From the Department of Cell Biology and the Vanderbilt Cancer Center, Vanderbilt University, Nashville, Tennessee 37232-6838
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ABSTRACT |
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Transforming growth factor (TGF-
) is the
prototype for an evolutionarily conserved superfamily of secreted
factors implicated in diverse biological phenomena. The pleiotropic
responses to TGF-
are initiated by a heteromeric receptor complex
that binds and phosphorylates downstream effectors. Among these, the
Smads have been extensively studied. However, less attention has been directed toward alternative downstream effectors and their
participation in TGF-
signal transduction. We show that TGF-
promotes accumulation of the labile monomeric GTPase RhoB by
antagonizing its normal proteolytic destruction, presumably via the 26 S proteasome. RhoB accumulates in its isoprenylated form. Transient
overexpression of wild type RhoB but not its dominant negative mutant
RhoB-N19 antagonizes TGF-
-mediated transcriptional activation. These
results suggest a novel mechanism of regulation by TGF-
and
implicate RhoB as a negative regulator of TGF-
signal
transduction.
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INTRODUCTION |
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Transforming growth factor-
(TGF-
)1 is the prototype
for a multifunctional superfamily of secreted factors. TGF-
has been implicated in diverse phenomena including growth control, production of
extracellular matrix, cell adhesion and motility, and modulation of
cell phenotype (1). As such, TGF-
has broad influence over normal
growth and developmental programs and over the pathologic sequelae that
arise from their dysfunction. TGF-
communicates with the cytoplasm
through a heteromeric complex composed of type I (T
RI) and type II
(T
RII) transmembrane serine/threonine kinase receptors. Upon ligand
binding, T
RII transphosphorylates T
RI in a highly conserved GS
domain, thereby activating T
RI kinase activity toward downstream
effectors. Ligand binding specificity is conferred by T
RII, whereas
signal specificity is conferred by T
RI (2).
Several studies have focused on the nature of the signal transduction
machinery downstream of the TGF- receptor complex. Genetic
approaches in Drosophila melanogaster and
Caenorhabditis elegans, along with supporting biochemical
evidence in Xenopus laevis and mammalian systems have
implicated the Smad family of proteins in signaling by TGF-
superfamily members. Evidence suggests that the Smads, through
ligand-dependent phosphorylation and
hetero-oligomerization, play crucial roles in growth and
transcriptional regulation by TGF-
(3). Other putative downstream
effectors in TGF-
signaling include FKBP12 (4),
farnesyltransferase-
(5), and TRIP-1 (6), each identified through
their specific interactions with TGF-
receptors. However, despite
extensive investigation, a clearly defined picture of the post-receptor
signaling machinery for TGF-
remains elusive.
Rho proteins, monomeric GTPases of the Ras superfamily (7), serve as
molecular switches that cycle between GTP-bound (active) and GDP-bound
(inactive) states. Rho proteins undergo a complex series of
post-translational modifications initiated by isoprenylation of a
C-terminal CAAX motif (8). Generally, Rho proteins are geranylgeranylated, although RhoB can be modified by either a geranylgeranyl or a farnesyl moiety (9). In most cases, Rho protein
function requires isoprenylation (8, 10). Rho proteins regulate
specific actin cytoskeletal structures, including stress fibers
(RhoA/C), lamellipodia (Rac1), and filopodia (Cdc42), and make manifest
the effects of growth factors and oncogenes on cell morphology (11).
Additionally, Rho family members have been implicated in cell growth
control as regulators of the G1/S transition and in
transformation, both alone and in cooperation with oncogenic Ras
(12-14). Recent evidence also suggests that Rho proteins, as regulators of the stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) signaling module, are components of the intracellular relay for transcriptional activation signals that originate from the
TGF- receptor complex (15).
