mad(3SA), prevents nuclear accumulation in the presence of activated TKV receptor, indicating that phosphorylation of Mad is essential for accumulation in the nucleus. Since Mad is expressed at extremely high levels in these cells, it is unlikely that stoichiometric levels of a Smad4-like protein are required for Mad to accumulate in the nucleus. To determine whether entrance of Medea into the nucleus might require physical association with Mad, the subcellular localization of both proteins was examined when coexpressed in the same cell. Although most cells show Medea localized to the cytoplasm when it is expressed alone, coexpression of this protein with Mad increases the number of cells with nuclear Medea staining. Coexpression with both Mad and activated TKV results in strong Medea nuclear staining. In contrast, when Medea is coexpressed with Mad(3SA), which does not accumulate in the nucleus and does not interact with Medea, no shift of Medea from the cytoplasm to the nucleus is detected. Thus, the entrance of Medea into the nucleus requires physical association with phosphorylated Mad (Wisotzkey, 1998).
Under conditions that result in Dpp receptor activation, Mad is able to translocate to the nucleus, while Medea remains cytoplasmic. In the presence of activated Mad, however, Medea translocates to the nucleus. These observations suggest that Mad, but not Medea, is a direct target of the receptor signal, and that the signal from the activated receptor complex to Medea is mediated by Mad. Thus it is likely that Medea, unlike Mad, does not interact with the type I receptor. The distinct responses of these two closely related proteins to stimulation in cell culture, provide a biochemical explanation for the genetic requirement for Mad and Medea in dpp signaling. The basis of this difference in response to receptor activation may lie in the major sites of phosphorylation for the Smads. The class I Smads have been shown to be phosphorylated in response to stimulus at C-terminal serines (consisting of the SSXS motif), an event that is important for signaling. This motif is absent in Medea and the other class II Smads, as well as in the class III Smads. From these observations, it is possible to draw a model whereby the activation of Mad occurs before the activation of Medea during Dpp signal transduction. The levels of Mad that become activated (Mad*) determine the potential of the next, equally important step, which is its hetero-oligomerization with Medea. Thus, the higher levels of signaling achieved by the Dpp/Punt/Tkv activation system in cell culture, yield higher levels of Mad*, and cause high levels of nuclear Medea, while the lower Tkv* stimulus yields low levels of Mad*, and hence undetectable levels of nuclear Medea. Since the formation of the
Mad*-Medea complex is important for signaling, from this
model it is also conceivable that a quantitative increase in the
levels of Medea protein can compensate for a reduction of
Mad, by increasing the likelihood of the hetero-oligomerization
of Mad* with Medea, thereby explaining the
ability of Ubi-Medea to rescue the maternal effect lethality of
Mad 12 /+ flies with dpp hr27 (Das, 1998).
Intracellular signaling of the TGF-beta superfamily is mediated by Smad proteins, which are now grouped into three classes. Two Smads have been identified in
Drosophila. Mothers against dpp (Mad) is a pathway-specific Smad, whereas Daughters against dpp (Dad) is an inhibitory Smad genetically shown to antagonize Dpp signaling. A common mediator Smad, Medea, is described, which is closely related to human Smad4. Mad forms a heteromeric complex with Drosophila Medea upon phosphorylation by Thick veins (Tkv), a type I receptor for Dpp (Inoue, 1998).
Dad stably associates with Tkv and thereby inhibits Tkv-induced Mad phosphorylation. Dad also blocks hetero-oligomerization and nuclear translocation of Mad. The effect of Dad on Mad phosphorylation by
Tkv was studied. Various combinations of Mad, Dad, and constitutively active Tkv
were introduced into COS cells. In the first experiment, cells were labeled with [32P]orthophosphate in vivo, and incorporation of radioactivity into Mad was detected. Dad inhibits phosphorylation of Mad by constitutively active Tkv. Next, anti-phosphoserine antibody was used. As in the
orthophosphate labeling, phosphorylation of Mad diminishes in the presence of Dad.
In vertebrates, inhibitory Smads such as Smad6 and Smad7 have been shown to stably associate with type I receptors. The interaction of Mad or Dad with Tkv was studied: cells were transfected with an appropriate combination of expression plasmids, affinity labeled with iodinated BMP-2, and subjected
to immunoprecipitation with antibodies against Mad or Dad. Pathway-specific Smads are known to associate with type I receptors upon ligand stimulation, but this
interaction is too brief to detect under natural conditions. The interaction
can be observed when the type I kinases are rendered inactive or when the C-terminal phosphorylation sites of the Smads are modified to be resistant to phosphorylation. Mad interacts with the kinase-defective form of Tkv, whereas the interaction of Mad with wild type Tkv is also detectable. The interaction of Mad with Tkv might thus be more stable than that of mammalian Smads with receptors. Dad interacts with wild-type Tkv as efficiently as with the kinase-defective
form of Tkv. Notably, almost the same amount of Tkv is immunoprecipitated with Mad and Dad, although the expression level of Mad is much higher than that of Dad. Thus the affinity of Dad with Tkv seems to be higher than that of Mad. It was found that the interaction of Mad with Tkv was hampered by expression of Dad. Dad thus inhibits
the Tkv phosphorylation of Mad by competing with Mad in association with the receptor. Constitutively active Tkv causes hetero-oligomerization of Mad with Medea. The effect of Dad on the hetero-oligomerization was examined: could Dad inhibit the constitutively active Tkv-induced complex formation of Mad and human Smad4? The hetero-oligomerization of Mad with Smad4 was shown to be efficiently blocked. Dad thus blocked a critical step in the activation of Mad (Inoue, 1998).
The following model is suggested for Dpp signaling by Mad, Medea, and Dad: Dpp
induces phosphorylation of Mad through Tkv and Punt. Mad then forms homo-oligomeric complexes
and/or hetero-dimerizes with Medea. Oligomers of Mad and Medea translocate into the nucleus where
they transactivate target genes, such as vestigial. Dad is one such target, and its expression is induced by Dpp. Dad stably binds to Tkv and interrupts phosphorylation of Mad by Tkv (Inoue, 1998).
In the visceral mesoderm, dpp is expressed in parasegment (ps) 7 under the control of the homeotic
gene Ultrabithorax (Ubx). In this cell layer, dpp stimulates its own expression
and the expression of Ubx. dpp also
stimulates the expression of wingless (wg), an extracellular signaling molecule of the Wnt family, in
the neighbouring ps8. wg in turn feeds back to stimulate Ubx and dpp expression in ps7. Thus, dpp is part
of a parautocrine feedback loop by which Ubx maintains its own expression indirectly through
controlling dpp and wg. Dpp also diffuses from its mesodermal source through the
endodermal cell layer of the embyonic midgut, where it stimulates the expression of D-Fos and of the homeotic gene labial. These inductive steps ultimately specify the differentiation of
distinct cell types in the larval midgut epithelium. In order to understand the mechanism by which dpp stimulates transcription, a short enhancer fragment of Ubx, called Ubx B, has been characterized that contains response sequences for
dpp and wg signaling in the embryonic midgut. The dpp response sequence
of this enhancer is bipartite, consisting of a tandem repeat of Mad binding sites and a cAMP response
element (CRE). The presence of the latter raised the
question whether the co-activator CBP (CREB-binding protein, binding to CREs) might participate in
Dpp-induced transcriptional activation (Waltzer, 1999).
Drosophila CBP loss-of-function mutants show specific defects that mimic those seen
in mutants that lack the extracellular signal Dpp or its effector Mad. CBP loss severely compromises the ability of Dpp
target enhancers to respond to endogenous or exogenous Dpp. CBP binds to the C-terminal domain of Mad. These results
provide evidence that CBP functions as a co-activator during Dpp signaling, and they suggest that Mad may recruit CBP to effect the
transcriptional activation of Dpp-responsive genes during development (Waltzer, 1999).
The embryonic midgut of nejire (nej) mutants (whose CBP function is reduced) show phenotypes related to
wg gain-of-function phenotypes: increased labial expression in the endoderm, and derepression
of the Ubx B enhancer in the visceral mesoderm. These phenotypes do not resemble those seen in dpp or Mad mutants: in Mad mutants,
labial expression is strongly reduced, and so is the beta-galactosidase
(lacZ) staining mediated by the Ubx B enhancer in the middle midgut. However, the narrow band of lacZ staining normally visible in the visceral mesoderm of the gastric
caeca (in ps3) is absent in nej mutant embryos. Indeed, closer inspection reveals that the gastric caeca frequently fail to elongate in these
mutants. A similar phenotype is observed in Mad and in dpp mutants. Thus nej, like dpp, is required for the formation of the gastric caeca, and also for the activity of the Ubx B enhancer in the caecal primordia. The activity of this
enhancer in these primordia coincides with Dpp expression and depends on dpp function. The formation of the first midgut constriction is often impeded. While this could reflect overactive Wg signaling, it also
mimics loss of glass bottom boat (gbb) signaling: Gbb is a Dpp homolog expressed in the
visceral mesoderm and whose function is required for the formation of the first midgut constriction (Waltzer, 1999).
The hypothesis that CBP is a co-activator of dpp-induced transcription was tested by
examining the Dpp response of the Ubx enhancer in nej mutants. Because it was expected that the repressive
effect of CBP on this enhancer would mask a possible activating effect of CBP in cells in which the
enhancer is stimulated by Wg signaling, a mutant
version of Ubx B, called B4, was used whose positive response to Wg is abolished.
B4 activity in the midgut is reduced compared with the wild-type enhancer; however, B4 still contains a
fully functional dpp-response sequence and can be efficiently stimulated by ectopic Dpp. B4 can thus be used to selectively monitor the stimulation of Ubx by Dpp in the visceral
mesoderm. The activity of Ubx B4 is significantly reduced in nej mutants. LacZ staining is particularly weak in ps6/7 (near the Dpp source), but also in ps10, and is
barely detectable in the gastric caeca. Furthermore, in nej mutant embryos
derived from nej mutant germlines (nej), lacZ staining mediated by B4 is even weaker than in
the zygotic nej mutants: although these nej GLC embryos are somewhat variable in
terms of their phenotypes the most severely mutant embryos show
lacZ staining in only a few cells in the ps8 region. Similarly, in Mad12 mutant embryos, lacZ staining is
much reduced, with some staining remaining in ps6 and ps8. This implies that CBP, like
Mad, is required for the Dpp response of the Ubx B4 enhancer (Waltzer, 1999).
The response of B4 to GAL4-mediated ectopic Dpp was examined in nej mutant embryos. If Dpp
is expressed throughout the mesoderm, B4-mediated lacZ staining is increased and detectable
throughout the midgut mesoderm. In nej mutants, this response
of B4 to ectopic Dpp is strikingly disabled: there is barely any lacZ staining
in the anterior midgut, and only a moderate increase of lacZ staining in the ps8/9 region, indicating a
residual Dpp response in this region. These results strongly support the conclusion that CBP is required
for the transcriptional response of the Ubx enhancer to Dpp signalling. They argue that CBP functions
downstream of the Dpp signal (Waltzer, 1999).
In the early blastoderm embryo, dpp mediates the subdivision of the dorsal ectoderm into two
embryonic tissues: the amnioserosa and the dorsal epidermis. High Dpp
levels in the dorsal-most cells specify amnioserosa while lower Dpp levels in dorsolateral regions
specify epidermis. Expression of the gene Race (related to
angiotensin converting enzyme; the earliest known marker for the amnioserosa) in the dorsal
blastoderm embryo depends on dpp signaling. Thus it was asked whether the
activity of the Race enhancer depends on CBP function. This enhancer mediates lacZ staining in the
presumptive amnioserosa and in the anterior midgut primodium: the former, but not the latter,
staining requires dpp. In nej GLC embryos, there is no detectable lacZ staining in the
presumptive amnioserosa, although staining remains, and is even slightly
enhanced, in the head and in the anterior midgut primordium. This
demonstrates that the Race enhancer depends on an activating function of CBP exclusively in a subset
of the blastoderm cells, namely in the dorsal-most cells of the embryonic trunk. It suggests that CBP is
required for the response of this enhancer to dpp (Waltzer, 1999).
