From the Center for Cancer Biology and Nutrition,
Alkek Institute of Biosciences and Technology, and Department of
Medical Biochemistry and Genetics, Texas A&M University System Health
Science Center, Houston, Texas 77030, the § College of
Life Sciences, Hunan Normal University, Changsha, Hunan 410081, People's Republic of China, and the ¶ Department of
Biochemistry and Molecular Biology, Baylor College of Medicine,
Houston, Texas 77030
Received for publication, August 30, 2002, and in revised form, December 11, 2002
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ABSTRACT |
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The Rho family of small GTPases, including Rho,
Rac, and Cdc42, play essential roles in diverse cellular functions. The
ability of Rho family GTPases to participate in signaling events is
determined by the ratio of inactive (GDP-bound) and active (GTP-bound)
forms in the cell. The activation of Rho family proteins requires the exchange of bound GDP for GTP, a process catalyzed by the Dbl family of
guanine nucleotide exchange factors (GEFs). The GEFs have high affinity
for the guanine nucleotide-free state of the GTPases and are thought to
promote GDP release by stabilizing an intermediate transition state. In
this study, we have identified and characterized a new
Rac/Cdc42-specific Dbl family guanine nucleotide exchange factor, named
GEFT. GEFT is highly expressed in the excitable tissues, including
brain, heart, and muscle. Low or very little expression was detected in
other nonexcitable tissues. GEFT has specific exchange activity for Rac
and Cdc42 in our in vitro GTPase exchange assays and
glutathione S-transferase-PAK pull-down assays with
GTP-bound Rac1 and Cdc42. Overexpression of GEFT leads to changes in
cell morphology and actin cytoskeleton re-organization, including the
formation of membrane microspikes, filopodia, and lamilliopodia.
Furthermore, expression of GEFT in NIH3T3 cells promotes foci
formation, cell proliferation, and cell migration, possibly through the
activation of transcriptional factors involved in cell growth and
proliferation. Together, our data suggest that GEFT is a
Rac/Cdc42-specific GEF protein that regulates cell morphology, cell
proliferation, and transformation.
The Rho-related GTP-binding proteins of the Ras superfamily
function as molecular switches in a variety of cellular signaling pathways and regulate diverse cellular functions, including control of
cell morphology, cell migration, cell growth and proliferation, actin
dynamics, transcriptional activation, apoptosis signaling, and neurite
outgrowth (1-7). Among the 18 known mammalian members of the Ras
superfamily, Rho A, Rac1, and Cdc42 are the most studied and well
characterized. Each of the three members has a distinct function in
cell actin cytoskeleton organization and responses (6). For example,
Rho has been shown to regulate the formation of actin stress fibers and
focal adhesion in fibroblasts (6, 8). In contrast, Rac1 specifically
induces membrane ruffling and lamellipodia formation (8, 9), and Cdc42
mediates the formation of filopodia and actin microspikes (9, 10).
Besides the roles in actin cytoskeleton reorganization, Rho, Rac, and Cdc42 seem to be involved in a number of other cellular functions, including gene expression and transcriptional regulation (11-15), cell
growth and cell cycle progression (16-21), the Jun amino-terminal kinase (JNK)1 signaling
pathway (22, 23), as well as axon guidance and neurite extension (7,
24-26).
Similar to all members of the Ras superfamily proteins, the
GTP-binding/GTP hydrolysis cycle of Rho family proteins is tightly controlled. The ability of Rho family GTPases to participate in signaling events is determined by the ratio of GTP/GDP-bound forms in
the cell. Like all GTPases, they exist in an inactive (GDP-bound) and
an active (GTP-bound) conformation. The activation of Rho family
proteins requires the exchange of bound GDP for GTP, a process
catalyzed by the Dbl family of guanine nucleotide exchange factors
(GEFs) and other specific GEFs (27-29). Like the ligand-activated seven-transmembrane receptors in activation of heterotrimeric G-proteins, the GEFs have high affinity for the guanine nucleotide-free state of the GTPases and are thought to promote GDP release by stabilizing an intermediate transition state (28, 30).
The Dbl family of oncoproteins is Rho-specific GEFs and contains ~60
distinct mammalian members (27, 28). All Dbl family proteins consist of
a Dbl homology (DH) domain (~200 amino acid residues) and a
pleckstrin homology (PH) domain (~100 amino acid residues)
immediately COOH-terminal to the DH domain (27, 31, 32). DH domains
interact directly with Rho GTPases to catalyze guanine nucleotide
exchange by preferentially binding to Rho GTPases depleted of
nucleotide and Mg2+ (33-38). Recent studies have
determined the structure of the DH and PH domains of Tiam-1 bound to
nucleotide-free Rac1 and the potential mechanism to stimulate guanine
nucleotide exchange of Rho GTPases by Dbl family GEFs (30, 36, 39). PH
domains have been found and invariably follow the DH domain in the Dbl family of proteins. PH domain contains ~100 amino acids and has been
found in a number of proteins (40, 41). Although DH-associated PH
domains promote the translocation of Dbl-related proteins to plasma
membranes (42, 43), PH domains have been shown to participate directly
in GTPase binding and regulation of GEF activity in the presence or
absence of phosphoinositides (28, 34, 44, 45).
