From the
Departments of Growth and Development, and Anatomy, Programs in Cell
Biology and Developmental Biology, University of California, San Francisco,
California 94143-0640 and the Department of Cell
Biology, Cellular and Molecular Biology Program, University of Alabama,
Birmingham, Alabama 35294-0005
Received for publication, February 19, 2003 , and in revised form, May 6, 2003.
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ubiquitylation results in attachment of one or several 76-amino acid long ubiquitin polypeptides to Lys residues of target proteins (6). This process requires the sequential actions of a ubiquitin-activating E11 enzyme, conjugating E2 enzyme, and E3 ligase. Ubiquitin is activated by the E1 enzyme in an ATP-dependent manner and transferred as a thiol ester intermediate to one of several E2-conjugating enzymes. Covalent attachment of ubiquitin to a target protein is then mediated by one of the many E3 ligases that define substrate specificity. Polyubiquitylated proteins are usually targeted for proteasomal degradation (6).
Sumoylation involves the covalent attachment of SUMO, a ubiquitin-related polypeptide, to a Lys residue (7). However, unlike ubiquitylation, sumoylation has not been reported to target a substrate for degradation. As with ubiquitylation, sequential activities of E1, E2, and E3 enzymes are involved in sumoylation, but the identities of the enzymes are different. The SUMO activating enzyme E1, a heterodimer of Aos1 and Uba2, transfers activated SUMO to the E2-conjugating enzyme Ubc9 that directly catalyzes the isopeptide bond formation between the C-terminal glycine residue of the activated SUMO to Lys residue of the target. PIAS family members have been implicated as E3 SUMO ligases, although their roles in the sumoylation of the diverse substrates need to be further defined (8). For example, RanBP2 can serve as E3 ligase for the HDAC4 deacetylase and Sp100 (9, 10), whereas ARIP3 is involved in sumoylation of the androgen receptor (11, 12). As with other post-translational modifications, sumoylation is thought to be a reversible and dynamic process.
Sumoylation can regulate the function of a protein by changing its
subcellular localization, protein-protein interactions, and/or stability,
depending on the identity of the protein. For example, SUMO-1 modification
targets the PML protein to nuclear bodies
(13), and the
homeodomain-interacting protein kinase 2 into nuclear speckles
(14). Sumoylation negatively
impacts the transcription activities of the transcription factors Sp3
(15), LEF-1
(16), and androgen receptor
(12), and may also affect
their subnuclear distribution, whereas other studies indicate a positive
effect of sumoylation on transcription factor activity such as heat shock
transcription factor-1 (17).
Signaling from the cell surface can also be directly impacted by sumoylation,
as illustrated in the case of IB, which sequesters NF
B in the
cytoplasm. Tumor necrosis factor-
-induced phosphorylation of I
B
allows for its ubiquitination, thus targeting it for proteasomal degradation
and enabling nuclear translocation of NF
B, whereas sumoylation of the
same Lys in I
B stabilizes it against degradation
(18). This dynamic balance
between sumoylation and ubiquitination of I
B can then regulate the gene
expression of the cell response mediated by NF
B. Taken together, the
effects of sumoylation on the function of the protein appear to depend on the
substrate and, unlike ubiquitylation, which frequently targets substrate for
proteasomal degradation, are not predictable.
TGF- and TGF-
-related proteins are key regulators of cell
proliferation and differentiation. They regulate development from nematodes
and flies to mammals (6), and
play important roles in tumor progression
(19). The central signaling
pathway from TGF-
-activated cell surface receptors to alterations in
gene expression involves a small class of signaling effectors, the Smads, as
key mediators of a response of the cell to TGF-
(20). Thus, following ligand
binding to type II/type I receptor complexes, the activated type I receptors
phosphorylate the C-terminal two serines of receptor-activated Smads,
e.g. Smad2 and Smad3 in response to TGF-
and Smad1, -5, and -8
upon stimulation by bone morphogenetic proteins. The phosphorylated Smads are
then released from the receptors to form a heterotrimeric complex with Smad4
as a common component of all Smad signaling pathways, activated by TGF-
family members (20). The Smad
complexes enter the nucleus where they interact at the promoter with other
transcription factors and co-regulators to regulate gene expression. In this
pathway, Smad4 acts as a central coactivator of all receptor-activated Smads,
presumably through its ability to stabilize the interaction of
receptor-activated Smads with the essential coactivator cAMP-response
element-binding protein/p300
(21) and its ability to
recruit yet another coactivator, SMIF1
(22). In addition to
C-terminal phosphorylation, the activation of the receptor-activated Smads is
also regulated by other phosphorylations, e.g. in response to
mitogen-activated protein kinase signaling
(23), and is counteracted by
inhibitory Smad6 and -7 (24,
25).