To see whether CBP may be required in other developmental contexts in which dpp functions, the developing tracheae were examined in nej mutant embryos. The tracheal system develops
from segmentally repeated clusters of ectodermal cells, the tracheal placodes. These cells undergo a
complex process of migration and fusion to generate the final branched structure of the tracheal
system. dpp signaling plays a crucial role in this process, and has been implicated in the dorsoventral migration of certain tracheal branches. For
example, in punt or thick veins mutants, the branches that normally migrate dorsally or ventrally (the dorsal and ganglionic branches, respectively) fail to develop, whereas the branches that grow out anteriorly (the dorsal trunk and the visceral branches) are essentially not affected. The tracheae in nej mutant embryos were examined using an antibody that stains the lumina of the
tracheal trees (2A12). The dorsal trunk and the visceral branches are essentially normal in these mutants. However, in most nej mutant embryos, branching defects are seen: usually, one or two dorsal branches fail to form at each side, and ganglionic branches fail to fuse. Essentially the same defects are
also seen in in nej GLC embryos. These defects resemble those found in punt
hypomorphs and in Mad12 mutant embryos, although the most apparent
defects in the latter mutants are the fusion defects in their ganglionic branches. Once again, the similarity of the tracheal phenotypes of nej mutants when compared to
dpp, punt and Mad mutants suggests that CBP may be required during Dpp signaling (Waltzer, 1999).
dpp promotes vein development during pupal stages, and a subclass of dpp mutant alleles cause loss of
veins. In particular, in dppS1
homozygous flies, vein 4 fails to reach the margin. This
weak dpp allele was exploited to see whether there would be a genetic interaction between dpp and nej. Indeed, while nej heterozygosity on its own shows no abnormality whatsoever in the wing, this condition clearly enhances the vein phenotype of dppS1 homozygotes: in many of the wings from flies of this genetic constitution, neither vein 4 nor vein 2 reaches the margin.
This synergy in the wing between nej and dpp loss-of-function alleles is consistent with the notion that
CBP functions during Dpp signaling. To clarify the position of CBP in this Dpp response in the wing, it was asked whether the mild dpp
overactivation phenotype due to overexpression of a constitutively active form of Sax (Sax*), a Dpp
type I receptor, depends on nej gene dosage. Expression of Sax* under the control of engrailed.GAL4
induces ectopic venation and overgrowth of the posterior part of the wing. Moreover, removal of one copy of genes required for Sax signaling, such as Mad or
Medea suppresses this phenotype. Likewise, nej heterozygosity suppresses to a considerable extent the wing phenotypes caused by Sax*.
This result is consistent with CBP being required for Sax signaling, and it indicates that CBP
functions downstream of this Dpp receptor (Waltzer, 1999).
Since Mad mediates transcriptional activation by dpp and appears to be a transcription factor required for every aspect of dpp signaling, it was asked whether CBP might be recruited by Mad as a transcriptional co-activator. To test
whether CBP might bind to Mad, the yeast two-hybrid system was used. When these binding
studies were begun, Drosophila CBP had not yet been discovered. So fragments of mouse CBP were used to test
whether these might bind to Mad, assuming that a putative interaction between the two proteins would
be conserved. Indeed, there is a strong degree of homology between mouse and Drosophila CBP. A set of fragments of mouse CBP were used that cover the whole
protein and these were fused to a transcriptional activation domain (the 'prey'). This series of prey was tested in
two-hybrid assays in yeast with full-length Mad protein fused to the LexA DNA-binding domain
(the 'bait'). The C-terminal domain of mouse CBP (CBP1678 to 2441) interacts specifically
with Mad in this assay. This interaction was confirmed using a similar set of prey with fragments from the Drosophila CBP
protein, which was tested against a series of baits containing different Mad domains. This reveals that a
fragment of Drosophila CBP that spans amino acids 2240-2608 (which overlaps the above mentioned
C-terminal domain of mouse CBP) interacts specifically with the MH2 domain of Mad
(Mad219-455). These specific interactions in the yeast two-hybrid assay between CBP fragments and
Mad almost certainly reflect direct binding since yeast does not encode any proteins homologous to
either of these. Interactions between CBP fragments and Mad are significantly
stronger if the N-terminal domain of Mad is removed, suggesting that MH1 inhibits the binding of
CBP to MH2. Inhibitory interactions between MH1 and MH2 have been described previously (Waltzer, 1999).
To confirm these results, direct binding between Mad and CBP in vitro were tested with pull-down
assays. In these assays, [35S]methionine-labelled Mad domains and various fragments from
Drosophila CBP expressed as GST fusion proteins and immobilized on GST-Sepharose beads were used. Either full-length Mad or its MH2 domain binds to the same Drosophila CBP
fragment that interacts with Mad in the yeast assay while Mad's MH1 domain does not bind CBP. Interestingly, deletion of the C-terminal of Mad's MH2 domain (amino acids 372-455)
abolishes CBP interaction, demonstrating that the C-terminal 84 amino acids of Mad are required for
binding to CBP. The linker domain (L) between MH1 and MH2 seems to be dispensable for Mad
interaction with CBP. Weak binding of full-length Mad to CBP2 (CBP2240-2507), the highly conserved domain of Drosophila CBP that overlaps the Mad-binding fragment
of CBP, was observed. However, the significance of this binding is uncertain, as this binding
activity could not be detected in the reciprocal assay nor in yeast. Finally, to characterize more precisely the mutual binding domains within Mad and Drosophila CBP, the reciprocal experiment was performed using [35S]methionine-labelled C-terminal fragments of CBP
and various GST-Mad fusion proteins. CBP binds to GST-MH2, but not to GST
alone, nor to GST-MH1+L fusion proteins, which include extended MH1 fragments (Mad1-241), nor to
GST-MH2C fusion protein in which the last 84 amino acids of Mad's MH2 domain are deleted.
Deletion mapping of the C-terminal region of CBP reveals a minimal fragment of CBP
(CBP2413-2608) that is sufficient for binding to Mad. This domain partially overlaps the
highly conserved CBP2 domain but in most binding assays, CBP2 by itself
is not sufficient for binding to Mad. Altogether, these experiments demonstrate that CBP and Mad bind to one another, and that the stretch
between amino acids 2507 and 2640 within Drosophila CBP is critical for CBP's binding to the MH2
domain of Mad (Waltzer, 1999).
The transcriptional activation potential of proteins can be assayed in chimeras containing a heterologous DNA-binding domain that
mediates their recruitment to reporter genes. This approach has been widely used in yeast and in transient mammalian cell assays.
This approach was applied to assay the transactivation potential of proteins in transgenic Drosophila embryos. A chimera
between the DNA-binding bacterial LexA protein and the transactivation domain from yeast GAL4 behaves as a potent synthetic
activator in all embryonic tissues. In contrast, a LexA chimera containing Drosophila Fos (Dfos) requires an unexpected degree of
context to function as a transcriptional activator. Evidence to suggest that this context is provided by Djun and Mad (a Drosophila Smad), and that
these partner factors need to be activated by signaling from Jun N-terminal kinase and decapentaplegic, respectively. Because Dfos behaves as an autonomous
transcriptional activator in more artificial assays systems, these data suggest that context-dependence of transcription factors may be more prevalent than previously
thought (Szuts, 2000).
Which factors provide the context for Dfos function? Several lines of evidence implicate JNK and Dpp signaling and their transcriptional target factors Djun and Mad
as the essential context. (1) The only embryonic cells in which LexFos functions reliably and robustly to stimulate transcription are the dorsal leading edge cells
which experience both of these signals. (2) Neither of the LexFos derivatives (LexFosN, LexFosC) function in these cells, strongly
implicating the basic leucine zipper domain of LexFos (the only domain absent from both derivatives) in its function. As this domain mediates dimerization with Djun,
the only known dimerization partner of Dfos in Drosophila, this indicates that the activity of LexFos depends on Djun. Recall that Djun is present and
activated by JNK signaling in the leading edge cells. (3) JNK signaling as mimicked by overexpression of constitutively acitive Drac* or Dcdc42*, potently synergizes with
LexFos to mediate widespread transactivation in the embryo. A very similar widespread synergy has also been seen between LexFos and Jun*, a mutant form of c-Jun
that mimics signal-activation of this protein. The embryonic territories in which these synergies are observed appear to correspond to sites of Dpp stimulation.
Consistent with this, a limited synergy between Dpp and LexFos has also been observed in some embryonic cells. These synergies strongly implicate JNK and Dpp as
necessary context signals for LexFos function. (4) LexFos activity strictly depends on the context sequence in the MadL target reporter; under no conditions does it
transactivate a reporter that contains four tandem LexA binding sites (albeit LexGAD very efficiently does so). The context sequence in MadL essentially consists of a binding site for the Dpp
response factor Mad, which is thus a likely partner for the putative LexFos/Djun* dimer (Szuts, 2000).
These results indicate that JNK-activated Djun and Dpp-activated Mad may be critical and widespread context partners of Dfos. Consistent with this, Dfos function is
required for dorsal closure of the embryo and, by implication, functions normally in cells that experience JNK and Dpp signaling. In the embryonic midgut,
Dfos functions in cells that experience Dpp and Egfr signaling. Because the LexFos/JNK synergy in the mesoderm implies that JNK signaling is normally
absent from this tissue, this suggests that the normal partner of Dfos in the midgut visceral mesoderm may be a factor, as yet unidentified, that is activated by Egfr
signaling. Interestingly, synergy between the c-Jun/c-Fos dimer and TGF-beta activated Smad has also been observed in mammalian cells.
Furthermore, Jun proteins have recently been shown to bind directly to Smad3/4. Thus, the partnership between signal-activated Jun/Fos dimers and Smads
may be fairly widespread and fundamental (Szuts, 2000).
Schnurri and Mad can interact directly through discrete domains. Smads characteristically contain an amino-terminal Mad homology
region 1 (MH1) and a carboxy-terminal Mad homology
region 2 (MH2) separated by a poorly conserved linker
region. Mad and Shn interactions were assayed using the yeast
two-hybrid assay. Prey plasmids were generated that express full-length Mad
(FL), Mad MH1, or the MH2 domain along with the linker
(MH21L). In the two-hybrid assay, interaction of the bait
and prey allows yeast expressing both proteins to grow on
selective media and activate transcription of a beta-galactosidase
reporter. Based on both these criteria, it was observed that Shn
associates with Mad FL and Mad MH21L, but not
with Mad MH1. The failure of Mad MH1 to interact
with Shn cannot be ascribed to lower levels of expression of
this domain, since antisera directed against an HA epitope
present in each prey fusion detect comparable levels of all
three Mad polypeptides on Western blots (Dai, 2000).
In order to delineate the domains in Shn that interact
with Mad, GST pull-down assays were used. Mad MH21L domain was expressed
as a glutathione S-transferase (GST) fusion protein. Sixteen
overlapping subclones encompassing the entire Shn coding
region were used to generate 35Smethionine-labeled protein
fragments using coupled in vitro transcription and
translation. The GST-Mad affinity matrix was tested
for its ability to retain the 35S-labeled Shn peptides. Mad MH21L interacts most strongly with two overlapping regions, Shn 1069-1776 and Shn
1463-2318. Further deletions have allowed the interaction to be narrowed to Shn 1441-1635, a region of 194 residues preceding the second set of paired
zinc fingers. Two additional nonoverlapping
fragments, Shn 341-1069 and Shn 1776-2529, show a
moderate interaction with Mad. Subdivision of these fragments
significantly reduce their association with Mad. Thus, a total of one strong and two weaker Mad interaction domains (MIDs) has been detected in Shn. Shn 1441-1776 shows strong binding to itself with an
affinity comparable to that displayed for Mad MH21L. In addition significant binding was detected to Shn 341-1069 and Shn 1776-2529, fragments that contain
the other MIDs. The binding of Shn 1441-1776 is specific
since it fails to bind either Shn 1-968 (a fragment that
lacks a MID). Thus these results suggest that regions of the protein
that are involved in association with Mad may also mediate homomeric Shn interactions (Dai, 2000).
Signals of Dpp are transmitted from the cell membrane to the nucleus by Medea and Mad, both belonging to the Smad protein family. Mad has been shown to bind to the
Dpp-responsive element in genes such as vestigial, labial, and Ultrabithorax. The DNA binding affinity of Smad proteins is relatively
low, and requires other nuclear factor(s) to form stable DNA binding complexes. schnurri (shn) was identified as a candidate gene acting downstream of Dpp receptors, but its relevance to Mad has remained unknown. The biochemical functions of Shn have been characterized in this study. Shn forms homo-oligomers. Shn is localized in the nucleus, and is likely to have multiple nuclear localizing signals. Shn interacts with Mad in a Dpp-dependent manner. The present results argue that Shn may act as a nuclear component of the Dpp signaling pathway through direct interaction with Mad (Udagawa, 2000).
Drosophila C-terminal binding protein (dCtBP) and Groucho have been identified
as Hairy-interacting proteins required for embryonic segmentation and
Hairy-mediated transcriptional repression. While both dCtBP and Groucho are
required for proper Hairy function, their properties are very different. As
would be expected for a co-repressor, reduced Groucho activity enhances the
hairy mutant phenotype. In contrast, reduced dCtBP activity suppresses it. dCtBP can function as either a co-activator or co-repressor of
transcription in a context-dependent manner. The regions of dCtBP required for
activation and repression are separable. mSin3A-histone deacetylase
complexes (see Drosophila Sin3A) are altered in the presence of dCtBP and dCtBP interferes with
both Groucho and Mad transcriptional repression. Similar to CtBP's role in
attenuating E1A's oncogenicity, it is proposed that dCtBP can interfere with
corepressor-histone deacetylase complexes, thereby attenuating transcriptional
repression. Hairy defines a new class of proteins that requires both CtBP and
Groucho co-factors for proper function (Phippen, 2000).