Two of the well characterized effectors of Rac/Cdc42 GTPases are the
PAK family of serine/threonine kinases and the WASP proteins. In
response to physiological stimuli, the active GTP-bound Rac and Cdc42
interact with the p21 (Rac/Cdc42)-binding domain of PAK,
resulting in PAK autophosphorylation and increased kinase activity, and
downstream activation of a variety of cellular functions (22, 46-50).
Activation of the WASP protein by GTP-bound Cdc42 leads to the
signaling cascades mediated by the WASP protein, resulting in the
polymerization of cytoskeletal actin filaments (51, 52). Therefore,
interaction between the Rac/Cdc42 and their effectors are reversible
and are dependent on the GTP/GDP binding states of the Rac and Cdc42 GTPases.
In this study, we have identified a guanine nucleotide exchange factor
in both human and mouse, named GEFT, a member of the Dbl family
proteins. GEFT is highly expressed in the excitable tissues, such as
brain, heart, and muscle. The protein exhibited potent guanine
nucleotide exchange activity on Rac1 and Cdc42, whereas little activity
was observed on RhoA. Overexpression of GEFT in NIH3T3 cells caused
transformed phenotypes similar to the activation of Rac1 and Cdc42 by
Vav. Furthermore, expression of GEFT induces the formation of
lamellipodia, actin microspikes, and filopodia, similar to the
activation of Rac and Cdc42 proteins. In addition, GEFT also stimulates
the transcriptional activities of SRE, Elk1, and the c-Jun
transcription factors. Taken together, our data suggest that GEFT is a
specific activator preferentially for Rac1 and Cdc42 GTPases and may
play important roles in cell morphology, growth, and proliferation.
DNA Constructs--
The mouse GEFT fragment was initially
identified by enhanced retroviral mutagen (ERM) strategy (53). Briefly,
we constructed enhanced retroviral mutagen (ERM) vectors that contained
several engineered sequences (e.g. an ERM tag and a splice
donor) controlled by a tetracycline-responsive promoter. The ERM
vectors were introduced into the NIH3T3 cells. Endogenous genes can
thus be randomly activated and tagged in a conditional system. NIH3T3
cells were used to screen for focus-forming genes using the ERM
strategy. Full-length cDNAs encoding human GEFTs were
obtained by screening human brain library
(Clontech). For mammalian expression, cDNAs
encoding GEFT were inserted into the HindIII and
SalI sites of pCMV-Tag2B (Stratagene), resulting in the
plasmid of pCMV-GEFT. For expression and purification of recombinant
GEFT in bacteria, GEFT was subcloned in-frame into pQE-31 (Qiagen),
generating His-tagged pQE-GEFT. The wild-type full-lengths of the Rho
family GTPases, Cdc42, Rac1, and RhoA, were subcloned into the
BamHI and SalI sites of pGEX-4T-1, a GST gene
fusion vector (Amersham Biosciences), respectively, to produce three
GST-fused pGEX vectors.
Expression and Purification of Recombinant GEFT and
GTPases--
Bacterially expressed His6-tagged GEFT
protein and GST fusion GTPases were purified according to the
standard procedures of the manufacturers. Escherichia coli
strain BL21 was transformed by pQE-GEFT and pGEX vectors,
respectively, grown to midlog phase at 37 °C, and then induced with
1 mM isopropyl-1-thio- Guanine Nucleotide Exchange Assays--
The effects of GEFT on
the dissociation of [3H]GDP from the Rho family GTPases
were assayed as described previously (37, 54, 55). Briefly, each 1 µM eluted GTPases was incubated with 1 µM
[3H]GDP at 25 °C in the buffer B containing 50 mM HEPES (pH 7.6), 100 mM NaCl, and 1 mM dithiothreitol in the presence or absence of purified
1.5 µM GEFT. To stabilize the [3H]GDP-bound
GTPases, the reaction mixtures were supplemented with 20 mM
MgCl2. After a binding equilibrium was reached (~60 min), the GDP/GTP exchange reactions were initiated by the addition of excess
free 400 µM GTP (final concentration). At different time
points, the reactions were terminated by filtration of 20 µl of the
mixtures through nitrocellulose filters. And the filters were washed
twice with the ice-cold buffer B. The amount of the radionucleotides
remaining bound to the Rho GTPases (RhoA, Rac1, and Cdc42) were
quantified by scintillation counting, and normalized as the percentage
of [3H]GDP bound at time 0. For each time point, the
samples were assayed in triplicate.
Cell Culture, Transfection, and Transformation Assays--
HeLa,
COS-7, and NIH3T3 cells were maintained in Dulbecco's modified
Eagle's medium supplemented with 10% fetal bovine serum. Cell
transfection was performed using LipofectAMINE (Invitrogene) as
previously described according to the manufacturer's instructions (49). Cells were then allowed to grow 48 h. For each assay, control vector encoding LacZ was used as a control.
For foci formation analyses, infected NIH3T3 cells were maintained in
growth medium for 12-14 days and assayed as previously described (56).
Briefly, NIH3T3 cells were infected with GEFT virus or a vector
(pMSCV2.1) control virus for 2 days. Cells were then washed with PBS,
counted, and plated as shown in 6-well plates coated with 1 µg/ml
collagen. 5 ml of Dulbecco's modified Eagle's medium with 10% fetal
bovine serum was added and changed every 3 days. Cells were allowed to
grow for 14 days in a 37 °C incubator with a 95:5, air/carbon
dioxide mixture. At the end of 14 days, cells were washed once with PBS
and stained with crystal violet (0.5%), and the number of foci of
transformed cells was then quantitated. Total number of foci in each
well was counted with a light microscope and foci numbers were averaged
for the three wells.