Whereas phosphorylation plays a central role in the activation of the Smad
pathway, recent studies have revealed an additional level of regulation of
Smad signaling through ubiquitylation. Smurf1 and Smurf2, two Hect family E3
ubiquitin ligases, target receptor-activated Smads for ubiquitylation and
proteasomal degradation, thus decreasing their availability
(26,
27). In addition,
ubiquitylation and consequent degradation of ligandactivated Smad2 and Smad3
in the nucleus may be involved in the termination of their functions in
transcriptional regulation
(28). In contrast to the
receptor-activated Smads, Smad4 levels are not regulated through
ubiquitin-mediated degradation. However, some mutations, found in human
tumors, destabilize Smad4 via ubiquitylation and consequent degradation
(29,
30). The role of Smad4 as the
central mediator of signaling by all receptor-activated Smads in response to
all TGF- family members, and its shuttling between cytoplasm and nucleus
(31) suggests that some
mechanism may be in place to allow for stabilization and re-utilization during
signaling.
In this report, we show that among the Smads known to be involved in
TGF- signaling, Smad4 is the only one that is preferentially subjected
to sumoylation. We identified two sumoylation sites in Smad4, and sumoylation
was enhanced in the presence of the conjugating enzyme Ubc9 and PIAS family E3
ligases. Sumoylation by the PIASy E3 ligase resulted in redistribution of
Smad4 to subnuclear speckles that colocalized with SUMO-1 and PIASy.
Replacement of the sumoylation target lysines with arginine, or increased
sumoylation, enhanced the protein stability and increased transcription in
mammalian cells and in Xenopus embryos. These results suggest a role
for Smad4 sumoylation in the regulation of TGF-
family signaling through
Smads.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell Culture, Transfections, and Reporter AssaysCOS-1,
HeLa, and MDA-MB-468 cells were cultured in Dulbecco's modified Eagle's
medium, supplemented with 10% heat-inactivated fetal bovine serum
(Invitrogen), 100 µg/ml streptomycin sulfate, 100 units/ml penicillin G.
COS-1 and HeLa cells were transfected using LipofectAMINE (Invitrogen) and
MDA-MB-468 cells were transfected with FuGENE 6 (Roche Diagnostics) according
to the manufacturer's instructions. In transcription reporter assays,
MDA-MB-468 cells were seeded onto 6-well culture dishes and 2 µg of DNA,
including 0.5 µg of (SBE)4-luc luciferase reporter plasmid, 0.1
µg of pRK5--gal, and other expression plasmids, as required, were
used for each transfection. The total amount of DNA was kept constant by
addition of pRK5 DNA. 1216 h after transfection, cells were treated
with or without 10 ng/ml TGF-
in Dulbecco's modified Eagle's medium
containing 0.2% fetal bovine serum for 20 h. At the end of stimulation, cells
were washed twice in cold phosphate-buffered saline and processed for
luciferase or
-galactosidase enzyme assays as described
(37).
Cell Lysis, Immunoprecipitation, and Western Blotting Analysis Cells were washed four times in phosphate-buffered saline and lysed in RIPA lysis buffer (150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0), supplemented with 20 mM N-ethylmaleimide (Calbiochem, 34115), protease inhibitor mixture (Sigma, P8340), and 5% glycerol. In co-immunoprecipitation experiments cells were lysed in RIPA buffer without the ionic detergents. Cell lysates were cleared by centrifugation at 20,000 x g at 4 °C for 30 min. The resulting supernatant was subjected to immunoprecipitation with anti-FLAG M2-agarose (Sigma, A2220) overnight at 4 °C. The agarose beads were washed five times in lysis buffer and the bound proteins were eluted in SDS-PAGE loading buffer. The samples were subjected to SDS-PAGE, followed by Western transfer and immunoblotting as described (38). Mouse monoclonal anti-FLAG M2 antibody (Sigma, F3165), anti-Myc tag 9E10 antibody (Covance, MMS-150P), anti-HA tag HA11 antibody (Covance, MMS-101R), anti-GMP-1 (SUMO-1) antibody (Zymed Laboratories Inc.), and rabbit polyclonal anti-Smad4 antibody H-552 (Santa Cruz, sc-7154) were used at 1 µg/ml in Western blotting. Peroxidase-linked anti-mouse (NA931) and anti-rabbit (NA9340) secondary antibodies and ECL detection reagents (RPN2106) were purchased and used according to the manufacturer's instructions (Amersham Biosciences).
Pulse-Chase Analysis of Smad4 StabilityTransfected COS cells were starved in methionine- and cysteine-free medium containing 0.2% dialyzed fetal bovine serum for 2 h. The cells were then labeled with 0.25 µCi/ml [35S]EXPRESS protein labeling mix (PerkinElmer Life Sciences, NEG072) in the same medium for 1 h. At the end of the labeling, the cells were washed three times in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum before incubating in the same medium for various times, as needed. Where indicated, lactacystin (Calbiochem, number 426100) was used at 30 µM during the pulse-chase. Cells were harvested and processed as above. The cell lysates were subjected to anti-FLAG immunoprecipitation for isolation of exogenous Smad4, and immunoprecipitates were analyzed by SDS-PAGE. Smad4 was visualized by autoradiography and analyzed by densitometry.
