dSmurf Selectively Degrades Decapentaplegic-activated MAD, and Its Overexpression Disrupts Imaginal Disc Development*
Yao-Yun Liang
,
Xia Lin
,
Min Liang
,
F. Charles Brunicardi
,
Peter ten Dijke ¶,
Zhihong Chen
,
Kwang-Wook Choi
|| ** and
Xin-Hua Feng

From the
Michael E. DeBakey Department of Surgery,
Department of Molecular & Cellular Biology,
||Ophthalmology, **Program
of Developmental Biology, Baylor College of Medicine, Houston, Texas 77030 and
the ¶Division of Cellular Biochemistry, The
Netherlands Cancer Institute, 1066 Cx Amsterdam, the Netherlands
Received for publication, January 21, 2003
, and in revised form, May 12, 2003.
 |
ABSTRACT
|
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MAD plays an important role in decapentaplegic (DPP) signaling throughout
Drosophila development. Despite a recent study describing the
restriction of DPP signaling via putative ubiquitin E3 ligase dSmurf
(1), the molecular mechanisms
of how dSmurf affects DPP signaling remain unexplored. Toward this goal we
demonstrated the degradation of phosphorylated MAD by dSmurf. dSmurf
selectively interacted with MAD, but not Medea and Dad, and the MAD-dSmurf
interaction was induced by constitutively active DPP type I receptor
thickveins. Wild type dSmurf, but not its C1029A mutant, mediated
ubiquitination-dependent degradation of MAD. Silencing of dSmurf using RNA
interference stabilized MAD protein in Drosophila S2 cells. Targeted
expression of dSmurf in various tissues abolished phosphorylated MAD and
disrupted patterning and growth. In contrast, similar overexpression of
inactive dSmurf(C1029A) showed no significant effects on development. We
conclude that dSmurf specifically targets phosphorylated MAD to
proteasome-dependent degradation and regulates DPP signaling during
development.
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INTRODUCTION
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Decapentaplegic
(DPP)1 is the
Drosophila ortholog of human bone morphogenetic protein (BMP) 2/4,
which belongs to the transforming growth factor
(TGF-
)
superfamily
(24).
DPP plays a central role in Drosophila morphogenesis. DPP signals
through cell surface receptors and intracellular Smad proteins. In
Drosophila, all three classes of Smads, R-Smad (MAD, dSmad2), Co-Smad
(Medea), and I-Smad (Dad), have been characterized
(2). MAD, the founding member
of Smad family (5), transduces
the signal of DPP, while dSmad2 mediates TGF-
/Activin signaling
(2).
Ubiquitin conjugation is catalyzed by an enzymatic cascade including
ubiquitin-activating E1, ubiquitin-conjugating E2, and specific ubiquitin E3
ligase. The specificity of ubiquitination derives from the highly specific
protein-protein interaction between E3 ligase and substrate
(6,
7). In TGF-
signaling,
HECT-class E3 ligases Smurf1 and Smurf2 specifically promote degradation of
Smad1, Smad2
(810)
and possibly I-Smads and TGF-
type I receptor
(11,
12). In Xenopus
embryos, Smurf1 showed inhibition of BMP signaling and affected pattern
formation (8). Recently, a
Drosophila ligase E3 dSmurf has been identified by genetic screen
(1); the null mutation of
dSmurf resulted in a lethal defect in hindgut organogenesis. However, the
mechanisms underlying dSmurf actions in DPP signaling and embryogenesis remain
unclear.