Transient Expression Reporter Gene Assays--
COS-7 cells were
transfected by using LipofectAMINE (Invitrogen) as described previously
(57). Analyses of the cell lysates of the transiently transfected cells
were performed using enhanced chemiluminescence reagents from Promega
as described previously (58). The reporter constructs for AP1-Luc and
c-Jun-Luc were obtained from Stratagene. Reporter constructs for
Elk1-luc and SAP1-luc were obtained from Dr. K. L. Guan at the
University of Michigan. The data presented are the mean of three
individual transfected wells and the experiments were performed at
least three times.
Northern Blotting Analysis of GEFT Expression in Human
Tissues--
To study the expression patterns of GEFT in different
human tissues, a RNA filter comprising poly(A)-selected RNAs of
multiple human tissues (Clontech, Inc.) was
hybridized with specific 32P-labeled cDNAs as described
previously (49, 58). In brief, human GEFT probe were radiolabeled with
[ Immunoprecipitation, Immunoblotting, Immunocytochemistry, and
Fluorescence Imaging--
Immunoprecipitation of individual proteins
was carried out as previously described (49). In brief, cell lysates (1 mg of protein) were incubated with antibodies (1-10 µg) at 4 °C
for 1 h in a final volume of 1 ml of modified RIPA buffer (10 mM sodium phosphate, pH 7, 1% Triton X-100, 0.1% SDS, 2 mM EDTA, 150 mM NaCl, 50 mM NaF,
0.1 mM sodium vanadate, 4 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride) with constant rocking.
After the addition of protein A-agarose beads, reactions were incubated at 4 °C for 1 h. Immune complexes were resolved by SDS-PAGE and subjected to immunoblotting for interacting proteins.
For fluorescence labeling of the cellular components and observing cell
morphology changes, 48 h after transfection, cells were plated on
10 µg/ml fibronectin-coated glass coverslips. Then, cells were fixed
with 4% paraformaldehyde for 20 min, blocked with 10% bovine serum
albumin, and incubated with monoclonal antibody against FLAG (M2
monoclonal, Sigma). Actin filaments were labeled with
rhodamine-conjugated phalloidin (Molecular Probes). Double-label immunostaining was done with appropriate fluorochrome-conjugated secondary antibodies. Fluorescent images of cells were captured on a
CCD camera mounted on an Olympus inverted research microscope using
Ultraview imaging software (Olympus, Inc.).
Binding of GEFT to Rho GTPases and GST-p21-binding Domain
Pull-down Assays--
To determine GEFT binding affinity to the Rho
GTPases, 20 µg of His-tagged protein was incubated at 4 °C
overnight with 20 µl of GSH-agarose beads loaded with 20 µg of each
GTPase, Cdc42, Rac1, or RhoA in the absence of guanine nucleotides. The
beads were washed three times with PBS. The bound proteins were
separated by SDS-PAGE, and His-tagged GEFT proteins were detected by
Western blotting using an anti-His6 monoclonal antibody
(Santa Cruz Biotechnology).
GTPase activation assays in the cells were performed by GST-p21-binding
domain pull-down assays as described previously (59-61). Briefly,
cells transfected with GEFT or a control plasmid (pCMV-LacZ) were
washed and lysed on the dish in 50 mM Tris (pH 7.5), 500 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.5% sodium
deoxycholate, 10% glycerol, 10 MgCl2, 10 µg/ml leupeptin
and aprotinin, and 1 mM phenylmethylsulfonyl fluoride.
GTP-bound Rac1 or Cdc42 was pulled down using the GST-p21-binding
domain of PAK1 immobilized on glutathione beads. The amount of active
Rac1 and Cdc42 (GTP-bound form) was detected by Western blot using
specific antibodies against Rac1 and Cdc42, respectively.
Cell Proliferation Assay--
Proliferation studies were carried
out using the CellTiter96 AQueous One solution cell proliferation assay
(Promega). Briefly, cells were transfected with GEFT or a control
plasmid. Cells were plated at 500 cells/well and allowed to adhere to
the plate. At the indicated time points, the AQueous One solution was
added to the samples and measured at 490 nm.
Boyden Chamber Cell Migration Assays--
Cell
migration/motility assays were examined using modified Boyden chambers,
as described previously (62, 63). Briefly, NIH3T3 cells were stably
transfected with GEFT or vector (pCMV-tag2B). The outside of the
filters was coated with 1 µg/ml collagen for 1 h and then washed
three times with PBS. Filters were then incubated with Dulbecco's
modified Eagle's medium with bovine serum albumin for 1 h.
Filters were then put into Dulbecco's modified Eagle's medium without
fetal bovine serum and with 0.5 ng of mouse basic fibroblast growth
factor. NIH3T3 cells expressing the receptor or vector were
seeded at 20,000/well on top of the filter. Plates were incubated for
6 h. Excess cells that did not migrate through the filter were
removed from the insides of the filters. Cells were then fixed with 4%
paraformaldehyde for 20 min, washed three times with PBS, and then
stained with crystal violet. Stained cells were examined under the microscope.