Decapentaplegic (Dpp), a homolog of vertebrate bone
morphogenic protein 2/4, is crucial for embryonic
patterning and cell fate specification in Drosophila. Dpp
signaling triggers nuclear accumulation of the Smads Mad
and Medea, which affect gene expression through two
distinct mechanisms: direct activation of target genes and
relief of repression by the nuclear protein Brinker (Brk).
The zinc-finger transcription factor Schnurri (Shn) has
been implicated as a co-factor for Mad, based on its DNA-binding
ability and evidence of signaling dependent
interactions between the two proteins. A key question is
whether Shn contributes to both repression of brk as well
as to activation of target genes. During embryogenesis, brk expression is derepressed in shn mutants. However, while Mad is essential for Dpp-mediated
repression of brk, the requirement for shn is stage specific.
Analysis of brk;shn double mutants reveals that
upregulation of brk does not account for all aspects of the
shn mutant phenotype. Several Dpp target genes are also
expressed at intermediate levels in double mutant embryos,
demonstrating that shn also provides a brk-independent
positive input to gene activation. Shn-mediated relief of brk repression establishes broad domains of gene activation, while the brk-independent input from Shn is crucial for defining the precise limits and levels of
Dpp target gene expression in the embryo (Torres-Vazquez, 2001).
Genetic evidence implicates both Shn and Mad in dpp-dependent
repression of brk. In the wing disc, cells that lack Mad or shn ectopically express brk and fail to activate the Dpp-responsive
genes optomotor-blind, vestigial, spalt and
Dad. Abolition of
shn or Mad activity results in upregulation of brk in the embryo
and in the absence of shn ectopic Dpp cannot suppress brk
expression. Since Shn and Mad interact directly, an attractive hypothesis is that a Shn/Mad complex is involved in the Dpp-dependent repression of brk. It
has recently been suggested that Dpp signaling bifurcates
downstream of Mad/Med into a Shn-dependent pathway,
leading to brk repression and a Shn-independent pathway that
triggers gene activation. According to
this model, Shn acts primarily as a dedicated repressor
that switches Mad from a transcriptional activator to a
transcriptional repressor on the brk promoter. However several lines of evidence from this study are incompatible with such an interpretation (Torres-Vazquez, 2001).
A strong argument that shn has additional roles beyond brk
repression comes from the fact that simultaneous loss of brk
and shn activity results in a phenotype that is distinct from that
of brk-null animals. If the sole function of shn is to mediate
brk repression, then shn activity should be redundant in a brk
mutant background. However, both at the overt phenotypic
level as well as in the regulation of individual target genes, brk;shn double mutants display defects consistent with lower levels
of Dpp signaling, compared with embryos that lack brk alone. These results indicate that shn participates in gene activation through brk-independent mechanisms as well. The finding that Shn is not obligately required to
suppress brk transcription prior to germband elongation, while
Mad is essential in this process, also argues against an
exclusive role for Shn as a Mad co-repressor. In dpp- and Mad-null
embryos, brk is upregulated at stage 8, while in embryos
lacking shn function, derepression occurs approximately 3
hours later than the transition of brk regulation from maternal
to zygotic control. Thus, brk transcription is insensitive to the absence of shn function at a time when it is responsive to Dpp and Mad. This idea is
reinforced by the fact that ectopic Dpp signaling (through a
constitutively activated form of Tkv called TkvA) can repress brk transcription at stage 5/6 in both wild-type
and shn- animals, but not in Mad-null embryos. Collectively these data provide compelling evidence that refutes a model in which all aspects of the shn mutant phenotype result from derepression of brk transcription (Torres-Vazquez, 2001).
The unexpected result that at high levels TkvA mediates
activation of brk promoter, while at low levels it causes
repression reveals a possible mechanism by which Shn
contributes to Mad activity. One
explanation for these concentration-dependent effects of TkvA
could be that the default mode of Mad action is transcriptional
activation, and interaction with a co-repressor (perhaps present
in limiting amounts) is crucial to bring about repression. Cells
that receive very high levels of signaling could experience
'squelching', owing to excess nuclear Mad that binds to the
brk promoter without recruitment of the co-repressor, thus
promoting activation rather than repression. Supporting this
idea, injection of TkvA into embryos that lack Mad does not
induce either brk activation or repression.
The increased frequency of ectopic brk expression in shn-
embryos could indicate that Shn stabilizes a Mad/co-repressor
complex on the brk promoter. It is worth bearing in mind that
even in shn- embryos, ectopic activation did not occur
independent of brk repression in the peripheral cells. Thus, it appears that Shn does not determine whether Mad acts
as an activator or as a repressor, but may promote its interaction
with other factors that determine the polarity of Mad
transcriptional activity (Torres-Vazquez, 2001).
Analysis of Dpp-responsive gene expression in brk; shn double
mutants has allowed an assessment the brk-independent input
from shn to gene activation at different developmental stages
in a range of tissues. Although it has not been demonstrated
that each of these markers is a direct target of Dpp signaling,
three categories of responses can be distinguished based on these
studies. In the first group (class A), exemplified by
dpp in the leading edge of the dorsal ectoderm, expression in
the double mutant is indistinguishable from that in brk-
embryos. Thus, shn contributes to class A gene
expression primarily by relief of brk repression. Promoters
belonging to class B include Dad and pnr in the dorsal
ectoderm during germband extension. Expression of class B
genes is downregulated in the double mutant compared with
brk- embryos, but is equivalent to wild-type levels. It is inferred
from this result that in the absence of Brk and Shn, Mad-mediated
activation may be sufficient for expression within the
normal domain, but cannot sustain the lateral expansion
encountered in brk mutants. A third category of responses
(class C) includes dpp and Ubx in the midgut, and sna in the
primordia of the wing/haltere imaginal discs. Genes in this
class show significantly reduced levels of expression in the
double mutant, not only relative to brk- but also compared with
wild-type animals. Class C promoters incorporate a brk-independent
positive input from shn that is necessary for wild-type
levels of expression. The inability of ectopic Dpp to induce sna expression in shn mutants demonstrates the essential nature of the requirement for Shn in activation of class C genes (Torres-Vazquez, 2001).
It is evident that repression of brk is crucial for expression of
all three classes of genes described, and as such accounts for a
significant part of the positive input from shn to gene activation.
In addition, the data suggest that Mad and Shn contribute
equally to repression of brk and regulation of class A genes. However, the fact that brk activity is only partially
epistatic with respect to class B and C promoters, indicates that
the majority of genes examined in this study integrate positive
inputs from shn, as well as negative inputs from brk. The near
wild-type expression of class B genes in double mutant embryos
suggests that the brk-independent input from shn may be crucial
at the margins of the expression domains and may be less
significant in regions of the embryo that receive moderate to
high levels of Dpp signaling. In contrast, the positive input from
shn to class C targets appears to be important throughout the
domain of expression. The observation that genes such as
dCreb-A and Scr, which are repressed by dpp signaling, and which are also
sensitive to loss of brk, raises the possibility that Dpp regulates
their expression indirectly. In this event, the dpp target genes
that mediate repression of dCreb-A and Scr would belong to
classes A and C, respectively (Torres-Vazquez, 2001).
The partial restoration of dpp target gene expression in the
double mutants relative to shn- embryos provides a basis for
interpreting the cuticle phenotype. Homozygous brk;shn
animals as well as brk;tkv mutants have an intermediate
phenotype in that they show rescue of the dorsal closure defect
observed in shn and tkv mutants, but they also display a reduced
dorsal epidermis compared with brk-null embryos. Both dpp and pnr have been implicated in dorsal closure,
which results from movement of the epidermal cells over the
amnioserosa and their suturing at the midline. In light of this, the recovery of their expression in the dorsalmost ectodermal cells in the double
mutants correlates well with the restoration of dorsal closure. Likewise, the compromised expression of dorsal ectodermal markers such as Dad and pnr in brk;shn embryos relative to brk null animals, provides molecular correlates for the ventralization observed in the double mutants (Torres-Vazquez, 2001).
The data presented in this study indicate that Shn can mediate
both gene activation and brk repression in response to Dpp
signaling. An important question is whether Shn has a Mad-independent
role in activation. Shn contains a potential
activation domain, and the human ortholog of Shn (PRDII-BF1)
can elicit a 10-fold increase in gene expression in
transfection assays. However, a Shn-Gal4
fusion protein does not activate transcription in yeast, and Shn
is only marginally effective in stimulating a Dpp-responsive
reporter in the absence of Mad in cell culture assays. Taken together these results suggest that Shn acts by promoting Mad binding to DNA and/or its interactions with
the transcriptional machinery. There is ample
precedent for such a mechanism, since several vertebrate DNA-binding
Smad partners such as FAST1, OAZ, Mixer and Milk,
do not have an innate ability to stimulate transcription, but
potentiate gene activation by Smads in a pathway specific
manner. A prediction from this data is that promoters of class B and class
C genes are likely to contain binding sites for Shn as well as
Mad, and that Shn increases Mad specificity by recruiting it to
a subset of promoters that contain binding sites for both
proteins. Analysis of gene expression in brk;tkv mutants
demonstrates that for class B and class C genes Mad provides
a greater brk-independent input compared with shn, consistent
with the idea that Mad plays a primary role in Dpp-dependent
gene activation and that shn facilitates Mad activity. Further
support comes from the observation that deletion of Mad sites
in the Ubx midgut enhancer had a more profound effect than
abolition of Shn binding (Torres-Vazquez, 2001).
It has been shown that Mad interacts with Nejire (Nej), the
Drosophila homolog of the co-activator p300/CREB binding
protein (CBP). Reduction in nej activity affects the expression of ush, pnr and Ubx, and disrupts events that are dpp and shn-dependent, like tracheal migration and imaginal disc patterning. It is interesting to speculate that Shn may interact directly with Nej and stabilize complex
formation between Mad/Medea and Nej (Torres-Vazquez, 2001 and references therein).
The requirement for Shn and Mad in both aspects of Dpp
signaling suggests that Shn does not confer the ability to
activate or repress transcription. It appears more likely that the
activity of the Mad/Shn complex is modulated in a promoter
specific fashion analogous to the mechanisms that convert Dl
from an activator to a repressor. Similarly, the presence of binding sites for
factors that bring co-repressors into proximity with Mad/Shn
could permit inhibition of transcription at the brk promoter
while target genes that lack these sites could be activated in the
same cells. It has been shown that Smad4 interacts
with the co-repressor TGIF and the co-activator CBP in a
mutually exclusive manner. Thus, the
ability to recruit co-activators as opposed to Smad co-repressors
(such as cSki and SnoN), or more general
transcriptional repressors like Groucho or CtBP, would be
crucial to determining whether Dpp stimulation resulted in
activation or repression of the target gene (Torres-Vazquez, 2001).
It is conceivable that in addition to repressing brk
transcription, Shn and Mad could prevent residual Brk protein
in the nucleus from binding to target gene promoters through
steric hindrance or direct competition for common binding
sites. Related anti-repression mechanisms have been postulated
for Smad1 and Smad2 that interact with the transcriptional
repressors Hoxc-8 and SIP1, respectively, triggering their
dissociation from the osteopontin and X-Bra promoters. Although such a
mechanism could potentially enhance the efficiency with
which Shn and Mad antagonize brk activity, it does not account
for the brk-independent input from shn observed in brk;shn embryos, since there is no Brk protein in the double mutants.
Despite the fact that shn transcripts are present from the precellular
blastoderm stage onwards, loss of shn activity does not affect
either brk repression or the expression of Dpp target genes until
germband extension. Germline clonal analysis and ds-RNAi
experiments indicate that the insensitivity of Dpp target gene expression to loss of shn during early embryogenesis is unlikely to result from
perdurance of maternal message. Thus, the 'weakness' of the
shn mutant phenotype may reflect a limited temporal
requirement for shn in dpp signaling, rather than a lesser
requirement for shn activity throughout development. The
functional redundancy of shn during early patterning could be
due to the presence of another protein that contributes a Shn-like
activity to Dpp signal transduction. Alternatively, Mad
activity alone could be sufficient for induction of early D/V
patterning genes if they contain promoter elements that are
more sensitive to Mad. It is also possible that the higher levels
of nuclear Mad resulting from the synergy between Scw and
Dpp in early embryogenesis renders the potentiation of Mad
by Shn unnecessary. Finally, given the conserved nature of the BMP signal
transduction pathway and the identification of Shn homologs
in humans, frogs and worms, it is possible that Shn-like
proteins in other systems potentiate Smad activity in an
analogous manner (Torres-Vazquez, 2001).