In this study, we present biochemical and genetic evidence for the
biological functions of dSmurf. The results revealed that dSmurf specifically
inhibited DPP signaling by targeting DPP-activated MAD, not dSmad2, Medea, or
Dad, for ubiquitination/proteasome-dependent proteolysis. Targeted
overexpression of dSmurf in imaginal discs strongly reduces the level of
phosphorylated MAD and consequently causes disruption of DPP-dependent
patterning of imaginal discs, demonstrating that dSmurf is a negative
regulator of DPP signaling in vivo.
 |
EXPERIMENTAL PROCEDURES
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Molecular CloningdSmurf were obtained by using PCR with
primers (forward: gGAATTC aat aaa ttg gat tac cca cgt and
reverse: ttcGTC-GAC tta ctc cac ggc aaa tcc aca) from total
fly cDNA (Clontech) or Lack cDNA clone (gift of Scott Hawley), and cloned into
the EcoRI and SalI of pXF1F or pXF1M, for making FLAG-tagged
or Myc-tagged dSmurf, respectively. His-tagged MAD is made by subcloning into
a mammalian expression vector pXF2RH
(9). The point mutation of
dSmurf of C1029A substitution was generated from two-step PCR. MAD(2SA) mutant
was made by mutating the two Ser into Ala in the SVS phosphorylation
motif.
Yeast Two-hybrid AssayA LexA-based yeast two-hybrid assay
was performed as described previously
(9). MAD, dSmad2, and Dad were
obtained by PCR from respective cDNA (gifts of Kohei Miyazono and Richard
Padgett) and cloned into a yeast bait vector pEG202. dSmurf wild type and the
dSmurf deletion mutants, obtained by PCR, were cloned into prey vector
pJG45 at sites of EcoRI and SalI. Smurf1 (gift of
Jeff Wrana) was also subcloned into pJG45.
-Galactosidase
activity is measured by liquid assay according to the method of Matchmaker
two-hybrid system 2 (Clontech).
Immunoprecipitation and Western BlotCulture and
transfection of human kidney 293T cells and Drosophila Schneider S2
cells, and coupled immunoprecipitation-Western blot assays were carried out as
described previously (13,
14).
For degradation assay, copper-inducible pMK33 expression plasmids for MAD,
dSmurf, and the constitutively active type I receptor Thickveins (caTKV)
(gifts of Richard Padgett) were transfected into Schneider S2 cells by calcium
phosphate. In the case of RNA interference experiment
(Fig. 4D), cells were
cotransfected with synthetic 21-nucleotide duplex RNA (double-stranded RNA) of
dSmurf (15). Cells were
induced by 0.5 mM CuSO4 for 24 h and then harvested for
Western blot analysis.

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FIG. 4. dSmurf degrades MAD proteins in cells. A, dSmurf induces
proteasome-dependent degradation of MAD. Drosophila S2 cells were
transfected with indicated MAD, with or without Myc-tagged dSmurf and treated
with MG132 as indicated. Cell lysates were subjected to SDS-PAGE and Western
blotting analysis using anti-MAD and anti-Myc antibodies. Note that caTKV
further promoted dSmurf-induced MAD degradation (lane 4). B,
MAD degradation requires the HECT catalytic activity of dSmurf. 293T cells
were transfected with expression plasmids for FLAG-tagged MAD and increasing
doses of Myc-dSmurf (wild type or C1029A mutant). Cell lysates from
transfected cells were subjected to anti-FLAG Western blot to detect MAD and
anti-Myc immunostaining to detect dSmurf. C, depletion of dSmurf
expression stabilizes MAD. Drosophila S2 cells were transfected with
Myc-tagged dSmurf and MAD and treated with increasing doses (20, 40, and 100
nM) of siRNA against dSmurf as indicated. Cell lysates were
subjected to SDS-PAGE and Western blotting analysis using anti-MAD and
anti-Myc antibodies. D, depletion of dSmurf expression stabilizes
pMAD. Drosophila S2 cells were transfected with caTKV and treated
with 100 nM si-dSmurf. Level of phospho-MAD was analyzed by Western
blotting using anti-P-Smad1 antibody.
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For ubiquitination assay, 293T cells were cotransfected by His-tagged MAD,
HA-tagged ubiquitin, and c-Myc-tagged dSmurf. Proteasome inhibitor MG-132 was
added to the transfected cells to prevent degradation of MAD. 48 h after
transfection, cell lysates were subjected to precipitation using
nickel-NTA-agarose beads and to detect ubiquitinated MAD as described
previously (9).