ImmunofluorescenceHeLa cells were grown on coverslips and
transfected with the indicated plasmid DNA. 24 h after transfection cells were
kept in Dulbecco's modified Eagle's medium containing 0.2% fetal bovine serum
for 3 h and subsequently treated with 5 ng/ml TGF- for 40 min. Cells
were then permeabilized with 0.5% Triton X-100 in CSK buffer (10 mM
Pipes, pH 6.8, 100 mM NaCl, 300 mM sucrose, 3
mM MgCl2, 2 mM EDTA) for 2 min on ice, and
fixed in 3.7% paraformaldehyde in CSK buffer for 5 min at 37 °C
(39). Cells were rinsed twice
in TBS (20 mM Tris, pH 8, 150 mM NaCl) and then blocked
in 3% bovine serum albumin in TBS containing 0.03% Triton X-100 for 1 h at
room temperature. After incubation with a primary antibody in blocking
solution, the cells were washed in blocking solution five times before
incubating with 1:500 diluted secondary antibody (Alexa Fluor 488 goat
anti-rabbit IgG (A11029
[GenBank]
) and Alexa Fluor 594 goat anti-mouse IgG (A-11034)
from Molecular Probes) for 1 h. After washings cells were stained in 300
nM 4,6-diamidino-2-phenylindole (Sigma, 32670) in TBS for 5 min,
followed by brief washes in TBS. The samples were finally mounted in FluorSave
Reagent (Calbiochem, 345789) and examined by fluorescence microscopy.
Mesoderm Marker Induction in Xenopus EmbryoscRNAs encoding
Smad4 (wild-type and mutants) and SUMO-conjugating enzyme Ubc9 and
-catenin were synthesized in vitro, using Ambion mMessage
Machine kit. Capped RNAs were injected into both animal poles of two-cell
stage embryos. The doses of cRNAs used were indicated in the legend to
Fig. 7. The ectodermal explants
(animal caps) of injected embryos were dissected at blastula stages (stage 9),
and total RNA was extracted from these caps at gastrula stages (stage 11). For
treatment with basic fibroblast growth factor (bFGF), the animal caps were
dissected at stage 9 and incubated in buffer containing 100 ng/ml bFGF. The
caps were harvested at stage 11 as other samples. Reverse transcription-PCR
was performed using the primers as described previously
(40).
|
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Although many gene expression responses of TGF- are mediated by Smad3
and Smad4 (41), Smad2 and
Smad1 can also be activated by TGF-
(42,
43), and Smad6 and Smad7 act
as inhibitory Smads for the TGF-
response
(25,
41). We therefore evaluated
whether any of these Smads are targeted by sumoylation. As shown in
Fig. 1B, only Smad4,
and not the other Smads tested, was sumoylated under this condition.
Lysines 159 and 113 Are Major Sumoylation Sites in Smad4 To localize the site of sumoylation within Smad4, we expressed several Smad4 segments, either in the presence or absence of coexpressed SUMO-1. Immunoprecipitation followed by Western detection of SUMO-1 indicated that the NL segment, corresponding to amino acids 1300 and containing the MH1 domain and linker segment, was sumoylated (Fig. 2A). Smad4C, i.e. amino acids 266552 containing the MH2 domain, was not detectably sumoylated. Smad4N, i.e. amino acids 1140 corresponding to the MH1 domain, showed only a minimal level of immunoreactivity for Myc-SUMO-1 (Fig. 2A). Direct Western blotting of the cell lysates for Smad4 detected small fractions of Smad4, Smad4N, and Smad4NL (Fig. 2A, lower panel) with the same mobilities as the sumoylated forms that were detected using the anti-Myc antibody for SUMO-1 (Fig. 2A, upper panel).
|
SUMO conjugation occurs on lysine (K) residues within a general minimal
consensus sequence KXE, in which
is a large hydrophobic
residue (7). Only 1 lysine
within such sequence context, i.e. Lys-159 within the VKDE sequence,
is present in Smad4 and is located within the Smad4NL, but not the Smad4N
segment. Within the Smad4NL sequence are two other lysines, i.e.
Lys-51 and Lys-106, that are located within a KXE sequence that lack
a preceding hydrophobic residue. To examine which lysine is sumoylated, we
individually replaced each of these three lysines within Smad4NL by an
arginine (R). As shown in Fig.
2B, the K51R and K106R mutants were sumoylated to a
similar extent as the normal Smad4NL. In contrast, the K159R mutation strongly
reduced, yet did not abolish the sumoylation. This result suggests that
Lys-159 is a major sumoylation site. The triple mutation of Smad4NL, resulting
in replacement of all three lysines with arginines further reduced the
reactivity in Western blotting for SUMO to background level. This result
suggests that Lys-51 and/or Lys-106 can serve as targets for sumoylation
enzymes, albeit with a much lower efficiency than Lys-159.
We also tested the effect of these lysine mutations on sumoylation of
full-length Smad4 (Fig.