RhoB, whose primary structure is 88% identical to the Rho family prototype RhoA, has several distinguishing features. Most notably, RhoB is inducible by a variety of stimuli, including the growth factors epidermal growth factor and platelet-derived growth factor, genotoxic stresses from UV light and alkylating xenobiotics, and activated tyrosine kinases such as v-Src and v-Fps (16-18). Unlike other Rho family members, both RhoB mRNA and protein are labile, with half-lives of approximately 20 min and 2 h, respectively (18, 19). Additionally, RhoB is subject to post-translational palmitoylation as a secondary membrane localization signal following isoprenylation (20). This contrasts with other Rho proteins, for whom this role is presumably accomplished by an intrinsic polybasic domain immediately proximal to the CAAX motif. Because of a high degree of homology, it has been hypothesized that RhoB and RhoA have similar biological activities. Both proteins promote transactivation of the serum response element of the c-fos promoter (10, 21). Also, both RhoB and RhoA can potentiate the transforming activity of oncogenic Ras (13, 14, 19). However, RhoB and RhoA are localized differently in mammalian cells. Furthermore, although RhoA subcellular localization is altered in response to lysophosphatidic acid, localization of RhoB is unaffected (22). In conjunction with differences in protein stability, post-translational modifications, and inducibility by exogenous stimuli, the physiologic functions of RhoB and RhoA appear at least partially distinct.
Because of its inducibility and the previously described effects of Rho
family members on TGF- signal transduction, we explored the
influence of TGF-
on RhoB and the subsequent influence of RhoB on
TGF-
-mediated gene expression. We demonstrate that RhoB turnover
occurs via ubiquitin-mediated destruction by the 26 S proteasome and
that TGF-
exerts a stabilizing influence toward RhoB, thereby
permitting its accumulation. Unlike other stimuli that induce RhoB gene
transcription, TGF-
-mediated accumulation of RhoB does not correlate
with changes in RhoB mRNA levels. Furthermore, transcriptional
activation of the TGF-
-responsive reporter p3TP-Lux is antagonized
by wild type RhoB in a dose-dependent manner but is not
affected by its dominant inhibitory point mutant, RhoB-N19. These data
suggest that TGF-
can regulate the abundance of some cytoplasmic
effectors by controlling their destruction. Additionally, these data
suggest that cells enlist multiple Rho proteins to coordinate
transcriptional activation signals initiated by TGF-
.
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MATERIALS AND METHODS |
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Cell Culture, Plasmids, Antibodies, and Reagents--
Mv1Lu
cells, obtained from ATCC (CCL-64), and R1B(L17) cells (28), obtained
from Dr. Joan Massagué, were subcultured in Dulbecco's modified
Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS).
The pcDNA3-HA:RhoB and pcDNA3-HA:RhoB-N19 were kindly provided
by Dr. George Prendergast (19). Dr. Mathias Trier provided pMT107,
encoding octameric, hexahistidine-tagged ubiquitin, which has been
previously described (27). pGE-I and pGE-IX (23) were generously
provided by Dr. Yoshiaki Monden (Banyu Pharmaceuticals, Inc., Ltd.,
Japan). pCMV5-TRI:HA (28) was the gift of Dr. Joan Massagué.
The
-HA monoclonal antibody 12CA5 was obtained from Boehringer
Mannheim. Remaining antibodies, including
-RhoB (119),
-RhoA
(26C4),
-Rac1 (C-14), and
-Rho-GDI (C-20), were purchased from
Santa Cruz Biotechnology, Inc. Donkey
-rabbit and donkey
-mouse
conjugates with horseradish peroxidase were obtained from Amersham
Pharmacia Biotech. TGF-
1 was generously provided by R & D Systems,
Inc. Lovastatin, obtained from Merck, was activated by alkaline
hydrolysis of its intramolecular lactone in 0.1 N NaOH/EtOH
prior to use. The proteasome inhibitor leucinyl-leucinyl-norleucinal (LLnL) and cycloheximide were obtained from Sigma. Protein A-Sepharose was obtained from Amersham Pharmacia Biotech. Oligo(dT) cellulose was
purchased from Collaborative Biomedical Products, Inc.