Drosophila Smurf1 is a negative regulator of signaling by the BMP2/4 ortholog Decapentaplegic during embryonic dorsal-ventral patterning. Smurf1 encodes a HECT domain ubiquitin-protein ligase, homologous to vertebrate Smurf1 and Smurf2, that binds the Smad1/5 ortholog in Drosophila Mothers against dpp (Mad) and likely promotes its proteolysis. The essential function of Drosophila Smurf1 is restricted to its action on the Dpp pathway. Smurf1 has two distinct, possibly mechanistically separate, functions in controlling Dpp signaling. Prior to gastrulation, Smurf1 mutations cause a spatial increase in the Dpp gradient, as evidenced by ventrolateral expansion in expression domains of target genes representing all known signaling thresholds. After gastrulation, Smurf1 mutations cause a temporal delay in downregulation of earlier Dpp signals, resulting in a lethal defect in hindgut organogenesis. The results suggest that Smurf1 provides an important mechanism to maintain the available pool of Mad at limiting concentrations, and may have additional functions in regulating the levels of Dpp receptors (Podos, 2001).
To identify novel negative regulators of BMP signaling in Drosophila, a genetic selection was conducted for mutations that result in elevated Dpp activity during embryonic D-V pattern formation. From 65,000 mutagenized genomes, six extragenic mutations were recovered that suppressed the lethal, partially ventralized embryonic phenotype caused by the hypomorphic maternal-effect Medea mutation, Med15. Both the molecular and genetic characterization of two of these mutations, 11R and 15C, which disrupt the previously unrecognized DSmurf locus, are presented in this study. Both mutations act as largely recessive maternal-effect suppressors that restore viability and wild-type pattern to Med15 embryonic progeny, either as homozygotes or in trans-heterozygous combination. Because these mutations act in the same fashion to suppress the partially ventralized embryonic phenotype caused by dpp haploinsufficiency, they effect a general elevation of Dpp signaling activity during embryonic D-V pattern formation (Podos, 2001).
2001).
The activities of vertebrate Smurf1 and Smurf2 in opposition to BMP and TGF-ß signals are mediated in part by direct binding interactions with their R-Smad substrates. Whether Drosophila Smurf1 interacts physically with the R-Smad encoded by Mad was examined. In a yeast two-hybrid assay, it was found that Smurf1 binds Mad but not its co-Smad Medea. Similar to the vertebrate Smurf-Smad interactions, the interaction between Smurf1 and Mad was disrupted by deletion of the PY motif from Mad. Smurf1 therefore shares substrate binding properties with its vertebrate homologs, likely reflecting a common function in restricting Dpp/BMP signals by promoting the proteolysis of the BMP-specific R-Smad proteins (Podos, 2001).
While mutation of Smurf1 does not cause overt alterations in D-V cuticular pattern, the cuticle presents a snapshot of embryonic development that does not necessarily reflect initial D-V pattern and does not incorporate the dorsal-most tissue, the amnioserosa. To obtain a direct readout of the Dpp activity gradient that is sensitive to subtle changes in its strength and spatial parameters, wild-type and mutant embryos were examined for changes in the spatial extent of staining with the phosphorylated form of MAD (P-Mad) antibody and changes in the expression domains of direct Dpp target genes (Podos, 2001).
In wild-type embryos at the onset of gastrulation, a stripe of P-Mad staining is visible in a dorsal subset of dpp-expressing cells and in the cells at either pole of the embryo. In Smurf115C mutant embryos, there is a small but statistically significant increase (28%, P < 0.001) in the width of the dorsal P-Mad stripe as well as a nonquantitated increase in the intensity of staining. In wild-type embryos at this stage, the Dpp target genes zen and Race are activated by high levels of Dpp signaling in the presumptive amnioserosa, while the intermediate threshold target gene u-shaped (ush) is activated in a broader domain by lower levels of Dpp activity. All three transcriptional domains showed significant lateral expansion in Smurf115C mutant embryos; a lesser but significant expansion of zen was also observed in Smurf111R mutant embryos. Later, Smurf115C mutant embryos differentiate a nearly 2-fold excess of amnioserosa cells compared to wild-type. A 2-fold increase in dpp gene dosage effects a similar expansion of zen transcription and a comparable increase in amnioserosa cell number. These observations indicate that disruption of Smurf1 gene activity elicits an expansion of multiple Dpp signaling thresholds in the early embryonic ectoderm comparable to the phenotype caused by a doubling of dpp gene dosage (Podos, 2001).
Despite the intensive study of Dpp-dependent developmental events, there are multiple reasons why the role of Smurf1 eluded prior notice: (1) both the maternal and zygotic components of Smurf1 must be mutated to uncover a lethal phenotype; (2) Smurf1 mutants are relatively dosage insensitive, as Smurf1 mutations do not exert significant dominant phenotypes even in sensitized backgrounds such as Med15, precluding their isolation in most genetic screens. Such dosage insensitivity might be a general feature of enzymes, as opposed to stoichiometric components of signaling pathways such as Mad and MED. (3) Smurf1 mutations do not cause overt defects in dorsal-ventral patterning. Possibly, the activity of Smurf1 is partially redundant with another ubiquitin-protein ligase. In support of this hypothesis, Dpp signals are eventually downregulated in Smurf1 mutants. Moreover, an analysis of human Smad2 turnover has implicated a ubiquitination activity that does not require the Smad2 linker domain and therefore is likely independent of the Smurf proteins (Podos, 2001).
The spatial modulation by Smurf1 of graded Dpp signaling is evident prior to the onset of gastrulation. The abrogation of Smurf1 activity causes a sensitization to Dpp signals at all positions in the D-V activity gradient, as indicated by expanded domains of P-Mad staining, target gene transcription, and tissue differentiation. Similar global expansions of Dpp-dependent territories have been observed in embryos with elevated dpp gene dosage. Since an increase in ligand concentration is likely to result in the phosphorylation of additional cytoplasmic Mad, this spatial control over Dpp target gene expression is likely to derive from the unregulated ubiquitin-mediated proteolysis of Mad throughout the embryo. Similar properties have been established for human Smurf1, from demonstrations that BMP receptor activation does not alter the rate of Smad1 ubiquitination and degradation mediated by Smurf1 (Podos, 2001).
The results suggest that Smurf1 provides an important mechanism to maintain the available pool of Mad at limiting concentrations, the necessity of which has been supported by previous genetic observations. Although not normally haploinsufficient, the Mad gene is rendered so when the activities of other components of the Dpp pathway, including dpp, zen, and sog, are reduced. More generally, limiting amounts of Smad protein might be an essential feature of all graded TGF-ß superfamily signaling systems. Cytoplasmic Smad pools are similarly limiting in Xenopus embryos, according to quantitative studies of activin signaling. Experimental elevations in Smad2 concentration cause proportionate increases in Smad activation, as represented by both nuclear Smad2 import and transcriptional readout. Therefore, it is predicted that Smurf enzymes will prove to be essential to maintain Smad proteins at limiting concentrations to ensure appropriate responses to all graded BMP and activin/TGF-ß signals (Podos, 2001).
The Med15 mutation is a missense lesion in the MH2 domain, within the L3 structural loop that has been implicated in the signal-dependent interaction between trimers of Mad and MED. Because an experimental elevation of wild-type Mad levels is sufficient to restore the specification of amnioserosa to embryos derived from Med15 mothers (Hudson, 1998), it is hypothesized that Smurf1 mutations suppress Med15 because the resulting elevation of Mad is sufficient to overcome its reduced affinity for the mutant MED protein (Podos, 2001).
Phenotypic analysis has identified a second requirement for Smurf1 in the temporal downregulation of Dpp signaling. With the exception of the amnioserosal cells, all of the descendants of cells with high levels of P-Mad at the onset of gastrulation downregulate P-Mad staining by stage 10. In contrast, Smurf1 embryos of the same stage retain P-Mad staining in all these cell types, leading to deleterious consequences in hindgut morphogenesis. A causal link has been established between the prolonged Dpp signaling in the dorsal hindgut primordium, ectopic zen transcription, and the subsequent breakdown in the epithelial integrity of the hindgut cells (Podos, 2001).
This second function of Smurf1 is distinct, and possibly mechanistically separable, from its ability to target Mad for destruction. Although the incremental spatial expansion of P-Mad staining along the dorsal-ventral axis at the blastoderm stage is consistent with an overall increase in the amount of Mad protein in Smurf1 mutants, the complete lack of temporal downregulation of P-Mad staining in stage 8-10 Smurf1 embryos is more consistent with a specific effect of Smurf1 on P-Mad. Because the level of P-Mad is a readout of both the amount of Mad protein within a cell and the intensity of Dpp signaling that the cell receives, one attractive hypothesis is that Smurf1 downregulates the level of P-Mad by antagonizing Dpp signaling, independent of Mad degradation. One possible mechanism is suggested by demonstrations that vertebrate Smurf1 and Smurf2 can use the I-Smad, Smad7, as an adaptor to promote the degradation of activated TGF-ß receptors. It is proposed that Smurf1 might similarly target activated Dpp receptors for degradation, using Mad or, more likely, the I-Smad protein DAD as an adaptor. Such an activity would serve as a feedback mechanism of attenuation, whereby receptors are targeted for degradation only upon activation by ligand. Feedback mechanisms are integral to many developmental signaling processes; the proposed feedback activity of Smurf1 on the Dpp receptors would be one of the few instances where temporal downregulation of a signaling system has been shown to be necessary for the proper differentiation of cells previously exposed to the signal (Podos, 2001).
Involvement of Smurf1 have been demonstrated in the control of multiple aspects of Dpp signaling. Further analysis will be required to determine whether Smurf1 acts bifunctionally to mediate the degradation of Mad and of activated Dpp receptors. Smurf1 might also have additional activities that have not been uncovered by these mutants. For example, although homozygous Smurf1 adults have normal cuticular morphology, an examination of Dpp target gene expression might reveal Smurf1 function in imaginal disc patterning. By analogy to the vertebrate Smurf proteins, Smurf1 also might modulate the activin/Smad2 signaling pathway, which has been implicated in the control of cell proliferation. Lastly, Smurf1 might promote the ubiquitin-mediated degradation of other substrates, independently of the Smads. More generally, further characterization of the relationship between Smurf1 and other modulators of Dpp signaling will yield insights into the precise regulation that underlies intercellular signaling systems during normal development (Podos, 2001).
Although Wishful thinking shows a strong overall similarity to the vertebrate BMP type II receptor, its signaling mechanism remains untested. To examine whether Wit actually mediates a BMP type signal, use was made of an antibody that specifically recognizes the phosphorylated form of Mad (P-Mad) (Tanimoto, 2000), the major transducer of BMP signals in Drosophila. In late embryos, this antibody reveals an intricate developmental pattern of BMP signaling, including P-Mad accumulation in the developing midgut and gastric cecae that closely parallels dpp expression and is thought to be indicative of cells receiving a Dpp signal. In addition to these sites of accumulation, a novel pattern of P-Mad accumulation is noted in a subset of neurons. Staining is first evident at late stage 15 and then becomes more elaborate and intense as the embryos continue to develop. Ultimately about 35-40 neurons per hemisegment show strong staining by stage 17. This expression continues into the first instar stage but is not detectable in late third instar larvae. The pattern of P-Mad accumulation in the CNS is completely abolished in wit mutant embryos whereas all other patterns of P-Mad accumulation appear normal. Conversely, with the exception of the CNS pattern, all other sites of P-Mad accumulation in late embryogenesis are absent in zygotic punt null mutants. Maternal null embryos cannot be examined since they do not develop an organized CNS. These observations are consistent with Wit function being required specifically in the CNS to transduce BMP signals whereas other sites of P-Mad accumulation result from signaling by Punt, the primary Dpp type II receptor (Marqués, 2002).