Targeted Expression and ImmunocytochemistrycDNAs for dSmurf
and dSmurf(C1029A) were cloned in pUAST-FLAG, a modified
pUAST containing a FLAG tag sequence to induce FLAG-tagged
proteins.2 Transgenic
flies carrying UAS-dSmurf and UAS-dSmurf (C1029A)
were generated by P-element-mediated germline transformation
(16). For targeted expression,
UAS-dSmurf flies were crossed to Gal4 strains. For
immunocytochemistry, imaginal discs from the progeny
Gal4>UAS larvae were double stained with mouse anti-Flag
(1:250) and rabbit anti-pMAD (1:250) with fluorescein isothiocyanate- and
Cy3-conjugated fluorescent secondary antibodies as described previously
(17).
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RESULTS
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WW2/WW3 Domains of dSmurf Meditates Its Specific Interaction
with MADBy homology probing, we identified dSmurf cDNA that
encodes a polypeptide of 1061 amino acid. dSmurf belongs to the HECT-class
ubiquitin E3 ligase family (Fig.
1A) and displays a close homology to human Smurf2
(911)
and Smurf1 (8). During the
course of this study, Podos et al.
(1) also identified dSmurf in a
genetic screen (1). As a first
step to determine whether dSmurf modulates DPP signaling in
Drosophila, we investigated the physical interaction of this putative
ubiquitin E3 ligase with members of the Smad family in Drosophila. We
found that dSmurf only interacted with MAD, but not with dSmad2, Medea, or
Dad. (Fig. 1B).
Therefore, unlike its mammalian homologs Smurf1/2 that bind to both R-Smads
and I-Smads, dSmurf specifically interacts with DPP-specific MAD.
To locate the interacting domains of dSmurf, we generated deletion and
point mutations in dSmurf and assessed their interaction of MAD. The result
showed that the entire WW2/WW3 region of dSmurf (amino acids 513602)
was required for the interaction with MAD, whereas the WW2 or WW3 domain alone
did not interact with MAD (Fig.
1C). Notably, the same WW2/WW3 region with point
mutations in WW2 domain (W541F/P544A) completely lost the interaction with
MAD. These data suggest that the interaction of dSmurf binds to MAD through
the WW2/WW3 domain.
DPP Receptor Activation Augments dSmurf-MAD InteractionWe
next assessed the MAD-dSmurf interaction in vivo. To prevent MAD
degradation, we used the C1029A mutant of dSmurf to determine its interaction
with Smads. The C1029A mutant carries a cysteine-to-alanine substitution at
amino acid residue 1029 that is essential for catalysis. As shown in
Fig. 2A, dSmurf
coimmunoprecipitated with MAD, but not with dSmad2, Medea, and Dad, in
agreement with the interaction pattern from yeast two-hybrid analysis. caTKV,
constitutively active form of type I receptor Thick-veins
(18,
19), enhanced the interaction
between MAD and dSmurf (Fig. 2, A
and B).

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FIG. 2. DPP stimulates dSmurf-MAD interaction. A, dSmurf binds to
MAD in cells. 293T cells were transfected with plasmids for FLAG-tagged Smad
(MAD, dSmad2, Medea, or Dad) and Myc-tagged dSmurf(C1029A). dSmurf complex was
immunoprecipitated using anti-Myc antibody, followed by a Western blot to
detect dSmurf-associated MAD. Expression levels of dSmurf and Smads in whole
cell lysates were shown as indicated. Asterisk indicates a
nonspecific band close to the molecular mass of Medea. Note that dSmurf-MAD
interaction is stronger in the presence of constitutively active TKV (compared
lane 7 with lane 2). B, mutation in the C-terminal
phosphorylation sites of MAD abolishes dSmurf-MAD interaction. 293T cells were
transfected with indicated plasmids. Immunoprecipitation-Western blotting was
similarly carried out to A, except that anti-MAD (Smad1, Santa Cruz
Biotechnology) antibody was used for immunoprecipitation and anti-FLAG
(dSmurf) for Western blotting to detect MAD-bound dSmurf. MAD(SA) contains
point mutations at Ser-453 and Ser-455 into Ala. Expression levels of dSmurf
and MAD in whole cell lysates were shown as indicated. Level of pMAD in the
immunoprecipitate was also shown. C, endogenous MAD binds to dSmurf.