Identification, Expression, and Domain Structures of GEFT--
To
identify genes responsible to tumorigenesis, an ERM strategy was
used to screen for foci-forming genes in NIH3T3 cells (53). One of the
novel genes, mutagenized by the ERM, has shown strong oncogenic
activity and was identified by reverse transcriptase-PCR and direct
sequencing. The gene product shows sequence homology to the Dbl family
of GEFs, and was named GEFT. Subsequently, we cloned the human and
mouse GEFT full-length open reading frame by reverse transcriptase-PCR
and by 5'- and 3'-rapid amplification of cDNA ends. The mouse GEFT
(mGEFT) sequence is 90% identical to the human GEFT
(hGEFT) (Fig. 1A).
In contrast to hGEFT, mGEFT possesses an extra NH2-terminal
domain. Like other family members of the Dbl proteins, GEFT has an
NH2-terminal Rho exchange factor domain (Dbl homology
domain, called DH domain) and is followed by a PH domain (Fig.
1B). Sequence alignment of Dbl domains from hGEFT and other
Dbl-containing proteins shows significant homology in this region,
suggesting that GEFT is a potential exchange factor for the Rho family
(RhoA, Rac1, and Cdc42) of GTPases (Fig. 1C). A data base
search found that GEFT shows 35% sequence identity with human
Huntingtin-associated protein-interacting protein (Duo protein) or the
spectrin-like Kalirin (64, 65). GEFT also shares 35% and 60% sequence
homology with the first and second DH domains of human protein Trio,
respectively (66). GEFT contains 13 exons and is localized in
chromosome 12q13.11, a region frequently amplified in sarcomas and
brain tumors. In searching the single nucleotide polymorphisms data
base, we found two single nucleotide polymorphisms in the coding region
of the GEFT protein: one at exon 10 with G to A substitution without an
amino acid (Leu) change; and the other in exon 12 with nucleotide
change A to G and an amino acid change Gln to Arg (Q401R). The
potential role of these single nucleotide polymorphisms in the protein
is not clear at this moment.
To examine the expression of GEFT in human tissues, a
Northern blot analysis with multiple tissue membrane
(Clontech) was performed. As shown in Fig.
2, we detected one main transcript of
~3 kb, with highest expression in human brain, heart, and muscle, and
less extent in small intestine, colon, liver, placenta, and lung. Weak
or no expression was found in the examined immune tissues (Fig. 2). In
accordance with the predicted size, a protein with a relative molecular
mass of 53,000 (53 kDa) was identified with anti-FLAG tag monoclonal
antibody (M2, Sigma) in cells transfected with GEFT protein (data not
shown).
Specific Activation of Rac/Cdc42 by GEFT via Direct
Interaction--
To identify the Rho family of proteins that are
activated by GEFT, we constructed, expressed, and purified a
bacterially expressed hexahistidine-tagged human and mouse GEFT protein
(DH-PH domain). Then, we analyzed the guanine nucleotide exchange
activity of GEFT protein on the incorporation of cold GTP into
[3H]GDP-loaded RhoA, Rac1, and Cdc42, respectively (Fig.
3). As shown in Fig. 3, RhoA, Rac1, and
Cdc42 alone did not show significant intrinsic GDP dissociation over
the time period tested (8 min). However, addition of GEFT to the
reaction stimulated rapid and complete dissociation of
[3H]GDP from Cdc42 and Rac1 within 2-5 min. In contrast,
only 10-15% of the [3H]GDP was released from RhoA after
the same time period. As a control, we also examined the GEF exchange
activities of Vav2 (DH domain) and Tiam-1 (DH-PH domain), members of
the Dbl family, on their respective exchange activities of GTPases,
Cdc42, and Rac1 (43, 56, 67). We found that GEFT had demonstrated
similar exchange activities on Rac1 and Cdc42, as did Vav2 and Tiam-1, respectively. However, GEFT had much less exchange activity to RhoA
compared with Vav2 (56). No guanine nucleotide exchange activity was
found for H-Ras in our control experiment (data not shown). Taken
together, our data suggest that GEFT preferentially activates the
release of GDP from Rac and Cdc42 proteins, and to a much less extent
to RhoA.
To further confirm that GEFT activates Rac1 and Cdc42 in the cells, we
compared the amount of GTP-bound forms (active status) of Rac1 and
Cdc42 in cells transfected with GEFT or a control plasmid.
To determine the level of GTP-bound Rac1 and Cdc42 in the cells, we
utilized a GST-PAK1 fusion protein containing the Rac1/Cdc42-binding
domain as an affinity reagent in a GST pull-down assay (59-61). PAK1
is a downstream effector of Rac1 and Cdc42, and PAK1 binds
preferentially to the active, GTP-bound forms of Rac1 and Cdc42
GTPases. As shown in Fig. 4A,
transfection of GEFT in COS-7 cells increased the Rac1-GTP
level at least 5-fold compared with cells transfected with a control
plasmid (pCMV-tag2B). We also found a ~3-fold increase in Cdc42-GTP
levels in cells transfected with expression plasmid encoding GEFT (Fig.
4A, right). Together, these results suggest that
GEFT activate Rac1 and Cdc42 in the cell by stimulating the guanine
nucleotide exchange of the two GTPases (Rac1 and Cdc42).