2C). These results confirmed Lys-159 as the major
sumoylation site, because the K159R mutation nearly abolished sumoylation. In
contrast, mutation of Lys-106 exerted only a minor decrease, and mutation of
Lys-51 had no effect. We also replaced Lys-392, which is located within the
CKGE sequence in the carboxyl MH2 domain of Smad4, by Arg. This mutation also
had only a minimal, if any, effect on Smad4 sumoylation
(Fig. 2C), consistent
with the lack of detectable sumoylation of Smad4C
(Fig. 2A). Finally, an
alternate, less common sumoylation target sequence, KXD, has
also been described (44).
Lys-122, within an LKCD sequence, was therefore mutated and the effect on
Smad4 sumoylation was assessed. As shown in
Fig. 2C, the K122R
mutation did not detectably decrease the level of Smad4 sumoylation. We also
examined the sumoylation status of double lysine mutants, K51R/L159R and
K122R/L159R and found that they had a low level of sumoylation like K159R
(Fig. 2C).
While this paper was under review, Lin et al. (45) reported that Smad4 is sumoylated on Lys-113, in addition to Lys-159. We therefore verified the possible sumoylation of this site (Fig. 2D). As evaluated in the presence of SUMO-1, mutation of Lys-113 to Arg reduced sumoylation of Smad4. Mutation of both Lys-113 and Lys-159 to arginines almost abolished Smad4 sumoylation.
Ubc9 and PIAS E3 Ligases Enhance Smad4 Sumoylation Sumoylation involves the E2-conjugating enzyme Ubc9 (46) and the recently identified E3 ligases of the PIAS family, such as PIASy or ARIP3 (47). We therefore examined the effect of coexpression of Ubc9 in the absence or presence of PIASy or ARIP3 on Smad4 sumoylation. As shown in Figs. 2D and 3A, coexpression of Ubc9 with SUMO-1 strongly enhanced the sumoylation of Smad4. Under these conditions, we also detected, in addition to the singly conjugated Smad4 species, a low level of a higher molecular weight form that is likely to correspond to doubly sumoylated Smad4. Mutating either Lys-113 or Lys-159 resulted in mostly singly sumoylated Smad4 species while the K113R/L159R mutant showed almost no sumoylation (Figs. 2D and 3A).
|
Overexpression of PIASy or ARIP3 in the absence or presence of coexpressed Ubc9 markedly increased Smad4 sumoylation. In these experiments, we additionally detected higher molecular weight forms of Smad4 (Fig. 3A) that likely result from sumoylation at multiple lysines in addition to Lys-113 and Lys-159. The K113R/K159R mutant of Smad4 showed almost no detectable sumoylation even when both Ubc9 and PIASy were expressed. Nevertheless, coexpression of Ubc9 and ARIP3 resulted in polysumoylation of this double mutant (Fig. 3A), consistent with the use of alternative lysines as substrates for sumoylation under these conditions.
Because Smad4 can be sumoylated at more than one site, particularly when Ubc9 was coexpressed, we tested the effect of overexpressed Ubc9 on Smad1, -2, -3, -6, and -7 sumoylation, which are normally not sumoylated (Fig. 1B). As shown in Fig. 3B, Ubc9 overexpression facilitates sumoylation of Smad1, -2 and -3, albeit at a much lower level than Smad4. As in Fig. 1, only Smad4 was sumoylated at endogenous levels of Ubc9.
Sumoylation Alters the Subnuclear Distribution of Smad4
Sumoylation of some proteins has been shown to correlate with targeting of the
modified proteins to a specific subcellular localization. For example,
sumoylation of the PML protein is required for its localization to subnuclear
bodies (13). Furthermore, the
E3 ligase PIASy itself is localized in a punctated pattern within the nucleus
and can target LEF-1 to these subnuclear bodies
(16). Because Smad4 is
translocated into the nucleus upon TGF- treatment, we examined the
possible role of sumoylation in the subnuclear localization of Smad4 in
transfected HeLa cells by immunofluorescence staining. Without increased
SUMO-1 or PIASy expression, Smad4 was visualized as a diffuse nuclear staining
in TGF-
-treated cells (Fig. 4A,
a). PIASy by itself exhibited a punctated, subnuclear
staining pattern (Fig. 4A,
b), whereas SUMO-1 alone also exhibited a somewhat punctated
staining in the nucleus (Fig. 4A,
c).
|
Increased expression of SUMO-1 and PIASy, a condition that enhances the sumoylation of Smad4 (Fig. 3), strongly affected the subnuclear localization of Smad4. Indeed, in contrast to its normally diffuse distribution (Fig. 4A, a), coexpression of SUMO-1 and PIASy, and consequent Smad4 sumoylation, correlated with a recruitment of Smad4 into a punctate subnuclear pattern (Fig. 4B, a), similarly to that of PIASy (Fig. 4, B, b, and A, b). In fact, under these conditions, the Smad4 and PIASy staining largely overlapped (Fig. 4B, c). Additionally, when SUMO-1, Ubc9, and PIASy were coexpressed with Smad4, we observed that Smad4 colocalized with SUMO-1 in a punctated pattern (Fig. 4C, a--c). In the absence of the E3 ligase, however, the immunofluorescence staining of Smad4 was more diffuse (data not shown). Together, these results suggest that the interaction with the E3 ligase and concomitant sumoylation enhance targeting of Smad4 to the SUMO-1 containing subnuclear bodies that also define the localization of PIASy.