GST-dSmurf-(513612) was used to pull down endogenous MAD from S2 cell
lysates, transfected with caTKV and/or treated with proteasome inhibitor
MG-132 (as indicated). dSmurf-bound MAD was detected by anti-Smad1 (upper
panels) or anti-phospho-Smad1 (lower panels) antibodies.
NS, a nonspecific band. D, dSmurf interacts with activated
dSmad2. 293T cells were transfected with indicated plasmids, and
immunoprecipitation-Western blotting was similarly carried out as described in
the legend to A. caActRIB, constitutively active ActRIB.
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To examine the importance of the phosphorylation motif SSXS, we
generated double mutations in MAD at Ser-433 and Ser-435, which were changed
into Ala and presumably unphosphorylatable by TKV. Result in
Fig. 2B clearly showed
that the interaction between this mutant, named MAD(SA), and dSmurf was not
detectable regardless of the presence of caTKV, whereas wild type MAD strongly
interacted with dSmurf in the presence of caTKV. Furthermore, dSmurf also had
stronger binding to endogenous phospho-MAD, which can be further enhanced by
proteasome inhibitor MG-132 (Fig.
2C). Interestingly, dSmurf also interacts with dSmad2 in
the presence of caActR1B (Fig.
2D), suggesting that dSmad2 phosphorylation is necessary
for its binding to dSmurf (despite the absence of such interaction in yeast
two-hybrid assay as shown in Fig.
1). Therefore, data presented in
Fig. 2 clearly demonstrate the
important involvement of SSXS phosphorylation in Smad interaction
with dSmurf.
dSmurf Mediates Ubiquitination of MADTo dissect the
function of dSmurf as a ubiquitin E3 ligase, we tested whether dSmurf
stimulated the ubiquitination of MAD. As shown in
Fig. 3, wild type dSmurf was
capable of enhancing ubiquitination of MAD protein (lane 3), while no
ubiquitination was observed with MAD alone (lane 1). Markedly, the
C1029A mutant of dSmurf could not conjugate HA-ubiquitin to MAD (lane
4). This suggests that the catalytic activity of dSmurf is required for
the ubiquitination of MAD.

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FIG. 3. dSmurf ubiquitinates MAD. 293T cells were transfected with
expression plasmids for His-tagged MAD, HA-tagged ubiquitin, and Myc-dSmurf.
His-MAD was pulled down from cell lysates using nickel-NTA-agarose beads
(Ni-NTA ppt) and then subjected to SDS-PAGE and Western blotting
analysis using anti-HA antibodies to detect the pool of ubiquitinated MAD
(upper). Levels of MAD in cells were detected using anti-His antibody
(bottom). dSmurf(CA) is the catalytically inactive mutant of dSmurf
with C1029A point mutation. Ubiquitinated MAD is marked.
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dSmurf Promotes Proteolytic Degradation of MADHaving
established that dSmurf mediated MAD ubiquitination, we next examined the MAD
degradation in Drosophila S2 and human 293T cells. In S2 cells, the
MAD protein level was reduced dramatically by overexpressed dSmurf
(Fig. 4A). Similarly,
dSmurf degraded MAD in 293T cells in a dose-dependent manner
(Fig. 4B). In sharp
contrast, the C1029A mutant did not decrease the steady state level of MAD
(Fig. 4B).