GEFs can be distinguished from other GTPase-interacting proteins by
their ability to bind preferentially to the nucleotide-depleted state
of small GTPases compared with GTP- or GDP-bound states (29, 36). To
test whether GEFT can directly bind to Rac1, Cdc42, and RhoA, GST
fusion protein pull-down assays were performed with GST-Rac1,
GST-Cdc42, and GST-RhoA, in the absence of GTP. As shown in Fig.
4B, Cdc42 and Rac1 bound His-tagged human GEFT protein in
the absence of GTP, but not GST-RhoA protein. These data suggest that
GEFT activate the guanine nucleotide exchange activities of Rac1 and
Cdc42 through direct protein-protein interactions.
GEFT Induces Formation of Filopodia and Lamillipodia, and Actin
Cytoskeleton Rearrangement by Activating Cdc42 and Rac1 GTPases in the
Cells--
Previous studies demonstrate that in fibroblasts, Rac and
Cdc42 induced the formation of lamellipodia and filopodia,
respectively, whereas RhoA promotes stress fiber formation (3). We
examined the effects of GEFT overexpression on the actin cytoskeleton
reorganization in HeLa cells. As shown in Fig.
5, overexpression of GEFT in HeLa cells
caused actin cytoskeleton rearrangement, an induction of membrane
spikes and filopodia, a characteristic of Cdc42 activation by the GEFT
protein (Fig. 5, A-C). In addition, cells
expressing GEFT displayed some membrane ruffling and formation of
lamellipodia (Fig. 5D), suggesting activation of the Rac1
protein in the GEFT-transfected cells. The fact that GEFT caused the
induction of filopodia, microspikes, and lamellipodia supported the
possibility that overexpression of GEFT is associated with constitutive
up-regulation of Cdc42 and Rac1 function.
GEFT Induces Foci Formation and Contact-independent Colony Growth
in NIH3T3 Cells--
By abrogating normal contact inhibition, some of
the tumorigenic proteins have the ability to form foci of piled up
transformed cells on a background monolayer of untransformed cells. To
determine whether GEFT protein has the transformation activity, we
infected NIH3T3 cells with retrovirus vector-encoding GEFT. As shown in Fig. 6, overexpression of GEFT induces
the transformed foci formation in NIH3T3 cells, whereas vector alone
has no effect on the cells (Fig. 6A). The GEFT-induced foci
contain densely packed nonrefractile cells, which is different from
foci induced by Ras, similar to foci formed by active Rho family of
proteins (Fig. 6, B and C). Similar to the foci
induced by another guanine nucleotide exchange factor, Vav, we observed
no multinucleated giant cells for GEFT-induced foci. The number of foci
formed by GEFT in NIH3T3 cells was examined at three different cell
densities. As expected, GEFT induced foci formation at all three
densities (Fig. 6D), suggesting a stronger transforming
ability for the GEFT protein. The transforming activity of GEFT
protein, together with its ability in activation of transcription factors, suggest that this protein may play an important role in cell
proliferation and tumorigenesis.
GEFT Expression Leads to Cell Proliferation and an Increase in Cell
Motility--
Proliferative signaling has been associated with
polypeptide growth factor receptors that possess an intrinsic
protein-tyrosine kinase activity as well as many G protein-coupled
receptors, including thrombin, bombesin, bradykinin,
substance P, endothelin, serotonin, acetylcholine, prostaglandin
F2
The effect of GEFT on cell morphology also prompted us to examine
whether expression of this protein changes cell migration and leads to
an increase in cell motility. In our experiments, we generated NIH3T3
cells stably transfected with GEFT and a control vector. When the cells
were placed in modified Boyden chambers coated with collagen, the cells
expressing GEFT migrated much faster than the ones expressing the
vector alone (Fig. 7B). The number of cells that migrated
increased 2-3-fold in NIH3T3 cells expressing GEFT, suggesting that
GEFT mediates cell motility via the activation of Rac/Cdc42 proteins.
GEFT Activate Rac/Cdc42-mediated Transcriptional
Activities--
To further examine the signaling pathways activated by
GEFT, we examined the ability of GEFT to stimulate Rac1- and
Cdc42-mediated signaling pathways and transcription factors. It has
been shown that activation of the Rho family of small GTPases leads to
the activation of a number of transcriptional factors in cell growth and proliferation, including the c-fos serum response
element (SRE) and other transcription factors. The SRE forms a ternary complex with the transcription factor serum response factor and ternary
complex factors, such as Elk1 and SAP1. Activation of the Rho family of
GTPases, RhoA, Rac1, and Cdc42 leads to transcriptional activation via
serum response factor and act synergistically at the SRE with signals
that activate ternary complex factors (12). To test whether GEFT
directly affect the activation of transcription factors at the SRE, we
measured the activation of GEFT on transcription factors in our
cell-based transfection assays. COS-7 cells were transfected with
luciferase reporter genes controlled by SRE, Elk1, and SAP1,
together with the expression plasmids
encoding Rho A, Rac1, and Cdc42. As shown in Fig. 8A,
transfection of GEFT, together with Cdc42 and Rac1, dramatically
increased SRE-luciferase activity ~100- and ~150-fold,
respectively. GEFT also moderately activated RhoA mediated SRE activity
in our transcriptional reporter assay (Fig. 8A). To further
examine the effects of GEFT on ternary complex factor-linked signaling
pathway, we measured the activation of Ets domain transcription
factors, Elk1 and SAP1, members of the ternary complex factor complex.