To further address the role of sumoylation in subnuclear targeting of Smad4, we examined the effect of increased expression of SUMO-1, Ubc9, and PIASy on the subnuclear localization of the K122R/K159R mutant of Smad4 that lacks one of the major sumoylation sites (Fig. 4C, eh). Unexpectedly, the K122R/K159R mutant showed similarly punctated subnuclear localization as wild-type Smad4 under these conditions. This is consistent with the observation that PIASy can target LEF-1 into subnuclear bodies, independently from sumoylation of LEF-1 (16). This targeting of the K122R/K159R mutant of Smad4 in the presence of PIASy may be because of residual sumoylation of this mutant, but could also be explained by a sufficiently strong physical interaction of PIASy with Smad4. Indeed, we found that PIASy interacted equally well with wild-type Smad4 and with the K122R/K159R or K113/159R mutants in coimmunopreciptation assays (Fig. 4D, data not shown). Thus, the coexpression of PIASy, and consequent physical interaction with Smad4, rather than Smad4 sumoylation per se, may be sufficient to target Smad4 into a punctate pattern of subnuclear bodies.
Sumoylation Enhances the Stability of Smad4 In contrast to
ubiquitylation, SUMO conjugation does not appear to target proteins for
degradation (48,
49). Furthermore, in the case
of IB
, sumoylation has been shown to protect against
ubiquitination of the same lysine residue, and thereby protect
I
B
against ubiquitin-mediated degradation
(18). We thus tested the role
of sumoylation on the stability of Smad4 using pulsechase experiments, in
which proteins were 35S-labeled, and the stability of
35S-labeled Smad4 was followed over time.
We first compared the stability of wild-type Smad4 with the K113R/K159R mutant that has the major sumoylation target Lys replaced by Arg. As shown in Fig. 5A, the K113R/K159R mutant, as well as the K159R mutant (data no shown), had a higher stability than wild-type Smad4. Whereas wild-type Smad4 is a relatively stable protein, replacement of Arg-100 by Thr (R100T), a mutation observed in tumor specimens, results in increased ubiquitination and a shorter half-life because of increased degradation (29). As reported previously (29), we also observed that Smad4 R100T has a shorter half-life than wild-type Smad4 and the protein was stabilized by treatment of cells with proteasomal inhibitor lactacystin (Fig. 5B). The K159R mutation introduced into Smad4 R100T similarly conferred a higher stability (Fig. 5B), as observed for wild-type Smad4. Finally, we evaluated the effect of increased sumoylation on the stability of Smad4 R100T. We found that coexpression of SUMO-1, Ubc9, and PIASy enhanced the stability of the Smad4 R100T mutant (Fig. 5C). These results suggest that sumoylation enhances the stability of Smad4, but that replacement of the targeted lysine 159 also confers increased stability.
|
Effect of Sumoylation on Smad4-mediated Transcriptional
ActivationSumoylation has been reported to affect the
transcriptional activity of p53, c-Jun, and the androgen receptor
(50,
51). We therefore evaluated
the effect of Smad4 sumoylation on the function of Smad4 as transcriptional
coactivator in TGF--induced, Smad3-mediated transcription. We used the
(SBE)4-luciferase reporter, in which transcription is directed from four
tandem Smad-binding elements, to evaluate the activity of Smad4 as a
coactivator (35).
Transcription from tandem SBE sites in response to TGF-
is mediated by
Smad3 in cooperation with Smad4 and the basal transcription machinery. As
reporter cell line, we used MDA-MB468 cells, which lack endogenous Smad4, thus
coexpression of Smad4 is required for Smad3-mediated transcriptional
activation in response to TGF-
(52). All transcription assays
were normalized against
-galactosidase to account for the effects of
sumoylation on non-Smad-mediated transcription. As shown in
Fig. 6A, expression of
Smad4 in MDA-MB468 cells conferred TGF-
responsiveness, yet also
enhanced transcription in the absence of exogenous TGF-
, presumably a
consequence of autocrine TGF-
responsiveness. Mutation of the major
sumoylation target site in Smad4 K159R resulted in a higher level of
transcription (Fig.
6A), consistent with the higher stability of Smad4 K159R,
when compared with wild-type Smad4 (Fig. 5,
A and B; data not shown). The K113R mutation
resulted in only a slight enhancement on Smad4-mediated transcriptional
activity, whereas the K113R/K159R mutant showed a similar or slightly higher
transcriptional activity as the K159R mutant
(Fig. 6B). Finally,
coexpression of Ubc9, SUMO-1, or both Ubc9 and SUMO-1 enhanced the
transcription coactivator function of Smad4, particularly in the presence of
added TGF-
(Fig.