Electrophoresis and Immunoblotting of Rho Proteins-- Mv1Lu cells treated as described in figure legends were washed with ice-cold PBS and lysed by scraping into Nonidet P-40 lysis buffer (50 mM Tris, 150 mM NaCl, 1% Nonidet P-40, 10 mM EDTA, 20 µM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 5 mM NaF, 0.5 mM Na3(VO4), 5 mM N-ethylmaleimide, pH 8.0). Crude lysates were incubated at 4 °C for 15 min with agitation and clarified by centrifugation for 15 min at 4 °C. Protein concentrations determined by Bradford protein assay were used to normalize gel loading. Proteins were resolved by Tricine SDS-PAGE essentially as described (24) and electrotransferred to polyvinylidene difluoride membranes (Millipore, Inc.) in 25 mM Tris, 192 mM glycine, 20% methanol, pH 8.35. Membranes were blocked in PBS containing 0.25% gelatin and 0.05% Tween-20 and probed with primary antibody as indicated for 2 h at ambient temperature. After washing with PBS + 0.05% Tween-20, membranes were probed for 1 h with appropriate secondary antibody conjugate. Proteins were visualized by chemiluminesence detection. Scanning densitometry was performed on an Alpha Innotech IS-1000 Digital Imaging System.
Transient Assays Using p3TP Luciferase Reporter--
Mv1Lu or
R1B(L17) cells in 6-well plates were transiently transfected with
pcDNA-HA:RhoB, pcDNA3-HA:RhoB-N19, and pCMV5-TRI:HA by the
DEAE-dextran method as shown in the legend for Fig. 5. The constitutive
reporter CMV-
gal was also included to normalize transfection
efficiency. After overnight recovery in DMEM + 10% FBS, cells were
incubated in DMEM + 0.2% FBS with or without 40 pM TGF-
for 16 h. For all assays, cells were washed with ice-cold 1× PBS.
Cell lysates were then prepared by rocking for 15 min in 100 mM
Na2HPO4/NaH2PO4, 1%
Triton X-100, 1 mM
-mercaptoethanol, 2 mM
EDTA, pH 7.8, and subsequently clarified by brief centrifugation. Luciferase and
-galactosidase activities were measured in an Analytical Luminescence Labs Monolight 2010 luminometer.
-Galactosidase activity was determined with the proprietary
Galacton-Plus luminescent substrate/accelerator system (Tropix, Inc.)
according to the manufacturer's instructions.
Northern Blot Analysis of Poly(A) RNA-- Mv1Lu cells were maintained in DMEM + 0.2% FBS for 24 h and then incubated as indicated in the legend for Fig. 2. Poly(A) RNA was purified using oligo(dT) cellulose according to standard methods (25). 4 µg of poly(A) RNA was resolved in a 1% formaldehyde-agarose gel and transferred to Hybond N+ filters. After 4 h of prehybridization (50% deionized formamide, 150 µg/ml salmon sperm DNA, 1× Denhardt's, 50 µg/ml poly(A), 0.1% SDS, 5× SSC) at 42 °C, filters were probed for 18 h with either the complete RhoB cDNA or a cyclophilin-specific probe, both 32P-labeled by random priming. Filters were washed sequentially, twice with 2× SSC + 0.1% SDS, then three times with 0.5× SSC + 0.1% SDS at 42 °C, and once in the same wash buffer at 65 °C. Results were visualized with a Molecular Dynamics, Inc. PhosphorImager 445 SI.
Reverse Transcriptase-coupled PCR--
Primers specific for mink
RhoB were based upon regions of absolute nucleotide conservation
from rat to human RhoB. Primer sequences were as follows:
5'-GAGAACATCCCCGAGAAGT-3' and 5'-TGCAGCAGTTGATGCAGCC-3'. A 304-bp
fragment is predicted based upon the human and rat RhoB sequences.
Primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the
internal control, were purchased from CLONTECH,
Inc. Mv1Lu cells were maintained in DMEM with 0.2% FBS. Total RNA was isolated from TGF--treated or time matched, untreated controls using
guanidinium isothiocyanate as described previously (26). Total RNA (0.5 µg) was reverse transcribed using an oligo(dT) primer. The resulting
cDNA was amplified by PCR with RhoB-specific and GAPDH-specific
primers simultaneously. PCR products were resolved in 2% agarose/0.5×
TBE (1× TBE = 90 mM Tris, 90 mM borate, 2 mM EDTA, pH 8.0) gels and visualized by ethidium bromide
staining and ultraviolet transillumination. The GAPDH PCR product
was used to normalize gel loading. PCR parameters permitted both RhoB
and GAPDH products to be analyzed within the linear range. The
RhoB-specific PCR product was isolated and subjected to automated
dideoxy sequencing to confirm its identity.