We next examined the role of phosphorylation of MAD in dSmurf-mediated
degradation. It was clear that activated type I receptor further enhanced the
degradation of MAD protein in dSmurf-overexpressing cells
(Fig. 4A, compare
lane 4 with lane 3), consistent with increased
phosphorylation level (data not shown). Most notably, addition of proteasome
inhibitor MG-132 inhibited MAD degradation caused by dSmurf
(Fig. 4A, lane
5).
We then took a loss-of-function approach to examine the MAD degradation by
depleting the dSmurf expression. S2 cells were transfected with MAD and
Myc-dSmurf and treated with or without a 21-nucleotide double-stranded RNA
that targets dSmurf (si-dSmurf). We could detect more MAD protein with
increased concentration (20, 40, and 100 nM) of the siRNA
(Fig. 4C).
Simultaneously, reduced dSmurf protein level was detected by anti-Myc antibody
(lower panel, Fig.
4C). Importantly, an increased endogenous phospho-MAD
protein level was detected after 100 nM si-dSmurf treatment
(Fig. 4D), suggesting
the silencing of endogenous dSmurf by si-dSmurf.
Overexpression of dSmurf Leads to Loss of Phospho-MAD and Disruption of
Imaginal Disc DevelopmentDPP signaling is essential for patterning
of imaginal discs. DPP is expressed along the anterior-posterior border of the
wing disc and activated phospho-MAD is expressed in wider area around the AP
border. Using UAS-Gal4 targeted expression system
(20), we tested whether
overexpression of dSmurf along the AP border can reduce or abolish
phospho-MAD. As expected, when dSmurf expression was targeted to the AP border
using ptc-Gal4, there was a strong reduction of phospho-MAD level in
the ptc expression domain (Fig.
5). ptc-Gal4;UAS-dSmurf (ptc>dSmurf)
flies failed to eclose due to severe developmental defects. Pharate adult
flies removed from the pupal case showed rudimentary wings
(Fig. 5L) and
truncated legs (not shown), indicating strong reduction of DPP signaling. In
contrast, overexpression of catalytically inactive dSmurf in
ptc>dSmurf(C1029A) showed no significant defects in
appendages. Wing discs from ptc>dSmurf(C1029A) showed
slight increases in the level of phospho-MAD
(Fig. 5, EG),
suggesting that dSmurfCA may titrate the function of normal
dSmurf.

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FIG. 5. dSmurf degrades MAD in vivo and disrupts wing disc
development. Wing discs from wild-type (AD),
ptc>dSmurf(C1029A) (EH), and
ptc>dSmurf (IL) were stained with
anti- -galactosidase (red) and anti-pMAD (green).
A, pMAD is distributed in the medial wing pouch region of the normal
disc. Its level is lower in the Ptc domain at the anterior-posterior
(AP) border (arrowhead). B and C,
schematic of Ptc (red) and pMAD (green) domains in the wing
disc. A and P indicate anterior and posterior, respectively.
D, wild type adult wing. EG, overexpression of
dSmurf(C1029A) using ptc-Gal4 causes no decrease in pMAD level.
Instead, pMAD level is slightly increased in the Ptc domain (arrow,
compare the Ptc domains in A and E). H, wings from
ptc> dSmurf(C1029A) adult flies are almost normal.
IK, overexpression of dSmurf using ptc-Gal4 results
in strong reduction of pMAD along the anterior-posterior border. L,
adults with ptc>dSmurf show only rudimentary wings.