6C).
|
Effect of Sumoylation on Smad4-mediated Gene Expression in Xenopus
Expression AssaysThe effects of Smad ubiquitylation on the
transcriptional activation of TGF- family- or Smad-responsive genes have
been studied using injection of cRNAs in Xenopus embryos and by
examining the induction of the downstream genes in ectodermal explants
(26,
53). In such assays, the
activin/nodal-responsive Smad2 and the bone morphogenetic protein-responsive
Smad1 determine the balance between dorsal and ventral mesoderm marker
expression. Generally speaking, Smad2 induces pan-mesoderm markers, and at
high doses enhances dorsal mesoderm marker expression, whereas repressing
ventral mesoderm formation
(54). In contrast, bone
morphogenetic protein-activated Smad1 enhances ventral mesoderm
differentiation while repressing dorsal mesoderm marker expression
(55). Smad4, which serves as
coactivator for all receptor-activated Smads, induces expression of the
pan-mesodermal marker Brachyury (Xbra)
(56), yet also has the
potential to enhance the responses of all receptor-activated Smads. We
therefore addressed the role of Smad4 sumoylation in these Xenopus
explant assays.
In one set of experiments, we compared the activities of Smad4 and the mutant Smad4 K159R, in which the major sumoylation target Lys-159 was replaced by Arg, for their abilities to induce Xbra expression. As shown in Fig. 7A, Smad4 induced the expression of Xbra mRNA, while Smad4 K159R, translated from an equal quantity of injected cRNA, induced a higher expression of Xbra (compare lanes 2 and 4). This result is in agreement with the data obtained from the transcriptional reporter assay shown in Fig. 6A and may reflect the increased stability of the Smad4 K159R mutant over wild-type Smad4 (data no shown). A similar effect of the K159R mutation was also seen in the Smad4 mutant, in which Arg-100 was replaced by Thr (compare lanes 3 and 5). These results suggest that substitution of Lys-159 by Arg can alter the activity of Smad4 in mesoderm induction.
In another set of experiments, we also compared the effects of the K113R, K159R, and K113R/K159R Smad4 mutants on their ability to regulate gene expression (Fig. 7B). In these experiments we used a more sensitive assay for Smad4 activity, based on the ability of Smad4 to enhance Smad2-mediated mesodermal marker expression. Similarly to activin, Smad2 induces the expression of various mesodermal and endodermal markers in a dose-dependent manner. At low doses, Smad2 induces ventral and ventrolateral mesodermal markers, such as Xwnt8. With increasing Smad2 levels, the expression of dorsal mesodermal and endodermal marker genes, such as chordin and mixer, is turned on (Fig. 7B (54, 55)). Smad4 enhances the mesodermal marker gene expression by Smad2, so that, even at low levels of Smad2, dorsal mesoderm marker expression is turned on efficiently (57). In our experiments, Smad2 induced the pan-mesodermal marker Xbra and the ventrolateral marker Xwnt8, whereas coexpression of Smad4 with Smad2 not only enhanced the expression of these markers, but also induced stronger expression of chordin and mixer (Fig. 7B, lane 3 versus 2). Either K113R or K159R mutation further increased gene activation by Smad4, which is evident in the levels of transcription of the dorsal mesodermal marker chordin and the endodermal marker mixer (Fig. 7B, lanes 4 and 5). The K159R mutant is slightly more active than the K113R mutant whereas the K113R/K159R double mutant has a comparable effect on activation of gene expression as the K113R mutant (Fig. 7B, lane 6).
To further address the role of Smad4 sumoylation on gene regulation, we examined the effect of Ubc9, which strongly enhances Smad4 sumoylation (Figs. 2D and 3A), on the ability of Smad4 to induce mesoderm marker gene expression (Fig. 7C). Again, we used the sensitive assay of enhancement of Smad2-dependent gene activation by Smad4. In our experiments, coexpression of Smad4 with Smad2 induced mixer, chordin, and Xbra expression to a higher level than Smad2 by itself (i.e. with endogenous Smad4), as illustrated in lanes 4 versus 2 in Fig. 7C. Coexpression of Ubc9 strongly enhanced the expression of the endodermal and dorsal mesoderm markers mixer and chordin, as well as the ventral mesoderm marker Xwnt8 and the pan-mesoderm marker Xbra (Fig. 7C, lanes 5 versus 4). Ubc9 also enhanced the marker induction by Smad2 (Fig. 7C, lanes 3 versus 2), possibly through its ability to drive sumoylation of endogenous Smad4, or of Smad2 itself.
It is conceivable, if not likely, that sumoylation has a general effect on
transcription through its effects on the general transcription machinery, and
thus may affect signaling mediated by other growth factors. We therefore
analyzed the effect of Ubc9 on marker induction by two unrelated signaling
molecules (Fig. 7D).
bFGF induced the mesodermal gene Xbra in Xenopus animal
caps, and coexpression of Ubc9 had only minimal effect on this marker
induction (Fig. 7D,
compare lanes 4 with 3). In contrast, the activity of
-catenin, a downstream signaling effector of the Wnt pathway, which
stimulates Siamois expression, was reduced by Ubc9
(Fig. 7D, lanes 6
versus 5). Our results therefore suggest that the enhancement of
Smad4-mediated transcription by sumoylation is primarily an effect of Ubc9 on
Smad4, and is not because of a general transcription activation by this
sumoylation enzyme.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Smads act as central effectors of TGF- family-induced gene expression
(58,
59). The receptor-activated
Smads relay the information from the ligand-activated receptors, while Smad4
acts as a common Smad that is required for ligand-induced activation of gene
expression. The receptor-activated Smads are activated through direct
C-terminal phosphorylation by the type I receptors
(60,
61), whereas additional
phosphorylation events by Erk mitogen-activated protein kinase, c-Jun
N-terminal kinase, CamKII, and protein kinase C further define their
activation state
(6266).