Similar to the activation of SRE, cotransfection of GEFT with Rac1 and
Cdc42 significantly stimulated the transcriptional activities of Elk1
and SAP1 (data not shown). Whereas the stimulation of GEFT on
RhoA-mediated activation was significantly lower compared with Rac1 and
Cdc42.
In most cell types, activation of Rac1 and Cdc42, but not RhoA, leads
to the stimulation of JNK activity, and consequently, the activation of
the AP-1 and c-Jun transcription factors (11, 22). To investigate the
effects of GEFT on the stress-regulated JNK signaling pathway, we
examined the ability of GEFT to stimulate c-Jun and AP-1
transcriptional activities using a transient transcriptional reporter
assay in COS-7 cells. We transfected the control vector or vectors
encoding wild type RhoA, Rac1, and Cdc42 in the presence or absence of
GEFT, and then assessed the transcriptional activation of c-Jun and
AP-1. As shown in Fig. 8B, coexpression of GEFT with Rac1
and Cdc42 significantly induced the activation of the c-Jun luciferase
reporter gene, whereas cotransfection of GEFT with RhoA had little
stimulation of the reporter genes. Also, similar activation of AP-1 was
obtained with AP1-luciferase reporter assays (data not shown).
Together, these data suggest that GEFT strongly stimulates the JNK
signaling pathway and its related transcriptional factors by activating
the small GTPases, Rac1 and Cdc42 in the cells.
GEFs regulate GTP-binding and regulatory proteins by converting
GTPases to their biologically active state by catalyzing the exchange
of bound GDP for GTP. The Dbl family of proteins are Rho-specific GEFs
that contain a Dbl domain followed by a PH domain. In our search of
genes involved in tumorigenesis using the ERM strategy, we identified a
new partial sequence encoding a new guanine nucleotide exchange factor
and strongly promoting foci formation in NIH3T3 cells, and the gene was
named GEFT (53). In this research, we cloned the full-length genes
encoding the human and mouse GEFT and characterized the
roles of this protein in the Rho family of small GTPases and their
signaling pathways.
We found that GEFT preferentially activated Rac1 and Cdc42 and promoted
the guanine nucleotide exchange of Rac1 and Cdc42 GTPases whereas
relatively low activity was observed with RhoA GTPase in our in
vitro exchange assays. Moreover, GEFT activated Rac1/Cdc42-mediated transcriptional activities of SRE, Elk1, and SAP1
in our transcriptional reporter gene assays. Furthermore, we
demonstrated that GEFT significantly induced the activation of c-Jun
and AP-1 transcription factors, downstream targets of the JNK
mitogen-activated signaling pathway that are activated by Rac1 and
Cdc42, but not RhoA (11, 22). Therefore, GEFT may function as a
specific activator for Rac1 and Cdc42 small GTPases in the cells.
The Rho family proteins have been shown to regulate actin cytoskeletal
re-organization, and therefore, influencing cell shape, morphology,
adhesion, cell migration, and motility (3, 6). For example, RhoA
promotes the formation of actin stress fibers and focal adhesions,
whereas Rac1 induces the formation of lamellipodia and membrane
ruffling. On the other hand, Cdc42 promotes formation of microspikes on
cell surface and filopodia development in the cells. In our
experiments, we observed that expression of GEFT promotes cell
morphology change and actin cytoskeleton reorganization. Like
activation of Cdc42 and Rac1, we found that GEFT induced the formation
of microspike, filopodia, and lamellipodia structure in cells
overexpressing the proteins, suggesting that GEFT activate Rac1- and
Cdc42-mediated signal pathways in the cells.
The fact that GEFT is highly expressed in the brain and the heart, and
that GEFT is localized on chromosome 12q13.11, a region frequently
amplified in sarcomas and brain tumors, suggest that the protein may
play a potential role in brain tumor and other tumors. In this study,
we demonstrated that overexpression of GEFT in NIH3T3 cells strongly
induced the formation of foci, similar in morphologic appearance to the
ones induced by activated Rac and Cdc42 signaling pathway, suggesting
the tumorigenic potential of the GEFT protein. We also demonstrated
that GEFT strongly activated a number of transcription factors that
mediated the expression of genes involved in cell growth,
proliferation, and survival. These cellular functions are important in
tumorigenesis. The exact roles of this protein in brain tumor and other
tumors are under further investigation.
There is a great deal of evidence that the Rho family of GTPases play
an important role in neuronal morphogensis (7, 11, 22). During neuronal
development, neuronal precursor cells migrate and then differentiate,
extending axons and dendrites to specific regions to form synapses with
appropriate target cells. Several GEFs, such as Tiam-1, Trio, Ephexin,
and Kalirin, have been implicated in neuronal morphogenesis, growth
cone guidance, and neuronal dendritic spines (68-72). We have
demonstrated that GEFT is highly expressed in human brain, and GEFT can
induce the formation of lamillipodia, microspikes, and filopodia in the
cells. The potential roles of this brain-specific GEF protein in
neuronal morphogenesis and differentiation, axon guidance, and
dendritic spines are under investigation.