The number and the positions of the phosphorylated form of Mad (P-Mad)-positive cells, per hemisegment at stage 17, indicative of cells undergoing Wit signaling, correlates well with the estimated number and positions of motoneurons at this stage. To better characterize the cells that accumulate P-Mad in the nervous system, wild-type embryos were double stained for P-Mad and several other markers, including Even-skipped and Lim-3, two transcription factors expressed in discrete subsets of embryonic motoneurons and interneurons. Staining was also performed for Fasciclin II (Fas II), a neural adhesion molecule expressed in a subset of interneurons as well as most motoneurons. There is colocalization of Eve and P-Mad in the RP2 and U/C motoneurons but not in the EL interneurons, while P-Mad and Lim-3 colocalize in a lateral cluster of cells that include motoneurons 28 and 14/30. In addition, Lim-3 and P-Mad colocalize in RPs 1, 3, 4, 5. The Fas II antibody highlights axon tracts and cell bodies of most motoneurons as well as some interneurons. Most, if not all, P-Mad cells also show colocalization of Fas II. These findings suggest that Wit is required to transduce BMP signals in motoneurons and is consistent with the ability of UAS-wit constructs to rescue wit mutants when expressed with a motoneuron-specific driver (Marqués, 2002).
dpp expression has been examined in two groups of dorsal ectoderm cells at the posterior end of the embryo, in abdominal segment 8 and the telson. These dpp-expressing cells become tracheal cells in the posterior-most branches of the tracheal system (Dorsal Branch10, Spiracular Branch10, and the Posterior Spiracle). These branches are not identified by reagents typically used in analyses of tracheal development, suggesting that dpp expression confers a distinct identity upon posterior tracheal cells. dpp posterior ectoderm expression begins during germ band extension and continues throughout development. The sequences responsible for these aspects of dpp expression have been isolated in a reporter gene. An unconventional form of Wingless (Wg) signaling, Dpp signaling, and the transcriptional coactivator Nejire (CBP/p300) are required for the initiation and maintenance of dpp expression in the posterior-most branches of the tracheal system. These data suggest a model for the integration of Wg and Dpp signals that may be applicable to branching morphogenesis in other developmental systems (Takaesu, 2002).
dpp expression in posterior
tracheal branch anlagen appears to be initiated by prior episodes of wg
and dpp expression in the undifferentiated dorsal ectoderm.
The maintenance of dpp expression in posterior tracheal
branches appears to require continuous input from wg and
from a dpp feedback loop. The initiation and maintenance
of dpp expression in posterior tracheal branches also requires
continuous nej activity. Overall, the data are consistent
with the following combinatorial signaling model. The transcriptional activator Medea (Med, signaling for the Dpp
pathway) interacts with the transcriptional activator Arm
(signaling for the Wg pathway) via the transcriptional coactivator
Nej. This multimeric complex initiates and, with
continuous signaling, maintains dpp expression in posterior
tracheal branches with the help of Zw3. These data extend
previous studies of dpp expression and Dpp signaling in
several ways. nej has been reported to participate in
Dpp signaling. Expression from Dpp responsive
enhancers is reduced in nej zygotic mutant
embryos. While they show that nej3 enhances dpp wing
phenotypes, this study shows that Mad1 enhances nej3 embryonic phenotypes. The Dorsal Trunk Branch
forms normally in Mad12 zygotic mutant embryos, and the Dorsal Trunk Branch appears normal in
Med1 mutants. nej is involved in mediating combinatorial signaling by the Wg and Dpp pathways and the involvement
of nej in morphogenesis of Dorsal Branch, Spiracular Branch, and the Posterior Spiracle is demonstrated. A region of the histone acetyltransferase domain
of Nej binds to Mad. Further study is needed to reveal the mechanisms used by Nej to interact with Wg and Dpp signaling. Several questions remain
about the regulation of dpp expression by Wg, Dpp, and Nej.
Two questions arise about the mechanism of signal integration:
how is zw3 involved and how is Nej recruited to bridge
the two pathways? It is tempting to speculate that, in
response to a Wg or a Dpp signal, Zw3 (a serine-threonine
kinase) is involved in Nej recruitment. Numerous studies
have shown that p300/CBP transcriptional coactivation
functions are stimulated by its phosphorylation, but the
site of phosphorylation has never been mapped. Other questions concern the molecular nature of the enhancers that direct dpp expression in the
posterior tracheal branches. A 54-nucleotide region has been identified that contains two sets of
conserved, overlapping consensus binding sites for dTCF
and Mad/Med. Analyses of DNA-protein interactions predicted
by the data involving this candidate combinatorial
enhancer have begun (Takaesu, 2002).
The CBP histone acetyltransferase plays important roles in development and disease by acting as a transcriptional coregulator. A small reduction in the amount of Drosophila CBP (dCBP) leads to a specific loss of signaling by the TGF-ß molecules Dpp and Screw in the early embryo. The expression of Screw itself, and that of two regulators of Dpp/Screw activity, Twisted-gastrulation and the Tolloid protease, is compromised in dCBP mutant embryos. This prevents Dpp/Screw from initiating a signal transduction event in the receiving cell. Smad proteins, the intracellular transducers of the signal, fail to become activated by phosphorylation in dCBP mutants, leading to diminished Dpp/Screw-target gene expression. At a slightly later stage of development, Dpp/Screw-signaling recovers in dCBP mutants, but without a restoration of Dpp/Screw-target gene expression. In this situation, dCBP acts downstream of Smad protein phosphorylation, presumably via direct interactions with the Drosophila Smad protein Mad. It appears that a major function of dCBP in the embryo is to regulate upstream components of the Dpp/Screw pathway by Smad-independent mechanisms, as well as acting as a Smad coactivator on downstream target genes. These results highlight the exceptional sensitivity of components in the TGF-ß signaling pathway to a decline in CBP concentration (Lilja, 2003).
These results suggest that several transcription factors that regulate expression of Dpp/Screw signaling components require the dCBP coactivator for their function in Drosophila embryos, and implicate dCBP in regulation of the Dpp/Screw pathway independently of an interaction with Smad proteins. An additional role of dCBP is to regulate Dpp-target genes, acting at a step downstream of Smad protein phosphorylation. It is likely that direct interactions between dCBP and Mad/Medea contribute to regulation of Dpp target genes). Such interactions have been observed in vitro, both in mammalian systems and using Drosophila proteins. However, a major cause of impaired Dpp/Screw signaling in dCBP mutant embryos is due to reduced tolloid expression. This prevents Dpp/Screw from initiating a signaling event in cells that would normally receive the Dpp/Screw signal, presumably by a failure to cleave the Dpp-Sog and/or Screw-Sog complexes. In fact, a majority of embryos that do not express the Dpp/Screw-target gene rhomboid in dorsal cells, also do not contain phosphorylated Smad proteins. Furthermore, the pattern of phosphorylated Smad proteins correlates closely with that of tolloid expression. For example, in many early, cellularizing dCBP mutant embryos, an anterior patch of both tolloid expression and phosphorylated Smad staining remains. At later stages, tolloid expression recovers in dCBP mutant embryos, as does Dpp/Screw signaling as revealed by Smad protein phosphorylation. This recovery of tolloid expression at later stages of development might explain the recovery of phosphorylated Smad proteins in dCBP mutant embryos, by allowing Dpp/Screw to signal. For these reasons, regulation of tolloid expression appears to be a major means of controlling Dpp/Screw signaling by dCBP (Lilja, 2003).
It is likely that reduced screw expression also contributes to the reduction of phosphorylated Smad proteins observed in dCBP mutant embryos. In both screw and tolloid mutants, phosphorylation of Mad is eliminated. Furthermore, progressive reduction in Screw activity leads to a corresponding progressive deletion of dorsal-most cell fate, the amnioserosa. Tsg is required together with Sog to generate peak Dpp activity in dorsal midline cells. Reduced tsg expression in dCBP mutants may therefore contribute to the lack of Dpp/Screw-target gene expression. However, it is not believe that this lack can explain the defects in dCBP mutants, because in tsg mutants, low levels of Dpp signaling persist in a broad dorsal domain, leading to expanded rhomboid expression in dorsal cells. By contrast, in dCBP mutant embryos, expression of genes in response to a low threshold of Dpp activity, such as U-shaped and the dorsal rhomboid pattern, is eliminated (Lilja, 2003).
These experiments do not address whether dCBP regulation of tolloid, screw, and tsg expression is direct or indirect. However, since expression of these genes begins at about the time when zygotic transcription initiates in the embryo, and the effect of dCBP is evident from the onset of expression of tolloid and screw, the notion is favored that dCBP is acting directly on the enhancers of these genes. It is not yet understood whether HATs such as CBP primarily act to acetylate large chromosomal domains, or are directed to specific genes. In the case of the tolloid gene, the results indicate that dCBP is being recruited to the enhancer by a DNA-binding protein, since the isolated enhancer removed from its normal chromosomal location requires dCBP for its activity (Lilja, 2003).
Given its central position in gene regulation and the great number of mammalian transcription factors shown to interact with CBP, relatively few genes are affected by the dCBP mutation. For example, activation and repression mediated by the Dorsal protein are unaffected in the dCBP mutant embryos, as demonstrated by the expression patterns of Dorsal target genes. Also, no defects in early segmentation gene expression could be observed in germline clone mutants. However, the nej1 mutation used in this study to create dCBP mutant germline clone embryos is a weak mutation that results in a very modest reduction in dCBP levels. Since other means of reducing the dCBP amount by approximately two-fold results in similar gene expression defects, Smad proteins and the unidentified activators of tolloid, tsg, and screw expression are particularly sensitive to a decline in dCBP concentration. It may not be a coincidence that screw, tsg, tolloid, and Dpp-target gene expression are all specifically affected by a small dCBP reduction. Perhaps components of the Dpp/Screw signal transduction pathway have evolved to be coordinately regulated by a common coactivator. Given the phylogenetic conservation of the CBP protein and the TGF-alpha signal transduction pathway, as well as the ability of CBP and Smad proteins to interact in vitro, CBP is likely to play an equally important role in TGF-ß signaling in other metazoans (Lilja, 2003).
The spatial and temporal control of gene expression during the development of multicellular organisms is regulated to a large degree by cell-cell signaling. A simple mechanism has been uncovered through which Dpp, a TGFß/BMP superfamily member in Drosophila, represses many key developmental genes in different tissues. A short DNA sequence, a Dpp-dependent silencer element, is sufficient to confer repression of gene transcription upon Dpp receptor activation and nuclear translocation of Mad and Medea. Transcriptional repression does not require the cooperative action of cell type-specific transcription factors but relies solely on the capacity of the silencer element to interact with Mad and Medea and to subsequently recruit the zinc finger-containing repressor protein Schnurri. These findings demonstrate how the Dpp pathway can repress key targets in a simple and tissue-unrestricted manner in vivo and hence provide a paradigm for the inherent capacity of a signaling system to repress transcription upon pathway activation (Pyrowolakis, 2004).
One of the primary events controlled by the Dpp morphogen gradient during growth and patterning of imaginal discs is the establishment of an inverse gradient of brk expression. brk expression is controlled by two opposing activities, a ubiquitous enhancer and a Dpp-dependent silencer. The minimal requirements for a functional silencer complex, both at the DNA and at the protein level, have been determined. Importantly, it has been demonstrated that the minimal element functions in vivo when assayed in the vicinity of a strong enhancer (the brk enhancer) or when present in a single copy in chimeric transgenes (brk enhancer-bamSE fusions) or from within an endogenous gene (gsb-enhancer lacZ fusions). The minimal functional silencer contains a distinct, single binding site for each of the two signal mediators, Mad and Med. Med binds to a GTCTG site, previously recognized as a high-affinity site for Smad binding. Mad binds to a different, GC-rich sequence. Upon binding of Mad and Med, the zinc finger protein Shn is recruited to the protein-DNA complex, bringing along a highly effective repression domain. Although ShnCT contains three essential zinc fingers, it does not bind the silencer element in the absence of Mad and Med. These data suggest that even in the triple protein complex, Shn might bind DNA with moderate sequence specificity, since only a single nucleotide position was identified that is essential for Shn recruitment. However, a number of other cis-regulatory elements that bind Mad and Med (derived from the vestigial, labial tinman, and ubx genes failed to recruit Shn, demonstrating the exquisite selectivity of the element defined in this study (Pyrowolakis, 2004).
Part of this selectivity is accounted for by the specific spacing and orientation of the Mad and Med binding sites in the silencer. Deletion and insertion of single base pairs between the two sites abolish Shn recruitment in vitro and Dpp-dependent repression in vivo, although such alterations still allow the efficient formation of a Mad/Med complex. These findings suggest that Shn recruitment requires a specific steric positioning of amino acid residues in the Smad signal mediators. Strikingly, GTCTG- and GC-rich elements were also found to be crucial for the activation of the Id gene by BMP signaling, but in this case the spacing between the GTCTG- and the GC-rich sites is much larger, and additional factors might be involved in the signal-dependent activation of the Id gene. A more recent study also links these two elements to transcriptional activation of the BMP4 synexpression group in Xenopus. It is tempting to speculate that simple sequence elements similar to the one identified here in several Drosophila genes might be involved in the repression of genes by BMP signaling. Interestingly, human Smad1/5 and Smad4 do form a complex with ShnCT on the Drosophila silencer element from brk; however, a mammalian protein sharing clear homology with Shn in the C-terminal three zinc fingers has not been identified (Pyrowolakis, 2004).