Furthermore, ubiquitin-mediated degradation regulates the levels of
receptor-activated Smads, thus providing an additional level of control of
signaling (26,
27). In contrast to this
extensive post-translational regulation of the receptor-activated Smads, Smad4
is not normally subject to regulation by phosphorylation or ubiquitylation.
Because of its central role as a common Smad, one would intuitively expect
that Smad4 needs to be stably available, a concept that would be consistent
with the apparent ability of Smad4 to shuttle between the cytoplasmic and
nuclear compartments (31).
Remarkably, sumoylation confers a unique modification to the common Smad4, not
present in the receptor-activated and inhibitory Smads.
Biochemical and mutation analyses have identified Lys-159 and Lys-113 as
major sumoylation sites in Smad4. The lysine 159 is located in the consensus
sequence KXE/D
(49), present in Smad4 but not
in the other Smads tested, i.e. Smad1, -2, -3, -6, and -7. In
agreement with the role of this sequence in recognition and binding of
E2-conjugating enzyme Ubc9
(67), sumoylation of Smad4 was
increased when Ubc9 was coexpressed. This effect was further enhanced by
coexpression with PIASy or ARIP3, members of the recently identified PIAS
family of SUMO E3 ligases (11,
16), suggesting that a PIAS E3
ligase is naturally involved in Smad4 sumoylation. Contrary to Lys-159, the
sequence around Lys-113 does not conform to the consensus sequence suggesting
that other factors, perhaps secondary structure features in the surrounding
sequence, may be involved in determining the choice of target lysine.
Interestingly the Lys-113 is flanked by two hydrophobic amino acids. The
hydrophobic amino acid preceding the lysine is apparently required for
sumoylation, because Lys-51, -106, and -392, which are located in a
KXE sequence lacking a preceding hydrophobic amino acid, were only
minimally if at all targeted for sumoylation. Additionally, we did not have
evidence that Lys-122, located in the
KXD sequence, was
sumoylated. These potential secondary sumoylation sites likely account for the
low level sumoylation, when Lys-159 and Lys-113 are mutated, and the
sumoylation of the lysine double mutant, when Ubc9 and E3 ligases are
overexpressed.
To address the role of sumoylation on Smad4 function, we mutated lysine 159, a major sumoylation site, either alone or in combination with other secondary lysine modification sites, to arginine and compared the functions of these mutants with wild-type Smad4. Arginine substitution preserves the charge while prohibiting the isopeptide bond formation with SUMO-1. Additionally, we examined the effects of increased sumoylation by E2 and/or E3 enzymes.
Without ectopic coexpression of SUMO-1 and PIASy, Smad4 was diffusely localized in the nucleus. Coexpression of the sumoylation enzymes relocated Smad4 into speckles, which colocalized with PIASy. A similar, punctated redistribution of the sumoylated proteins in the presence of SUMO-1 or sumoylation enzymes has also been observed in the case of the nuclear proteins LEF-1, PML, and HIPK2 (14, 16, 68). Under similar conditions, the Smad4 K122R/K159R mutant also displayed a speckled nuclear distribution. This redistribution of Smad4 is likely because of the ability of PIASy to interact with Smad4, independent of the presence of Lys-159 or Lys-113, and the punctated, subnuclear localization of PIASy. Whereas some protein interactions have been shown to help define the distribution of transcription factors inside the nucleus (16), the subnuclear distribution of receptor-activated Smads is so far only known to be affected by Runx proteins, which can directly interact with Smads (69). Such an effect has not been reported for Smad4. The ability of PIASy to define the subnuclear localization of Smad4 in speckles thus represents the first example of intranuclear redistribution of Smad4. The role of the punctated sequestration of Smad4, or any other protein with similar subnuclear distribution, is as yet unclear, but is likely related to the regulation of the transcriptional activity and/or nuclear metabolism (70, 71).
Sumoylation also modestly enhanced the stability of Smad4, as assessed
using pulse-chase analyses. The effect of sumoylation on protein stability was
better appreciated using the Smad4 R100T mutant than using the more stable,
wild-type Smad4. Smad4 R100T is one of the few Smad4 mutants found in cancer
cells that predisposes Smad4 to increased ubiquitinmediated degradation, thus
significantly decreasing its stability
(29). Coexpression of SUMO-1
and sumoylation enzymes enhanced the stability of Smad4 R100T, suggesting that
it may offset ubiquitin-mediated degradation. As both sumoylation and
ubiquitylation occur at Lys residues, it is conceivable that sumoylation could
compete with ubiquitylation at the same Lys, thereby preventing degradation.