Determination of RhoB Half-life in Mv1Lu Cells--
Mv1Lu cells
were maintained in DMEM + 10% FBS. In parallel cultures, growth medium
was changed to either DMEM + 0.2% FBS or that same medium supplemented
with 40 pM TGF-1. Cultures were incubated in these
conditions for 4 h. At the end of this 4-h incubation,
corresponding to time 0, growth medium was changed to DMEM + 0.2% FBS
and 30 µg/ml cycloheximide, with or without coincident 40 pM TGF-
1. At the indicated times, cells were lysed in
Nonidet P-40 lysis buffer and clarified as above. Equivalent amounts of
total cellular protein were analyzed by Tricine SDS-PAGE and
RhoB-specific immunoblot analysis. Scanning densitometry was performed
as above. To ensure abrogation of new RhoB synthesis during the
cycloheximide chase, control cultures were maintained in
M
/C
medium for 1 h then incubated for
4 h in M
/C
medium supplemented with
200 µCi/ml [35S]methionine/cysteine with or without
coincident 30 µg/ml cycloheximide. Cell monolayers were washed and
lysed in Nonidet P-40 lysis buffer as described above. Crude lysates
were clarified by centrifugation and precleared with protein
A-Sepharose. Supernatants were then incubated for 16 h with either
1 µg of RhoB-specific antibody or nonspecific rabbit serum. Immune
complexes were collected with protein A-Sepharose and subjected to
SDS-PAGE as described above. Labeled proteins were detected in fixed
gels by autoradiography.
Determination of RhoB Ubiquitination and Turnover by the 26 S Proteasome-- Mv1Lu cells maintained in DMEM + 10% FBS were transfected by the DEAE-dextran technique with pMT107 and pcDNA3-HA:RhoB in the combinations indicated in Fig. 4. Cultures were incubated for 36 h in DMEM + 10% FBS and then switched to DMEM + 0.2% FBS with or without coincident 50 µM LLnL. After 24 h of incubation, cells were lysed in 100 mM NaH2PO4/Na2HPO4, 6 M guanidinium-HCl, 20 µM phenylmethylsulfonyl fluoride, 10 mM N-ethylmaleimide, pH 8.0, and ubiquitinated proteins purified by Ni2+/NTA-agarose chromatography essentially as described (27). Ubiquitinated proteins were resolved by Tricine SDS-PAGE and subsequently examined by RhoB-specific and HA epitope tag-specific immunoblot analysis as above. The degree of ubiquitin conjugation was estimated by mobility in SDS-PAGE relative to the nonubiquitinated RhoB protein and molecular weight markers.
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RESULTS |
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TGF- Promotes Accumulation of Isoprenylated RhoB--
Several
stimuli have been shown to promote accumulation of RhoB mRNA (16,
17, 18). However, although more closely aligned with function, RhoB
protein levels have been examined in a more limited context. We
examined the abundance of RhoB, the Rho family members RhoA and Rac1,
and the Rho-specific regulator Rho-GDI in Mv1Lu cells (Fig.
1A, a-d) in the
presence and absence of TGF-
. Lovastatin, which abrogates isoprenoid
synthesis by inhibition of hydroxymethylglutaryl-coenzyme A reductase,
was used to distinguish between prenylated (*) and nonprenylated forms
based upon mobility differences in Tricine SDS-PAGE. Prenylated and
nonprenylated RhoB were easily distinguished, but differentially
prenylated forms of RhoA and Rac1 were not readily resolved. As
anticipated, no mobility differences were noted for Rho-GDI, because it
is not a prenylated protein.