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DISCUSSION
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In this study, we have elucidated the mechanism underlying dSmurf-MAD
interaction and characterized the biological function of dSmurf in
Drosophila. dSmurf preferentially interacts with phosphorylated MAD
(pMAD). The preferential binding of dSmurf to pMAD is similar to a previously
reported Smurf2 binding to phosphorylated Smad2
(9). Furthermore, for the first
time we demonstrate that mutation in the SSXS motif of MAD abolished
its binding to dSmurf, highlighting the important role of MAD phosphorylation
in its degradation. This phosphorylation-dependent MAD-dSmurf interaction is
contrary to a previously proposed model that Smurfs target non-phosphorylated
Smad1/MAD for degradation and thus reduces the cell competence to BMP/DPP
signals (1,
8). The
phosphorylation-dependent mechanism, as also discussed
(21), is consistent to the
disappearance of pMAD in dSmurf-overexpressing transgenic flies
(Fig. 5) and also agrees with
the increased pMAD level in dSmurf-null mutants
(1). While at present it is not
clear how the phosphorylation controls the dSmurf-mediated MAD degradation,
three possible mechanisms can be envisioned. First, phosphorylation of the
SSXS motif disrupts the intrinsic MH1-MH2 interaction and results in
an exposed PPXY motif in the linker region for dSmurf binding. Second, dSmurf
interaction with MAD depends on the nuclear entry of MAD, which is induced by
ligand. The third possibility is that the phosphorylated SSXS motif
may be directly involved in MAD binding to dSmurf. Besides PPXY motif,
phosphoserine (Ser(P)) residues also serves as ligand for WW domain
(22). It will be interesting
to determine whether one of WW domains of dSmurf can directly bind to the
phosphorylated SSXS motif of MAD.
dSmurf also interacts with dSmad2 dependent of receptor activation. The
binding specificity of dSmurf is similar to that of its structurally related
Smurf2 which binds both BMP- and activin-activated R-Smads in vertebrates
(9,
10). One remaining question is
whether dSmurf also targets dSmad2 for degradation. Since activin signaling
through dSmad2 appears to have no effects on patterning of wing development
(23), the hypothetical
dSmurf-mediated dSmad2 degradation in the wing imaginal disc should have no
contributions to the severe phenotype observed in the dSmurf transgenic flies.
It is the disappearance of phophos-MAD that completely shuts down DPP
signaling during wing development.
Unlike its vertebrate homologs, dSmurf fails to interact with DAD, the
inhibitory Smad in fly. In vertebrates, Smurf1 and Smurf2 can form a strong
association with Smad6 and Smad7
(12), which may promote the
degradation of TGF-
receptors
(11,
12). While the physiological
role of mammalian Smurf in controlling receptor homeostasis remains to be
clarified, dSmurf has no apparent functions in the proteolytic degradation of
DPP receptors.
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FOOTNOTES
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* This work was supported by American Cancer Society Grants RSG-02-145-01-CCG
(to X. L.) and RPG-00-214-01-CCG (to X.-H. F.), Dutch Cancer Society Grant NKI
2000-2217 (to P. t. D.), and National Institutes of Health Grants R01 CA95731
(to F. C. B.), R01 EY11110 (to K.-W. C.), and R01 GM63773 (to X.-H. F.). The
costs of publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact. 

To whom correspondence should be addressed: Dept. of Molecular & Cellular
Biology, Baylor College of Medicine, One Baylor Plaza, Rm. 137D, Houston, TX
77030. E-mail:
xfeng{at}bcm.tmc.edu.
1 The abbreviations used are: DPP, decapentaplegic; BMP, bone morphogenetic
protein; TGF
, transforming growth factor
; E1,
ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; E3,
ubiquitin-protein ligase; HA, hemagglutinin; NTA, nitrilotriacetic acid;
sidSmurf, 21-nucleotide double-stranded RNA that targets dSmurf; pMAD,
phosphorylated MAD. 
2 S. Izaddoost and K.-W. Choi, unpublished data. 
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ACKNOWLEDGMENTS
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We thank Rik Derynck for critical reading of the manuscript, Kohei
Miyazono, Richard Padgett, Scott Hawley, and Jeff Wrana for reagents, and
Jianluo Jia for technical assistance.
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REFERENCES
|
---|
- Podos, S., Hanson, K., Wang, Y., and Ferguson, E.