The Dpp-dependent SE allows cells in the developing organism to read out the state of the Dpp signaling pathway. This readout is relatively straightforward because the SE participates in a single switch decision, that is, either to repress (bind Mad/Med and recruit Shn along with its repression domain) or not to repress (not bind Mad/Med, thus failing to recruit Shn). This decision is critically dependent upon one major parameter: the amount of available nuclear Smad complex. For the SE to be functional in vivo, it only needs to interact with a Mad/Med heteromer in those regions of the genome that are actively transcribed; genes that are not active in a given tissue do not need to be repressed by Dpp signaling. This might be one of the main characteristics explaining why such a simple sequence element can have operator-like function in vivo; the element only needs to be recognized by the relevant trans-acting factors in open and active chromatin regions (Pyrowolakis, 2004).
A minimal Dpp-dependent silencer element derived from the brk gene has been identified and demonstrated to function in vivo in a single copy. Its interaction with relevant trans-acting factors have been identified. Based on the results of this analysis, it was possible to derive a consensus sequence, GRCGNCN(5)GTCTG, which allowed scanning of the entire Drosophila genome for potential additional elements. Approximately 350 sites were identified, that, when assayed using transgenic approaches in vivo or in cell culture, should function in a manner analogous to the SEs isolated from the brk regulatory region. Strikingly, and likely significantly, in silico search revealed that the brk gene contains a total of ten SEs, three of them in regions that have been shown to respond to Dpp-dependent repression. Since brk transcription responds to (or can respond to) Dpp signaling in all tissues examined so far, brk might require a SE in the vicinity of each of the different enhancers driving expression in distinct tissues. Alternatively, the readout of the Dpp morphogen gradient might require several SEs, each contributing to the graded repression by Dpp signaling (Pyrowolakis, 2004).
Interestingly, subsequent analysis of two genes containing such Dpp-dependent SEs has demonstrated that these elements function in these transcription units the same way as they do in the brk regulatory region. Therefore, the same molecular principle underlies morphogen readout (brk repression), germline stem cell maintenance (bam repression), and restriction of gene expression to the ventral side of the developing embryo (gsb repression). When the SEs from these three genes are aligned, all the parameters determined to be important for complex formation and for repression are conserved; at all other positions, different base pairs were found in different SEs. In addition, several genes harboring silencer elements are expressed in the wing imaginal disc in a pattern similar to brk or are known to be repressed by Dpp signaling. In contrast, SEs were not found in the vicinity of enhancers known to be activated by Dpp signaling (Pyrowolakis, 2004).
Clearly, these findings implicate that Dpp-induced, Shn-dependent repression via SE elements is a key aspect of development. The readout of the brk gradient contributes to growth and patterning of appendages, and the repression of bam in the germline is essential for the maintenance of germline stem cells. To what extent the repression of gsb contributes to proper cell fate determination along the dorsoventral axis will have to be determined by rescuing the gsb phenotype with a transgene lacking the gsbSE. However, it has been observed that wingless (wg) expression expands from ventral positions to the dorsal side in shn mutant embryos. Since gsb activates wg transcription, the expansion of gsb (in the absence of the gsbSE) possibly leads to the expansion of wg and subsequently to the alteration of dorsoventral cell fate assignments (Pyrowolakis, 2004).
It is important to note that genes repressed by a signaling pathway will not easily be identified in genetic screens because the loss-of-signaling phenotype does not correspond to the loss-of-function phenotype of a repressed gene; in the absence of the signal, such genes are ectopically expressed, leading to a locally restricted gain-of-function phenotype of the corresponding gene. Moreover, since these specific, local patterns of misexpression are likely to result in different phenotypes than widespread overexpression would, simple gain-of-function screens for candidate targets of signal-mediated repression are unlikely to offer straightforward results. Since the target sequence of Dpp/Shn-mediated repression have been identified, the genome can now be scanned and potential target genes can be identified by expression studies and enhancer dissection. It is likely that additional Dpp-repressed genes will be identified using this approach, and this will allow the painting of a much clearer picture of the gene network controlled by Dpp signaling (Pyrowolakis, 2004).
Only a few cases of signal-induced repression have been studied at the molecular level. In most of these cases, repression relies on cooperative action of cell type-specific transcription factors with nuclear signal mediators. The DNA elements that have been demonstrated to mediate repression of particular genes have not been demonstrated to be important for the regulation of other genes, and genome-wide identification of potential target genes using a bioinformatic approach might therefore be difficult, if not impossible (Pyrowolakis, 2004).
The Dpp-dependent repression system identified in this study relies on the organization of Smad binding motifs into Smad/Shn complex-recruiting SEs. The simplicity of these SEs and their capacity to repress transcription in different tissues argues that they function in the absence of tissue-restricted factors. The simple consensus sequence of the SE provides a signature for Dpp-dependent repression, allowing for a genome-wide analysis of potential target genes. Confirmed Dpp-repressed target genes can then be expressed ectopically under the control of the appropriate SE-mutated enhancers to assess the biological importance of repression in a given tissue (Pyrowolakis, 2004).
Phosphorylation of the SSXS motif of Smads is critical in activating the TGF-ß and bone morphogenetic protein (BMP) pathways. However, the phosphatase(s) involved in dephosphorylating and hence inactivating Smads has remained elusive. Through RNA interference (RNAi)-based screening of serine/ threonine phosphatases in Drosophila S2 cells, pyruvate dehydrogenase phosphatase (PDP) was identified as required for dephosphorylation of Mothers against Decapentaplegic (MAD), a Drosophila Smad. Biochemical and genetic evidence suggest that PDP directly dephosphorylates MAD and inhibits signal transduction of Decapentaplegic (DPP). The mammalian PDPs are important in dephosphorylation of BMP-activated Smad1 but not TGF-ß-activated Smad2 or Smad3. Thus, PDPs specifically inactivate Smads in the BMP/DPP pathway (Chen, 2006).
In Drosophila S2 cells, the level of DPP-induced phosphorylation of MAD remains in the continuous presence of DPP. However, upon removal of DPP, the amount of phospho-MAD decreases rapidly while the total protein level of MAD is unchanged. Thus, it was reasoned that phospho-MAD is dephosphorylated once the DPP signal subsides. To identify phosphatase(s) for MAD, a library of double-stranded RNAs (dsRNAs) against all 44 Ser/Thr phosphatases in the Drosophila genome was screened using RNA interference (RNAi). The dsRNA against CG12151 (CG12151-A, targeting the Drosophila PDP) consistently impeded dephosphorylation of MAD. A similar observation was made with a second dsRNA for PDP (CG12151-B) targeting a different region. It was noticed that eventually (i.e., 2 h after removal of DPP), the level of phospho-MAD decreased substantially even when PDP was knocked down. This could reflect activities from residual PDP or other phosphatases that can compensate partially. Another possibility is that if phospho-MAD constitutes a very small fraction of the total MAD, then degradation of phospho-MAD specifically may also result in reduction of phospho-MAD without grossly changing the level of total MAD (Chen, 2006).
In parallel to the decrease in phospho-MAD, the expression of Daughters against Decapentaplegic (DAD), a DPP-dependent target gene, dropped substantially from the peak DPP-induced level 2 h after DPP removal. dsRNA against PDP significantly reduced such decrease in DAD expression, consistent with its ability to delay dephosphorylation of MAD (Chen, 2006).
To further determine the importance of PDP in regulating the C-terminal phosphorylation state of MAD, Drosophila strains carrying genetic lesions that affect the PDP locus on the X chromosome were examined. The strain PBacRBCG12151e02351 (PBacCG12151) harbors a piggyback transposon 7 base pairs upstream of the first exon of the PDP gene. Another strain, Df(1)ct4b1, carries a chromosome deletion that includes the PDP gene. Whole embryos were stained with the PS1 antisera that specifically recognize phospho-MAD in Drosophila embryos. In wild-type blastoderm-stage embryos, phospho-MAD is distributed only in the dorsal-most cells and was predominantly nuclear. In the PBacCG12151 and Df(1)ct4b1 embryos, in addition to the dorsal nuclei, punctate staining was observed for phospho-MAD throughout the embryo. The ectopic PS1 staining was mostly outside the nucleus, and was detected in embryos at most, if not all, developmental stages. In cleavage-stage embryos, which undergo rapid mitosis, aggregated signals were detected for phospho-MAD in PBacCG12151 and Df(1)ct4b1 embryos. Double staining with DAPI showed that in these mutant embryos, phospho-MAD accumulated on both sides of the condensed chromosomes (Chen, 2006).
Genetic analysis showed that the ectopic phosphorylation of MAD is a maternal effect phenotype. All embryos from crosses between heterozygous mutant females and wild-type males displayed phenotypes. When heterozygous males were crossed with wild-type females, no embryos showed any ectopic PS1 staining. Therefore, haploinsufficiency of PDP has a pronounced maternal effect on the level and distribution of phospho-MAD in the early embryos, suggesting that PDP is a critical regulator of the C-terminal phosphorylation state of MAD in vivo (Chen, 2006).
Thickveins (TKV) and Punt are the receptor kinases upstream of MAD that are themselves activated by phosphorylation. Conceivably, phosphatases toward either TKV or Punt could also impact the level of phospho-MAD. It was therefore determined if PDP directly dephosphorylates MAD as its substrate. Recombinant GST-PDP effectively dephosphorylated phospho-MAD, at as low as 0.3 microM. Removal of the GST moiety by thrombin did not affect the efficiency of dephosphorylation. Asp93 in PDP is highly conserved and critical for metal ion chelating. When Asp93 was mutated to Ala, the phosphatase activity toward MAD was largely abolished. Therefore, C-terminally phosphorylated MAD is a bona fide substrate of PDP. In eukaryotes, Ser/Thr phosphatases are grouped into PPP and PPM families. PDPs belong to the PPM family whose catalytic domains are similar to that of PP2C, and pyruvate dehydrogenase was the only known substrate of PDPs. This study has thus identified a novel substrate and function for PDP (Chen, 2006).
The C-terminal phosphorylation is critical for MAD to regulate gene transcription. Therefore whether PDP could inhibit MAD-mediated transcriptional activation was tested. In the Drosophila S2R+ cells, 2xUbx-lacZ, a reporter controlled by a DPP response element from the Ultrabithorax promoter, was activated upon expression of MAD, Medea (MED), Punt, and TKV. The expression of this reporter was significantly repressed when PDP was overexpressed in a dose-dependent manner. Moreover, dsRNA against PDP, but not dsRNAs against PP1-like (CG8822) or PP4-like (CG11597) Ser/Thr phosphatases, enhanced the expression of 2xUbx-lacZ upon activation of MAD. Either overexpression or knockdown of PDP had little effect on the basal 2xUbxlacZ expression. Thus, the level of PDP is an important determinant of the strength of DPP signaling (Chen, 2006).
After DPP stimulation, endogenous phospho-MAD was readily coimmunoprecipitated with V5-tagged PDP. In S2 cells transfected with Flag-MAD and PDP-V5, anti-Flag immunoprecipitation brought down PDP-V5, demonstrating that the coimmunoprecipitation of MAD and PDP works in reciprocal order. Anti-V5 immunoblotting revealed two V5-containing proteins in cells transfected with PDP-V5, suggesting the presence of different forms of PDP. The contribution of C-terminal phosphorylation of MAD in its interaction with PDP was further investigated. Phospho-MAD was bound equally well by wild-type and the D93A mutant form of GST-PDP. Unphosphorylated MAD interacted with PDP. However, when compared with the input, ~3% of phospho-MAD was bound by GST-PDP; while <1% of unphosphorylated MAD was bound. Thus, while not being required for interaction, the C-terminal phosphorylation substantially enhanced MAD interaction with PDP (Chen, 2006).
In live S2 cells, PDP-GFP not only distributed throughout the cell, but also exhibited a punctate pattern that overlapped with mitochondria as revealed by
Mito-Tracker. Such distribution of PDP was not changed upon activation of the DPP pathway. Overexpression of PDP-GFP did not apparently prevent nuclear accumulation of MAD, possibly because the receptor activation overrode PDP activity under the condition or because changes in the kinetics of MAD nuclear import/export require more sensitive and quantitative methods to measure. These observations suggest that PDP has broad subcellular localization and can therefore gain access to substrates in mitochondria, cytoplasm, and nucleus. Indeed, in both the cytosolic and nuclear fractions, coimmunoprecipitation of phospho- MAD and PDP could be detected. Less phospho-MAD was coimmunoprecipitated with PDP in the nuclear fraction, which could be due to the fact that less PDP was present in the nucleus and that the buffer used to extract nuclei contained a higher salt and detergent concentration, which is more stringent for protein-protein interaction (Chen, 2006).