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RhoB mRNA Levels Are Unaffected by TGF---
Growth factors
such as epidermal growth factor and platelet-derived growth factor are
known to increase the level of RhoB mRNA (16, 17). To address the
possibility of changes in RhoB mRNA level, we examined the
abundance of RhoB mRNA by both Northern hybridization of poly(A)
RNA (Fig. 2A) and by RT-PCR
(Fig. 2B). Mv1Lu cells were first incubated for 24 h in
low serum medium to limit the potential confounding influences of serum
components. Parallel cultures were then given a fresh aliquot of low
serum medium with or without 40 pM TGF-
and incubated
for the indicated times. Northern analysis of poly(A) RNA using the
RhoB cDNA as a probe yielded a single band of 2.6 kilobases
characteristic of RhoB mRNA (16-18). As an internal control, we
employed the 700-bp 1B15 probe specific for cyclophilin. Once again, a
single band at 1 kilobases, characteristic of cyclophilin mRNA, was
observed. The filter was probed sequentially with the RhoB and
cyclophilin probes and analyzed by PhosphorImager. Fig. 2A
demonstrates that RhoB mRNA levels are unchanged by TGF-
throughout the 24-h exposure. Concerned that subtle changes in RhoB
mRNA levels might be missed by direct hybridization, we employed
RT-PCR to amplify subtle differences if they existed. Regions of
absolute nucleotide conservation within the RhoB gene from rat to human
and maximally divergent from other Rho family members were used to
produce RhoB-specific primers. The identity of the predicted 304-bp
RT-PCR product was confirmed by automated dideoxy sequencing (data not
shown). Quantitation of the RhoB-specific RT-PCR product was normalized
to GAPDH. As in Fig. 2A, the time course of RhoB mRNA
levels in the presence and absence of TGF-
was preceded by
incubation in low serum medium. Consistent with the Northern
hybridization, we observed no change in RhoB mRNA levels.
Additionally, TGF-
had no effect on luciferase reporter activity
driven by a complete RhoB promoter (pGE-I, see "Materials and
Methods"), which was inducible by 10% FBS (data not shown). These
results suggested that the observed modulation of RhoB protein level
induced by TGF-
did not involve changes in the abundance of RhoB
mRNA.
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TGF- Stabilizes RhoB Protein from Turnover by the 26 S
Proteasome--
Regulation of protein stability has emerged as an
important mechanism for controlling biological function (29). Labile
proteins are excellent candidates for such regulation, because they can be rapidly increased in response to exogenous stimuli, yet their effects rapidly reversed by proteolysis. The short to intermediate half-life of RhoB suggests that its levels could be controlled by
regulating its destruction. To address the stability of RhoB in the
context of TGF-
, we examined its fate in Mv1Lu cells in the presence
and absence of TGF-
using cycloheximide to abrogate new RhoB
synthesis (Fig. 3). Parallel cultures of
Mv1Lu cells were incubated for 4 h in low serum medium with or
without 40 pM TGF-
to create permissive environments
characteristic of each treatment. Cells were then incubated in chase
medium identical to the corresponding pretreatment mediums but
supplemented with 30 µg/ml cycloheximide. At the times indicated,
extracts of total protein were prepared and analyzed by
-RhoB
immunoblot. Total RhoB signal was estimated by scanning densitometry
and normalized to RhoB present at the start of the chase. In low serum
medium, RhoB displays an apparent half-life between 60 and 90 min,
consistent with previously published results (19). However, RhoB
remains stable in the presence of TGF-
. To confirm that RhoB
detected during the chase reflected pre-existing protein rather than
new RhoB synthesis, we subjected Mv1Lu cells to labeling with
[35S]methionine/cysteine during the chase period either
in the presence or in the absence of cycloheximide. RhoB protein
synthesized during the chase was isolated by immunoprecipitation. Fig.
3C demonstrates that new RhoB synthesis was effectively
abolished by cycloheximide. In concert, these data suggest that RhoB
accumulation in response to TGF-
reflects antagonism of the
mechanisms required for its turnover.
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RhoB Turnover Occurs via Ubiquitin-mediated Proteolysis--
The
majority of protein turnover outside the lysosomal compartment occurs
via the 26 S proteasome by ubiquitin-dependent proteolysis (29). To gain additional insights into the mechanism of RhoB turnover
in Mv1Lu cells, we examined its conjugation to ubiquitin and its
subsequent elimination by the 26 S proteasome. Mv1Lu cells were
transiently transfected with expression constructs encoding HA-tagged
RhoB (HA-RhoB) and octameric, hexahistidine-tagged ubiquitin (H6-ubiquitin), in the combinations shown in Fig.