(2001) Dev. Cell
1,
567578[Medline]
[Order article via Infotrieve]
- Raftery, L., and Sutherland, D. (1999) Dev.
Biol. 210,
251268[CrossRef][Medline]
[Order article via Infotrieve]
- Roberts, A. B., and Derynck, R. (2001) Science's STKE
http://stke.sciencesmag.org/cgi/content/full/sigtrans;2001/113/PE43
- Padgett, R., and Patterson, G. (2001) Dev.
Cell 1,
343349[Medline]
[Order article via Infotrieve]
- Sekelsky, J., Newfeld, S., Raftery, L., Chartoff, E., and Gelbart,
W. (1995) Genetics
139,
13471358[Abstract/Free Full Text]
- Hershko, A., and Ciechanover, A. (1998)
Annu. Rev. Biochem. 67,
425479[CrossRef][Medline]
[Order article via Infotrieve]
- Ciechanover, A., Orian, A., and Schwartz, A. L. (2000)
Bioessays 22,
442451[CrossRef][Medline]
[Order article via Infotrieve]
- Zhu, H., Kavsak, P., Abdollah, S., Wrana, J. L., and Thomsen, G.
(1999) Nature
400,
687693[CrossRef][Medline]
[Order article via Infotrieve]
- Lin, X., Liang, M., and Feng, X.-H. (2000)
J. Biol. Chem. 275,
3681836822[Abstract/Free Full Text]
- Zhang, Y., Chang, C., Gehling, D., Hemmati-Brivanlou, A., and
Derynck, R. (2001) Proc. Natl. Acad. Sci. U. S.
A. 98,
974979[Abstract/Free Full Text]
- Kavsak, P., Rasmussen, R. K., Causing, C., Bonni, S., Zhu, H.,
Thomsen, G., and Wrana, J. (2000) Mol.
Cell 6,
13651375[Medline]
[Order article via Infotrieve]
- Ebisawa, T., Fukuchi, M., Murakami, G., Chiba, T., Tanaka, K.,
Imamura, T., and Miyazono, K. (2001) J. Biol.
Chem. 276,
1247712480[Abstract/Free Full Text]
- Feng, X.-H., Lin, X., and Derynck, R. (2000)
EMBO J. 19,
51785193[Abstract/Free Full Text]
- Feng, X.-H., Liang, Y.-Y., Liang, M., Zhai, W., and Lin, X.
(2002) Mol. Cell
9,
133143[Medline]
[Order article via Infotrieve]
- Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber,
K., and Tuschl, T. (2001) Nature
411,
494498[CrossRef][Medline]
[Order article via Infotrieve]
- Spradling, A., and Rubin, G. (1982)
Science 218,
341347[Medline]
[Order article via Infotrieve]
- Cho, K. O., and Choi, K. W. (1998)
Nature 396,
272276[CrossRef][Medline]
[Order article via Infotrieve]
- Brummel, T., Twombly, V., Marques, G., Wrana, J., Newfeld, S.,
Attisano, L., Massague, J., O'Connor, M., and Gelbart, W. (1994)
Cell 78,
251261[Medline]
[Order article via Infotrieve]
- Nellen, D., Affolter, M., and Basler, K. (1994)
Cell 78,
225237[Medline]
[Order article via Infotrieve]
- Brand, A., and Perrimon, N. (1993)
Development (Camb.) 118,
401415[Abstract/Free Full Text]
- Arora, K., and Warrior, R. (2001) Dev.
Cell 1,
441442[Medline]
[Order article via Infotrieve]
- Lu, P., Zhou, X., Shen, M., and Lu, K. (1999)
Science 283,
13251328[Abstract/Free Full Text]
- Brummel, T., Abdollah, S., Haerry, T., Shimell, M., Merriam, J.,
Raftery, L., Wrana, J., O'Connor, M. (1999) Genes
Dev. 13,
98111[Abstract/Free Full Text]