Such a mechanism has been implicated in the regulation of the IB
stability by sumoylation and ubiquitylation
(18). In the case of Smad4
R100T, however, Smad4 ubiquitylation was as efficient as Smad4 R100T/K159R
(data not shown), suggesting that a mechanism other than competition is
involved. Similarly, sumoylation has been shown to enhance the stability of
c-Myb independent of an effect on ubiquitin-mediated degradation
(72). The overall higher
stability of Smad4, even though at any time only a fraction is sumoylated, is
consistent with the dynamic and reversible nature of the sumoylation process.
Remarkably, replacement of Lys-159 and Lys-113 with Arg also enhanced the
stability of Smad4 independent of sumoylation at these residues, supporting
the critical nature of these Lys residues in Smad4 stability and that
sumoylation may alter protein stability indirectly such as via a
conformational change. Further study is required to identify the detailed
molecular basis.
Finally, we also evaluated the role of Smad4 sumoylation in Smad-mediated transcription. Smad4 is known to function as a coactivator of receptor-activated Smads, thereby enhancing Smad-mediated transcription. In one set of experiments, Smad4 enhanced Smad-mediated transcription in transfected mammalian cells that lack endogenous Smad4. In the second system, i.e. Xenopus embryo explant assays, defined amounts of mRNA were injected, thus overcoming any possible effects of sumoylation on transcription from the transfected plasmid. Additionally, the gene expression monitored in Xenopus assays is exquisitely sensitive to quantitative differences in Smad signaling. In both systems we found that replacement of Lys-159 by Arg enhanced the coactivator function of Smad4. In addition, increased sumoylation, resulting from Ubc9 or Ubc9 and SUMO-1 coexpression, also enhanced transcription.
These results need to be interpreted with great caution. Indeed, substitution of Lys-159 by Arg is not necessarily equivalent to lack of sumoylation of Lys-159, and may result in a conformational change. On the other hand, increased Ubc9 expression may affect other components of the transcription machinery, and result in low level sumoylation of receptor-activated Smads. Nevertheless, ectopic Ubc9 expression either did not affect or decreased non-Smad-mediated signaling in Xenopus, in contrast to the enhanced Smad signaling (Fig. 7). Also, increased sumoylation by Ubc9 and SUMO conferred increased Smad4-dependent transcription in luciferase reporter assays that were normalized for nonspecific effects on general transcription. The basis for the increased transcription may be related to the increased Smad4 stability, which was observed when sumoylation enzymes were overexpressed or when Lys-159 was replaced with Arg.
Additionally, the redistribution of Smad4 into speckles may also affect its
function. However, in the cases of other transcription factors studied,
sumoylation and redistribution into speckles correlate frequently, but not
always, with decreased transcription
(12,
16,
73). An additional possibility
would be that sumoylation of Lys-159 and Lys-113 affect, through
conformational changes or protein-protein interactions, the function of Smad4.
A previously identified short sequence, the "synergy control"
motif, that is found within negative regulatory regions of several
transcription factor such as glucocorticoid receptor and C/EBP transcription
factors, contains the KXE sumoylation consensus sequence
identical to the one at Lys-159 in Smad4
(74). The lysine residue
within this motif in the C/EBP-transcription factors has been shown recently
to be sumoylated (75,
76). Mutations of this
sequence in the synergy control motif selectively increases the transcription
activity of glucocorticoid receptor
(74) and C/EBP transcription
factors (75,
76) at compound but not in
single response element, suggesting a regulatory role of sumoylation in higher
order interactions among transcription regulators. In addition, sumoylation at
Lys-159, which resides in the linker segment (aa 136322) of Smad4, may
somehow affect the function of the SAD domain (Smad4 activation domain, aa
274322) that is also located in the linker segment of Smad4
(52,
77). This SAD domain is
required for efficient function of Smad4 as coactivator, presumably through
its ability to recruit the SMIF1 coactivator
(22) and cAMP-response
element-binding protein/p300
(77). Further studies will be
required to define the effect of sumoylation on the intrinsic activity of
Smad4.
![]() |
FOOTNOTES |
---|
Supported by a fellowship from the Tobacco-related Disease Research Program
of California.
¶ Supported by a fellowship from the National Institutes of Health.
|| To whom correspondence should be addressed: Dept. of Growth and Development, University of California, San Francisco, CA 94143-0640. Tel.: 415-476-7322; Fax: 415-476-1499; E-mail: derynck{at}itsa.ucsf.edu.
1 The abbreviations used are: E1, ubiquitin-activating enzyme; E2, ubiquitin
carrier protein; E3, ubiquitin-protein isopeptide ligase; SUMO-1, small
ubiquitin-related modifier-1; TGF-, transforming growth factor-
;
aa, amino acid(s); HA, hemagglutinin; bFGF, basic fibroblast growth factor;
Pipes, 1,4-piperazinediethanesulfonic acid.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|