4. Transfected cells were then incubated
in the presence or the absence of 50 µM LLnL, an
inhibitor of proteolysis by the 26 S proteasome. Ubiquitin-conjugated proteins were collected under denaturing conditions by
Ni2+/NTA-agarose chromatography and subjected to SDS-PAGE
and immunoblot analysis using either an -RhoB or an
-HA antibody.
These conditions permit detection of only covalent associations between
ubiquitin and its targets. When 26 S proteasome activity is antagonized by LLnL, ubiquitinated forms of RhoB, confirmed by detection with both
the RhoB- and HA-specific antibodies, are noted in cells transfected
with both expression plasmids. No such bands are detected in mock
transfected cells or in cells transfected only with a H6-ubiquitin
expression construct. Electrophoretic mobilities of the ubiquitinated
RhoB forms suggest that conjugation by one, two, and three ubiquitins
could be easily discerned, whereas higher order conjugates were not
resolved. The stabilization of RhoB in response to TGF-
in
conjunction with this data suggests that TGF-
may exert negative
regulatory influence toward some aspect of ubiquitin-mediated protein
turnover by the 26 S proteasome.
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RhoB Antagonizes TGF--mediated Transcriptional
Activation--
Members of the Rho family can transactivate the
TGF-
-responsive promoter p3TP-Lux in a ligand-independent manner. To
explore the impact of RhoB on TGF-
signal transduction, we examined
p3TP-Lux transactivation in Mv1Lu cells and their T
RI deficient,
TGF-
-nonresponsive derivative, R1B(L17). Cells were transfected with
p3TP-Lux reporter and either HA-RhoB or HA-RhoB-N19 expression plasmids
as shown in Fig. 5. R1B(L17) cells were
additionally transfected with a T
RI expression plasmid to restore
TGF-
responsiveness. Luciferase activity in cell extracts was
determined in the presence and absence of TGF-
. Equivalent
expression of HA-RhoB and HA-RhoB-N19 was confirmed by
-HA
immunoblot analysis in Mv1Lu cells in a parallel experiment (data not
shown). In both Mv1Lu and R1B(L17) cells, HA-RhoB antagonized
TGF-
-induced p3TP-Lux activation in a dose-dependent manner. HA-RhoB-N19 failed to attenuate TGF-
-mediated p3TP-Lux activity in either Mv1Lu or R1B(L17) cells. These data suggest that
RhoB GTPase function is required to inhibit TGF-
-mediated transcriptional activation.
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DISCUSSION |
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The signal transduction machinery downstream of the TGF-
receptor complex has been the focus of intense investigation.
Considerable evidence suggests that Smad proteins are essential
regulators of TGF-
-mediated growth inhibition and transcriptional
activation (3). However, recent data also suggest a critical role for Rho proteins in TGF-
-mediated transcriptional activation (15). GTPase deficient, constitutively active mutants of RhoA, Rac1, and
Cdc42Hs can transactivate the TGF-
-responsive reporter p3TP-Lux in
the absence of ligand. Additionally, Rac1 and to a lesser extent RhoA
can potentiate the positive influence of TGF-
on the same reporter.
Dominant negative derivatives of Rac1, RhoA, and Cdc42Hs individually
antagonized but collectively abolished TGF-
-mediated transcriptional
activation. Furthermore, dominant negative versions of MEKK1, MKK4,
SAPK/JNK, and c-Jun, components of the SAPK/JNK pathway, each abolished
the positive influences of constitutively active Rho proteins on
p3TP-Lux transactivation. In concert, these results suggest that
TGF-
responsiveness may require the cooperation of the Rho/SAPK/JNK
and Smad signal transduction cascades.