Whether mammalian PDPs has similar functions to their Drosophila counterpart was investigated. Two orthologs of Drosophila PDP, PDP1 and PDP2, have been identified in the human genome, and both share ~40% identity with the Drosophila PDP in amino acid sequences. Coimmunoprecipitation between PDP2 and Smad1 was detected in 293T cells, suggesting that the functional interaction between PDP and Smad is conserved in mammalian cells. To further test this, two siRNA duplexes were designed for each of PDP1 and PDP2; these correspondingly reduced the mRNA levels of PDP1 and PDP2. The TGF-ß receptor kinases can be inhibited by the compound SB431542, while SB202190 was shown to block BMP receptor kinase activity toward Smad1. In HeLa cells, TGF-ß- and BMP2-induced phosphorylation of Smad2/3 and Smad1 decreased considerably after SB431542 or SB202190 treatment, respectively, reflecting dephosphorylation of these Smads. Therefore, these two kinase inhibitors were used to monitor phosphatase activities toward Smad1, Smad2, and Smad3 (Chen, 2006).
Compared with the control, siRNA against PDP1 or PDP2 resulted in reduced Smad1 dephosphorylation. However, the same siRNA treatment did not significantly change the dephosphorylation of Smad2 and Smad3. Even when siRNAs against PDP1 and PDP2 were combined, dephosphorylation of Smad2 and Smad3 was largely unaffected. Moreover, the expression of one BMP target gene, Smad6, was enhanced in cells transfected with siRNA against PDP1 and PDP2, both at the basal state and after BMP2 stimulation. The increase in Smad6 expression without added BMP2 could reflect enhancement of low-level autocrine BMP signaling. These observations suggest that PDP1 and PDP2 are important for dephosphorylation of Smad1. Therefore, the mechanism of Smad dephosphorylation in vertebrates is similar to that in Drosophila. BMP is more closely related to DPP, and Smad1 is more similar to MAD than are Smad2/3. Interestingly PDP1 and PDP2 did not appear to be rate-limiting in dephosphorylation of Smad2 and Smad3. This raised the possibility that different phosphatases are used to inactivate different R-Smads (Chen, 2006).
The activation state of various signal transduction pathways is often dictated by phosphorylation-dephosphorylation control of key signaling molecules. This study reports that PDPs are phosphatases for R-Smads in the DPP pathway in Drosophila and the BMP pathways in mammals. PDP can act to reduce the concentration of phospho-R-Smads in the nucleus and consequently weaken the transcriptional responses to BMP. Moreover, these findings also raised the possibility that MAD may be phosphorylated by kinases outside of the DPP pathway, and the role of PDP is to remove such aberrant phosphorylation and prevent ectopic DPP signaling. TGF-ß/BMP cytokines control the biology of many cell types in different physiological contexts. In addition to the receptors and Smads, other factors must participate to modify and fine-tune the signaling. The identification of phosphatases that inactivate R-Smads provides a new angle to study how TGF-ß/BMP signaling is modulated (Chen, 2006).
Drosophila Nemo is the founding member of the Nemo-like kinase (Nlk) family of serine/threonine protein kinases that are involved in several Wnt signal transduction pathways. Nemo performs a novel function in the inhibition of bone morphogenetic protein (BMP) signaling. Genetic interaction studies demonstrate that nemo can antagonize BMP signaling and can inhibit the expression of BMP target genes during wing development. Nemo can bind to and phosphorylate the BMP effector Mad. In cell culture, phosphorylation by Nemo blocks the nuclear accumulation of Mad by promoting export of Mad from the nucleus in a kinase-dependent manner. This is the first example of the inhibition of Drosophila BMP signaling by a MAPK and represents a novel mechanism of Smad inhibition through the phosphorylation of a conserved serine residue within the MH1 domain of Mad (Zeng, 2007).
This study demonstrates a novel regulatory role for the
Drosophila Nlk family member Nemo in a TGF-ß-superfamily signal
transduction pathway. Evidence is provided that Nemo is an antagonist of BMP
signaling in Drosophila by examining its role in wing development
through genetic analysis and monitoring of BMP-dependent gene expression. The
genetic interaction studies show that phenotypes caused by activation of the
BMP pathway can be suppressed by ectopic nmo and enhanced by loss of
nmo. The data suggest that Nemo participates in the BMP pathway by
modulating Mad activity. This is seen in the inhibition by Nemo of
Mad-dependent gene expression and in the elevated expression of Mad target
genes observed in nmo mutant clones. Nemo can bind to and
phosphorylate Mad and this phosphorylation has direct consequences on the
nuclear localization of Mad in cell culture. The single Nemo target residue maps to serine 25 within the MH1 domain of Mad, a site distinct from those previously implicated in the regulation of Mad activity and nuclear localization (Zeng, 2007).
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. Such nucleocytoplasmic shuttling is observed with R-Smads
participating in both BMP and TGF-ß signaling. The
shuttling provides a tightly regulated mechanism for monitoring the activation
status of the receptors. 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. 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 (Zeng, 2007).
From these findings it is concluded 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 (Zeng, 2007).
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. This research
presents Nemo as the first MAPK-like protein to attenuate Drosophila
BMP pathway activity through phosphorylation of Mad. It has also been found that
murine Nlk can bind to Mad, raising the intriguing
possibility that this mechanism is conserved across species (Zeng, 2007).
MAPK can repress TGF-ß-superfamily signaling by targeting several
Smads. 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. Ras-stimulated Erk also phosphorylates two R-Smads involved in TGF-ß/Activin signaling and prevents their nuclear accumulation.
The phosphorylation sites within these Smads differ, thus providing a
mechanism for preferentially selective inhibition of one subtype. Thus, the
distinct Nemo phosphorylation site in the MH1 domain represents an additional
level of regulation of these proteins (Zeng, 2007).
Interestingly, in these studies, 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 (Zeng, 2007).
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. 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. The
second alpha-helix contains the predicted nuclear localization sequence (NLS). 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 the finding that mutation of serine to alanine renders Mad
constitutively nuclear. Interestingly, a similar constitutively nuclear localization has been observed when the
Erk phosphorylation sites is mutated 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 (Zeng, 2007).
In addition to the biochemical and cell culture evidence that Nemo targets
the MH1 domain of Mad to promote its nuclear export, in vivo
evidence is presented that 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 (Zeng, 2007).
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. Studies with leptomycin B (LMB), which acts to inhibit Crm1-dependent nuclear export of Smads, demonstrate that Nemo can
affect the nuclear localization of Mad. Thus, it is proposed 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 (Zeng, 2007).
It is proposed 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. It is consistently observed that the genotypes in which wing width is
affected do have a mild effect on the spacing of pMad stripes, and it is suggested
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 (Zeng, 2007).
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 (Zeng, 2007).
It has been demonstrated that Nemo can antagonize
Drosophila Wg signaling during wing development. In
this study it was demonstrated 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 (Zeng, 2007).
An intriguing but still incomplete picture is emerging regarding the
regulation of both Nlk expression and activity; this regulation represents a potential
point of crosstalk between signaling pathways. nmo
is transcriptionally regulated by Wg signaling. The kinase activity of Nlk is stimulated by Tak1 after Wnt induction and that Tak1 can be activated by BMP signaling. Activated Nlk can inhibit Tcf/Lef proteins and modulate Wnt-dependent gene expression. In this study, it was 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 (Zeng, 2007).
Cell-to-cell communication at the synapse involves synaptic transmission as well as signaling mediated by growth factors, which provide developmental and plasticity cues. There is evidence that a retrograde, presynaptic transforming growth factor-β (TGF-β) signaling event regulates synapse development and function in Drosophila. This study shows that a postsynaptic TGF-β signaling event occurs during larval development. The type I receptor Thick veins (Tkv) and the R-Smad transcription factor Mothers-against-dpp (Mad) are localized postsynaptically in the muscle. Furthermore, Mad phosphorylation occurs in regions facing the presynaptic active zones of neurotransmitter release within the postsynaptic subsynaptic reticulum (SSR). In order to monitor in real time the levels of TGF-β signaling in the synapse during synaptic transmission, a FRAP (fluorescence recovery after photobleaching) assay was established to measure Mad nuclear import/export in the muscle. Mad nuclear trafficking is shown to depend on stimulation of the muscle. These data suggest a mechanism linking synaptic transmission and postsynaptic TGF-β signaling that may coordinate nerve-muscle development and function (Dudu, 2006).
Molecular signals are often transmitted in the reverse direction across the synapse to regulate the structure and function of the presynaptic neuron. There is now good evidence that BMP-type TGF-β ligands are essential retrograde signaling ligands in the Drosophila nervous system and that they control synaptic growth, synaptic function, and in some cases the specificity of the particular neurotransmitter released at the synaptic terminal. These data show that there is an additional postsynaptic TGF-β signaling cascade at the NMJ that is consistent with anterograde and/or autocrine signaling. Four lines of evidence support the existence of a postsynaptic signaling event: (1) core transduction components of the BMP-type signaling pathway, such as the receptor Tkv and the transcription factor Mad, are present in the muscle where they accumulate in the subsynaptic reticulum (SSR), (2) phosphorylation of Mad takes place in the PSD, opposite to the presynaptic active zones where exocytosis of neurotransmitter takes place, (3) a FRAP-based assay that monitors signaling-dependent nuclear import/export and nuclear retention of Mad revealed that Mad-mediated signaling can take place in the muscle, and (4) upon stimulation of the muscle, Mad targeting to the terminal is enhanced and the levels of postsynaptic Mad signaling are increased, as monitored with the FRAP assay (Dudu, 2006).
Taken together, these data are consistent with the existence of an anterograde signaling event that is initiated in the presynaptic terminal through exocytosis of a TGF-β-type ligand. Binding of the ligand to the Tkv receptor at the PSD would then lead to activation of the receptor and thereby phosphorylation of Mad. Phosphorylation of Mad causes a decrease in the nuclear export of the transcription factor and thereby its accumulation in the muscle nuclei. Nuclear P-Mad in turn mediates transcriptional control of the target genes of the signaling pathway. Thus, while retrograde BMP signaling instructs the neurons about the physiological and developmental state of the muscle, such an anterograde signaling event may provide the muscle with information about the activity of the neuron in the medium/long term (Dudu, 2006).
However, these observations do not rule out an autocrine BMP signaling event: a ligand (perhaps, again, Gbb) released from the muscle at the synaptic site could activate signaling in the muscle itself. Clearly, further analysis needs to be carried out to discriminate between these two possibilities (Maitra, 2006).
A FRAP assay was established to determine in real time the state of activation of TGF-β pathway in the muscle. This assay is useful to study rapid events such as synaptic transmission, where it is desirable to capture fast changes in the state of signaling. The FRAP experiments allow determination the rates of nuclear import/export of Mad and the size of the nuclear Mad pool that does not exchange (or exchanges slowly) with the cytosol: the immobile fraction. Experiments in which the levels of signaling are affected reveal that both the effective import/export rates and the size of the immobile fraction correlate with the signaling state: higher levels of signaling can be associated with a larger Mad immobile pool in the nucleus and higher import-to-export ratio (Dudu, 2006).
What is the molecular meaning of the correlation between signaling and the immobile pool? It is first considered that the nuclear P-Mad binds/unbinds to the DNA with rates that are slower than other rates during signaling (phosphorylation/dephosphorylation, import/export) and does not significantly contribute to the recovery in the 1000 s time of the experiments. Bound to the DNA, P-Mad could not exchange with the cytosol, and the relatively slow rates of binding/unbinding would generate an immobile fraction as shown in the FRAP experiment. Further experiments, however, did not support this hypothesis. After bleaching GFP-Mad in a small region within the nucleus in a TkvQD-expressing muscle (where 67% of the nuclear Mad does not exchange with the cytosol), the fluorescence fully recovered within this bleached region, revealing that Mad is not immobilized within the nucleus. Is then the DNA moving within the nucleus? FRAP experiments with Histone2A-GFP discard the possibility that the DNA in the nucleus is moving rapidly and show that it remains immobile during the time scale of the FRAP experiments (Dudu, 2006).
What could then explain the correlation between signaling and immobile pool? It is speculated that P-Mad associates with other cofactors in the nucleus with slow binding and unbinding rates. Engaged in a macromolecular complex, P-Mad would be unable to exit the nucleus through the nuclear pore and thereby would generate the immobile pool revealed in the FRAP experiments (Dudu, 2006).
Retrograde signaling has been proposed to convey information to the neuron about the development of the muscle, so that the presynaptic terminal and the muscle grow synchronously and the increase of the SSR is coordinated with the appearance of new boutons and active zones. What could then be the role of anterograde Mad signaling in the muscle? It is speculated that upon synaptic transmission, a quantum of neurotransmitter is released together with a “quantum” of growth factor from the presynaptic vesicles. If this is true, postsynaptic signaling coupled to synaptic activity could endow the muscle with information about the activity of the neuron and thereby control the growth and development of the NMJ. It will be interesting, therefore, to study at the ultrastructural level whether the same vesicles that contain the neurotransmitter contain the TGF-β growth factor (Dudu, 2006).
These data show that postsynaptic TGF-β signaling occurs at the NMJ. Furthermore, the data suggest a coupling between muscle stimulation and postsynaptic TGF-β signaling. Such coupled processes may confer information to the muscle about the activity of the neuron during development (Dudu, 2006).