A variety of extracellular stimuli have been found to activate the Rho
family of GTPases, including growth factors, cytokines, lysophosphatidic acid, interleukins, and matrix components via receptor
tyrosine kinases, G protein-coupled receptors, and integrin receptors,
respectively. How different signaling pathways directly link the
extracellular stimuli to the intracellular signal change and
consequently, gene expression, is one of the key questions. Protein
phosphorylation or membrane association and protein-protein interaction
through the PH domain of GEFs has been shown to activate GEF proteins
upon stimulation by extracellular stimuli (44, 45, 73-75). Domain and
motif analysis of the GEFT protein indicates that GEFT protein has a PH
domain directly after the guanine nucleotide exchange domain and
several potential protein phosphorylation sites that can be regulated
by pathways coupled to different cellular protein kinases.
In summary, we have identified and characterized a guanine nucleotide
exchange factor, preferentially for Rac1 and Cdc42, named GEFT. The
protein is highly expressed in human brain and heart, but very weakly
in other tissues. Overexpression of GEFT in the cell induces cell
morphology change, cytoskeleton reorganization, and the formation of
microspikes, filopodia, and lamilliopodia, characteristics of
Rac1/Cdc42 activation. Furthermore, overexpression of GEFT in NIH3T3
cells promotes the induction of foci formation and tumorigenesis,
possibly by activating Rac1 and Cdc42 signaling pathways and
transcription factors in the cells. The mechanism of GEFT activation
and its roles in neuronal cells and tumors need to be established in
future studies.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside for 3-4 h. For His6-tagged GEFT, the protein was purified
by nickel-nitrilotriacetic acid-agarose (Qiagen). For GST fusion
GTPases, the proteins were purified by GSH-agarose (Sigma). The GST
fusion proteins were in the beads or eluted in the solution containing
50 mM Tris (pH 8.0), 10 mM reduced glutathione
(Sigma). All the proteins used in the assays were visualized by
Coomassie Blue staining after SDS-polyacrylamide gel electrophoresis.
The content of each protein was at least 90% pure.
-32P]CTP by nick translation using random primers.
Probes (~4 × 107 cpm/µg) were hybridized with the
RNA filter and analyzed according to manufacturers protocol.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (37K):
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Fig. 1.
GEFT domain structure and comparison of DH
domains. A, sequence comparison of human and
mouse GEFT proteins. Identical amino acids are marked with an
asterisk (*). GenBankTM accession numbers are
AF487514 for human and AF487515 for mouse. B, domain
structure of human GEFT, including the NH2-terminal DH
domain (Rho GEF domain, also called DH domain) followed by the PH.
Mouse GEFT has similar domain structure and an extra
NH2-terminal region (not shown). C, multiple
sequence alignment of DH domains from GEFT and other members of the Dbl
family: Tiam-1, Kalirin (Duo), and UNC-73 (64, 65, 67, 76). Bold
letters indicate identical amino acids.
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Fig. 2.
Expression of GEFT in human tissues.
Northern blot analysis of GEFT expression in multiple human
tissues using multiple tissue Northern blot membrane containing premade
poly(A)+ RNA (Clontech Inc.). The
membrane was hybridized with 32P-labeled GEFT and -actin
probes, respectively. A single band at ~3 kilobases (kb) was
detected.
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[in a new window]
Fig. 3.
GEFT preferentially activates the guanine
nucleotide exchange activities of Rac1 and Cdc42. Stimulation of
GDP dissociation from RhoA, Rac1, and Cdc42 by GEFT was performed using
purified bacterial expressed proteins. Time-dependent study
for the dissociation of [3H]GDP from purified recombinant
GST-RhoA (A), GST-Rac1 (B), and GST-Cdc42
(C) in the presence or absence of His-tagged GEFT.
Experiments were performed in triplicate. Shown are representatives of
three independent assays.
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Fig. 4.
GEFT activates and binds to Rac1 and
Cdc42. A, activation of Rac1 and Cdc42 by increasing
the levels of GTP-bound forms of Rac1 and Cdc42, respectively, in cells
expressing GEFT. The amounts of activated Rac1-GTP and Cdc42-GTP were
determined by GST pull-down assays using GST-PAK1 domain (59-61).
Cells transfected with GEFT (+) and a control plasmid
(pCMV-Tag2B) ( ) were lysed and the amount of GTP-bound
Rac1 and Cdc42 were precipitated with GST-PAK. The proteins were
separated by 12% SDS-polyacrylamide gel electrophoresis. The amounts
of Rac1 and Cdc42 proteins were visualized by Western blot analysis
using specific anti-Rac1 and Cdc42 antibodies, respectively. Western
blot was also performed to verify equal amounts of endogenous Rac1 and
Cdc42 expression used in the assays (A, bottom,
Rac1 and Cdc42). B, GEFT directly binds to Cdc42 and Rac1
in vitro. Bacterial expressed and purified GST-RhoA, Cdc42,
and Rac1 were assayed for in vitro binding of His-tagged
GEFT without the addition of GTP as described under "Materials and
Methods." His-tagged GEFT binds to Rac1 and Cdc42 in the pull-down
assays. Bottom of B shows that equal amounts of
GST fusion proteins (GST-RhoA, GST-Rac1, and GST-Cdc42) were used in
the assays.
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[in a new window]
Fig. 5.