RhoB, like other members of the Ras superfamily, acts as a binary
switch that cycles between GTP-bound (active) and GDP-bound (inactive)
states. RhoB expression can be induced by a variety of stimuli, and
evidence suggests that both RhoB mRNA and RhoB protein are labile,
with half-lives of approximately 20 min and 2 h, respectively (18,
19). The inducibility and lability of RhoB, in conjunction with its
post-translational modifications and its regulation by a bound guanine
nucleotide, permit control of its function at several levels and as
such present unique regulatory opportunities. We demonstrate that
TGF- promotes the accumulation of isoprenylated RhoB by antagonizing
its destruction. Interestingly, accumulation of RhoB in its
isoprenylated form may reflect a connection between TGF-
signaling
and protein prenylation, suggested from the observed interaction of
farnesyltransferase-
and the TGF-
type I receptor (5). The
ability of TGF-
to regulate prenyltransferase activity toward RhoB
is currently being explored. In contrast to other stimuli, we see no
correlation between TGF-
stimulation and the abundance of RhoB
mRNA. Although this cannot be eliminated as a contributing factor,
our data suggest that regulated RhoB turnover is primarily responsible
for TGF-
-induced RhoB accumulation. By extension, regulated protein
stability might reflect a more general mechanism by which TGF-
controls the activities of its downstream effectors to produce its
diverse biological responses.
A small fraction of total cellular protein undergoes rapid turnover.
Frequently these proteins are components of regulatory pathways. Their
limited life span, in concert with other control mechanisms, provides
restrictions on their biological influence. By extension, controlling
the turnover rate of regulatory proteins, thereby influencing their
steady-state levels, could dramatically affect signal transduction via
the pathways in which these proteins participate. Several examples of
controlled proteolysis as a means for regulating protein function have
been described (29). The majority of selective protein turnover in
eukaryotic cells is achieved by obligatory conjugation between lysine
residues of the target protein and the C terminus of ubiquitin.
Sequential ubiquitin additions yield multiubiquitin chains that direct
the target protein for destruction by the 26 S proteasome (29). We show
that RhoB protein is conjugated to ubiquitin in Mv1Lu cells and that
ubiquitinated RhoB is subsequently eliminated by the 26 S proteasome.
The participation of the 26 S proteasome and the coincident influence
of TGF- on RhoB stability suggests that TGF-
may regulate a
component of the ubiquitin-dependent proteolytic
machinery.
The reporter p3TP-Lux is frequently used as a measure of TGF-
responsiveness in mammalian cells. To explore the influence of RhoB on
TGF-
signaling, we examined the impact of its expression or that of
its dominant-negative mutant, RhoB-N19, on p3TP-Lux transactivation. In
either Mv1Lu cells or R1B(L17) cells rendered TGF-
-responsive by
T
RI cotransfection, wild type RhoB antagonized p3TP-Lux activation
by TGF-
. RhoB-N19 failed to attenuate and instead perhaps
potentiated p3TP-Lux activity in these cells, consistent with dominant
interference with endogenous RhoB function. These data suggest that
RhoB exerts negative regulatory influence on TGF-
-induced
transcriptional activation. Functionally distinct negative regulation
of TGF-
signaling has also been described for FKBP12, Smad6, and
Smad7 and correlated with binding to T
RI. In light of the
potentiating influence of other Rho family members toward p3TP-Lux
transactivation and the labile nature of RhoB, it is attractive to
speculate that RhoB accumulation in response to TGF-
imposes
negative but inherently self-limited restriction on the Rho-mediated
aspects of TGF-
signal transduction.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge Mary Aakre for technical assistance and Dr. George Prendergast, Dr. Joan Massagué, Dr. Mathias Trier, and Dr. Yoshiaki Monden for provision of reagents. Also, we thank Dr. Brian Law and Maureen McDonnell for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants CA42572 and CA48799.The costs of publication of this article were defrayed in part by the payment of page charges. The 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: Vanderbilt Cancer
Center, 649 MRBII, Nashville, TN 37232-6838. Tel.: 615-936-1786; Fax:
615-936-1790; E-mail: hal.moses{at}MCmail.vanderbilt.edu.
1
The abbreviations used are: TGF-,
transforming growth factor
; T
RI, transmembrane serine/threonine
kinase receptor type I; T
RII, transmembrane serine/threonine kinase
receptor type II; SAPK/JNK, stress-activated protein kinase/c-Jun
N-terminal kinase; DMEM, Dulbecco's modified Eagle's medium; FBS,
fetal bovine serum; HA, hemagglutinin; LLnL,
leucinyl-leucinyl-norleucinal; PBS, phosphate-buffered saline; PAGE,
polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; RT,
reverse transcription; bp, base pair; GAPDH, glyceraldehyde-3-phosphate
dehydrogenase; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; NTA,
nitrilotriacetic acid.
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REFERENCES |
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