A screen for modifiers of Dpp adult phenotypes led to the identification of the Drosophila homolog of the Sno oncogene (dSno; termed snoN in FlyBase). The SnoN locus is large, transcriptionally complex and contains a recent retrotransposon insertion that may be essential for SnoN function. This is an intriguing possibility from the perspective of developmental evolution. SnoN is highly transcribed in the embryonic central nervous system and transcripts are most abundant in third instar larvae. SnoN mutant larvae have proliferation defects in the optic lobe of the brain very similar to those seen in baboon (Activin type I receptor) and Smad2 mutants. This suggests that SnoN is a mediator of Baboon signaling. SnoN binds to Medea and Medea/SnoN complexes have enhanced affinity for Smad2. Alternatively, Medea/SnoN complexes have reduced affinity for Mad such that, in the presence of SnoN, Dpp signaling is antagonized. It is proposed that SnoN functions as a switch in optic lobe development, shunting Medea from the Dpp pathway to the Activin pathway to ensure proper proliferation. Pathway switching in target cells is a previously unreported mechanism for regulating TGFß signaling and a novel function for Sno/Ski family proteins (Takaesu, 2006).
Studies in mammalian cells showed that, when overexpressed, Sno is an antagonist of TGFß/Activin signaling. Overexpression of dSno with A9.Gal4 (throughout the presumptive wing blade) resulted in small wings with multiple vein truncations at 100% penetrance. A9.Gal4:UAS.dSno pupal wing discs were examined for Drosophila serum response factor (dSRF) expression, an intervein marker repressed by Dpp signaling. In A9.Gal4:UAS.dSno pupal discs, dSRF expression is highly irregular with no obviously downregulated regions corresponding to vein primordia. These wing and disc phenotypes are strongly reminiscent of those expressing the dominant-negative allele Mad1 (DNA binding defective but competent to bind Medea) with a variety of drivers, including A9.Gal4 (100% penetrant) and 69B.Gal4. Mad1 dominant-negative effects are due to the titration of Medea into nonfunctional complexes. The similarity of dSno and Mad1 phenotypes suggests that overexpression of dSno antagonizes BMP signaling (Takaesu, 2006).
This was further tested this by coexpressing dSno with Medea or Mad or dSmad2. Coexpression of dSno with Medea or Mad rescues the dSno phenotype to nearly wild type in size and vein pattern. In dSno and Medea coexpressed wings, reduced size was completely eliminated and multiple vein defects were reduced to 28% penetrance. In dSno and Mad coexpressed wings, reduced size was completely eliminated and multiple vein defects were reduced to 19% penetrance. Alternatively, coexpression of dSno with dSmad2 has little effect on the dSno phenotype. In dSno and dSmad2 coexpressed wings, reduced size and multiple vein defects remained 100% penetrant. Coexpression of Mad1 and dSno significantly enhanced the dSno phenotype. One hundred percent of Mad1 and dSno coexpressing the wings are more abnormal than those expressing either dSno or Mad1. The coexpressing wing is very small and veinless and resembles wings expressing UAS.Dad (Dpp antagonist) or dpp class II disc mutants (e.g., dppd5). The enhanced phenotype suggests that dSno and Mad1 antagonize BMP signaling in distinct ways that have additive effects (Takaesu, 2006).
Experiments with a constitutively activated form of the Dpp type I receptor Thickveins (CA-Tkv) are also consistent with this hypothesis. One hundred percent of A9.Gal4:UAS.CA-Tkv wings are overgrown and have numerous ectopic veins as well as vein truncations. This phenotype is suppressed in 98% of the individuals when UAS.dSno is coexpressed with UAS.CA-Tkv. In fact, the coexpression phenotype is not much different from A9.Gal4:UAS.dSno alone, indicating that dSno antagonism of Dpp signaling is fully epistatic to activated Tkv. Finally, ubiquitous overexpression of dSno in the embryonic ectoderm with 32B.Gal4 resulted in discless larvae—a phenotype seen in Mad and Medea null genotypes and in dpp class V disc mutants ( e.g., dppd12. It is concluded that overexpression of dSno antagonizes BMP signaling (Takaesu, 2006).
In Drosophila, as in vertebrates, two TGFß subfamilies are present. The
bone morphogenetic protein (BMP) subfamily member Dpp signals through its type I receptor Thickveins to its dedicated transducer Mad (Smad1 homolog) and the Co-Smad Medea (Smad4 homolog). The TGFß/Activin subfamily member activin signals through its type I receptor Baboon to its dedicated transducer dSmad2 and Medea. This study shows that dSno binds Medea and then functions as a mediator of Activin signaling by enhancing the affinity of Medea for dSmad2. Antagonism for BMP signaling likely arises as a secondary consequence of dSno overexpression. Examination of dSno loss-of-function mutants shows that dSno is required in cells of the optic lobe of the brain to maintain proper rates of cell proliferation. Given that Dpp signaling is essential for neuronal differentiation in the optic lobe, these data suggest that dSno functions as a switch that shunts Medea from the Dpp pathway to the Activin pathway to ensure a proper balance between differentiation and proliferation in the brain (Takaesu, 2006).
To resolve the apparent contradiction between the effect on signaling of dSno overexpression (antagonism) and the role identified in dSno mutants (mediation), dSno was examined biochemically. Expression constructs encoding Flag or T7 epitope-tagged Medea, Mad, and dSmad2 were used and various combinations were co-expressed in COS1 cells. It was possible to clearly detect interaction of Medea with both dSmad2 and Mad by co-immunoprecipitation. A T7-tagged dSno expression construct was generated and coexpressed with Flag-dSmad2, Mad, or Medea or with a control vector. Complexes were isolated on Flag agarose and analyzed for the presence of coprecipitating dSno by T7 Western blot. T7-dSno was readily detectable in complexes isolated from cells expressing Medea, but not Mad or dSmad2 (Takaesu, 2006).
Since Medea is a shared partner for both Mad and dSmad2, whether co-complexes containing Medea and dSno together with either Mad or dSmad2 was tested. COS1 cells were transfected with T7-dSno and Flag-Mad or dSmad2 with or without T7-Medea. T7-dSno was present in a complex with Flag-dSmad2 only when T7-Medea was also present. Interestingly, approximately equal amounts of Medea and dSno appeared to coprecipitate with dSmad2, suggesting that much of the Medea that interacts with dSmad2 in this assay is also bound to dSno. In contrast, dSno was not detected in complex with Mad, even when Medea was present, even though Medea clearly interacted with Mad in this assay. These results suggest that dSno interacts specifically with Medea and that the dSno-Medea complex can interact with dSmad2 but not with Mad (Takaesu, 2006).
To test whether incorporation of dSno affected formation of the Medea-dSmad2 complex, Flag-Medea and T7-dSmad2 were coexpressed with or without dSno. The amount of dSmad2 that coprecipitated with Flag-Medea was clearly increased in the presence of dSno. In the reverse of this experiment, it was also observed that an increase in T7-tagged Medea present in Flag-dSmad2 precipitates when dSno was coexpressed. These results suggest that dSno may promote the formation of Medea-dSmad2 complexes (Takaesu, 2006).
To test whether dSno has any effect on the formation of Mad-Medea complexes, similar experiments were performed in which Flag-Medea and Western blotted was isolated for coprecipitating T7-Mad or dSmad2 in the presence or absence of coexpressed dSno. The interaction of Medea with Mad was more readily detectable than with dSmad2. However, inclusion of dSno again increased the interaction between Medea and dSmad2. In contrast, no increase was seen in the Medea–Mad interaction when dSno was coexpressed and it appeared that increasing dSno expression decreased the amount of Mad that coprecipitated with Flag-Medea. Thus it appears that dSno not only may promote interaction of Medea with dSmad2, but also may compete with Mad for Medea interaction, suggesting that dSno may play a role in determining the pathway specificity of Medea (Takaesu, 2006).
Nuclear translocation of Smad proteins is a critical step in signal transduction of transforming growth factor β (TGF-β) and bone morphogenetic proteins (BMPs). Using nuclear accumulation of the Drosophila Mad as the readout, a whole-genome RNAi screening was carried out in Drosophila cells. The screen identified moleskin (msk) as important for the nuclear import of phosphorylated Mad. Genetic evidence in the developing eye imaginal discs also demonstrates the critical functions of msk in regulating phospho-Mad. Moreover, knockdown of importin 7 and 8 (Imp7 and 8), the mammalian orthologues of Msk, markedly impaired nuclear accumulation of Smad1 in response to BMP2 and of Smad2/3 in response to TGF-β. Biochemical studies further suggest that Smads are novel nuclear import substrates of Imp7 and 8. Thus, evolutionarily conserved proteins have been identified that are important in the signal transduction of TGF-ß and BMP into the nucleus (Xu, 2007).
Genome-wide RNAi screening in this study offers a genetic approach to uncover new elements in TGF-ß signal transduction. Msk and its mammalian orthologues Imp7 and 8 are critical components in transporting TGF-ß-activated Smads into the nucleus. Biochemical evidence further suggests that Msk/Imp7/8 directly import phospho-Smads as cargoes (Xu, 2007).
Although there appears to be some discrepancy between these new findings and previous reports that importins are dispensable for the nuclear import of Smads, these observations can be reconciled. The present and previous studies, based on different approaches, may have revealed different nuclear import mechanisms used by basal and activated Smads to enter the nucleus. There are important differences comparing Smads import with or without TGF-ß stimulation. Unphosphorylated Smads are monomers, but phosphorylated Smads are assembled into complexes with Smad4 and are thus much larger in size. Moreover, as phospho-Smads accumulate in the nucleus they have to move across the nuclear pore against an ascending concentration gradient of Smads already in the nucleus, whereas unphosphorylated Smads never reach a higher concentration in the nucleus than in the cytoplasm. Thus, importing phospho-Smad complexes and unphosphorylated Smad monomers may entail different mechanisms, with or without the participation of importins. Indeed, RNAi data in both Drosophila and mammalian cells suggest that nuclear import of the two forms of Smads is very different regarding the requirement of Msk/Imp7/8. This type of differential requirement for import factors is not unique to Smads. In fact, STATs (signal transducers and activators of transcription) in the interferon pathway are another example in which the latent STATs are imported by an importin-independent mechanism, whereas the phosphorylated STATs depend on importins to accumulate in the nucleus. It is also interesting to note that phospho-Smads were still detected in the nucleus upon RNAi-mediated knockdown of Msk/Imp7/8. Although the trivial explanation that this may be due to incomplete depletion of the targeted proteins cannot be ruled out, this observation may also suggest additional import mechanisms for activated Smads. It is recognized that the previous finding of importin-independent nuclear import of Smads was largely based on an in vitro reconstituted nuclear import assay. Although this in vitro system is widely accepted, it may not fully recapitulate nuclear import of activated Smads in cells. Based on RNAi data, regarding the requirement of importins, the conclusion drawn from the in vitro import assay may not apply to phospho-Smads in intact cells. However, the current study does not necessarily contradict the previous suggestions that direct Smad-nucleoporin interaction is critical for nuclear import of Smads (Xu, 2007).
The data showed that Msk/Imp7/8 interacted with Smads regardless of their phosphorylation status; thus, additional factors must be involved to explain why only TGF-ß/BMP-activated Smads can accumulate in the nucleus. Because basal-state Smads are actively exported out of the nucleus, it is possible that retaining only phospho-Smads in the nucleus requires blocking Smads nuclear export, a scenario that has been demonstrated for Smad4. This hypothesis would be consistent with findings in live cells, in which TGF-ß signaling led to reduced mobility of Smad2 in the nucleus (Xu, 2007).
Because Msk, Imp7, and Imp8 are shown to be critical for targeting phospho-Smads into the nucleus, it is conceivable that regulatory inputs to this nuclear import factor would impact TGF-ß signaling. Although no changes were observed in subcellular localization of Msk or Imp7/8 in response to TGF-ß in cultured cells, during Drosophila embryonic development, Msk distribution changed between cytoplasm and nucleus in a dynamic fashion. Moreover, Msk is phosphorylated on tyrosine residues with yet-unknown functional consequences. If and how Msk localization is regulated and by what signals are completely open questions at present (Xu, 2007).
A number of mitogen-induced phosphorylation events in the linker region of Smad have been suggested to inhibit TGF-ß-induced nuclear translocation of Smads in Xenopus and mammalian cells. Because part of the Imp7/8 binding was mapped to the linker region of Smad3, it will be interesting to determine if linker phosphorylation would affect the interaction between Smads and Imp7/8 and hence the rate of nuclear import. It is also worth noting that Msk has been genetically implicated in the nuclear import of activated ERK in Drosophila. Such convergence on the same molecule for nuclear import raises the possibility of cross-talk between MAP kinase and TGF-ß pathways at the level of nuclear translocation of key signal transducers (Xu, 2007).
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