GEFT induces the formation of membrane
microspikes, filopodia, and lamellipodia in the cells.
A and B, formation of membrane microspikes in
cells transfected with FLAG-tagged GEFT. C and D,
induction of filopodia and lamellipodia in transfected HeLa cells.
Immunostaining of GEFT and actin cytoskeleton in HeLa cells transfected
with pCMV-Flag-GEFT are shown. Cells were plated on fibronectin-coated
coverslips, then fixed and immunostained with specific anti-FLAG
monoclonal antibody M2 (Sigma) for the FLAG-GEFT (left).
Actin cytoskeleton was stained with rhodamine-phalloidin
(middle). Shown in the right are overlay pictures
of GEFT (left) and actin cytoskeleton
(middle).
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Fig. 6.
Induction of foci formation in NIH3T3 cells
by GEFT expression. NIH3T3 cells were infected with a retrovirus
vector encoding GEFT or a control plasmid (pMSCV2.1,
Clontech). A, cells expressing the
control plasmid did not form any foci in the assays. B and
C, induction of foci formation in GEFT-infected cells.
Photographs of foci formation were taken 10 days after NIH3T3 cells
were infected with GEFT. The experiments were performed three times and
shown are representatives from foci formed by the GEFT-infected NIH3T3
cells. D, the number of foci formed in GEFT-expressing cells
and vector control cells. Three individual experiments with different
numbers of cells were plated and the numbers of foci formed were
counted for individual cell populations. Shown are the mean ± S.E. of three independent assays.
, and lysophosphatidic acid, in a variety of cell
types (reviewed by Gutkind (77)). The effect of GEFT on cell
transformation prompted us to examine the role of GEFT in cell
proliferation and cell motility/migration. To understand the potential
role of GEFT in cell proliferation and tumorigenesis, we examined cell
proliferation in cells stably transfected with the mouse GEFT vector
using the CellTiter 96 (Promega) assay. Fig.
7A shows that expression of
GEFT in NIH3T3 cells significantly increased cell proliferation
compared with cells expressed in vector only, suggesting that GEFT can
induce cell proliferation and tumorigenesis.
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Fig. 7.
GEFT promotes cell proliferation and cell
migration. NIH3T3 cells infected with either control vector or
GEFT were used in cell proliferation and migration assays.
A, expression of GEFT-induced cell proliferation. Cells were
transfected with GEFT and a control vector, respectively. Cells were
plated at 500 cells/well and allowed to adhere to the plate. At the
indicated time points, the AQueous One solution was added to the
samples and measured at 490 nm 2 h later. The experiments were
repeated three times and data shown are the mean of three independent
repeats. B, cell migration/motility assay using a modified
Boyden chamber approach. Chemoattractant was placed into the base wells
and separated from the top wells by an 8.0-µm pore Nucleopore PVP-F
polycarbonate membranes (Corning Separations), pre-coated 1 h with collagen (100 µg/ml) (Collaborative Biomedical). 2 × 104 cells were pipetted into each upper well of the
chamber, which was then incubated for 6 h at 37 °C in a 5%
CO2 humidified incubator. Nonmigrated cells were removed
and membranes were fixed and stained. The number of cells migrating per
well was counted microscopically, with three wells per condition. Then,
the mean ± S.D. or S.E. were calculated.
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Fig. 8.
Activation of transcriptional factors by GEFT
in the cells. A, GEFT activates the SRE mediated by the
Rho family GTPases. Rho family of proteins moderately activates the
SRE-Luc reporter gene, whereas co-transfection of GEFT dramatically
stimulates the Cdc42- and Rac1-mediated SRE in the reporter assays.
B, activation of Rac1- and Cdc42-mediated c-Jun
transcriptional activities by GEFT. COS-7 cells were transfected with
individual reporter plasmid, wild type or dominant-active
(DA, dominant active) RhoA, Cdc42, and Rac1.
pCMV-Flag-tagged GEFT is used in the transcriptional reporter assays.
Data shown are an average of three repeats. Similar experiments were
performed at least three times.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank members of the Center for Cancer Biology and Nutrition for insightful discussions and Dr. Kuanliang Guan for SAP1 and Elk1 reporter plasmids.
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FOOTNOTES |
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* This work was supported in part by NHLBI National Institutes of Health Grant 5R01 HL64792 and a grant from the Department of Defense (to M. L.).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF4487514 for human and AF4487515 for mouse.
Basil O'Conner Scholar of the March of Dimes Foundation. To
whom correspondence should be addressed: Center for Cancer Biology and
Nutrition, Alkek Institute of Biosciences and Technology, Texas A&M
University System Health Science Center, 2121 Holcombe Blvd., Houston,
TX 77030. Tel.: 713-677-7505; Fax: 713-677-7512; E-mail:
mliu@ibt.tamu.edu.
Published, JBC Papers in Press, January 23, 2003, DOI 10.1074/jbc.M208896200
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ABBREVIATIONS |
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The abbreviations used are: JNK, Jun NH2-terminal kinase; GEFs, guanine nucleotide exchange factors; PH, pleckstrin homology; GST, glutathione S-transferase; Dbl, diffuse B-cell lymphoma; DH, Dbl homology; ERM, enhanced retroviral mutagen; PBS, phosphate-buffered saline; SRE, serum response element; PAK, p21-activated protein kinase.
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