From the The Rho family GTPases are involved in a variety
of cellular events by changing the organization of actin cytoskeletal
networks in response to extracellular signals. However, it is not
clearly known how their activities are spatially and temporally
regulated. Here we report the identification of a novel guanine
nucleotide exchange factor for Rac1, STEF, which is related in overall
amino acid sequence and modular structure to mouse Tiam1 and
Drosophila SIF proteins. STEF protein contains two
pleckstrin homology domains, a PDZ domain and a Dbl homology domain.
The in vitro assay showed that STEF protein specifically
enhanced the dissociation of GDP from Rac1 but not that from either
RhoA or Cdc42. Expression of a truncated STEF protein in culture cells
induced membrane ruffling with altered actin localization, which
implies that this protein also activates Rac1 in vivo. The
stef transcript was observed in restricted parts of mice,
including cartilaginous tissues and the cortical plate of the central
nervous system during embryogenesis. These findings suggested that STEF
protein participates in the control of cellular events in several
developing tissues, possibly changing the actin cytoskeletal network by
activating Rac1.
Members of the Rho family GTPases, which include RhoA, Rac1, and
Cdc42, act as molecular switches that control the organization of actin
cytoskeleton in response to extracellular signals (1). Experiments
using cultured cells showed that each member of the Rho family has a
different influence on the cytoskeletal structure and eventually on
cellular morphology. RhoA induces stress fibers associated with focal
adhesions, Rac1 produces lamellipodia or membrane ruffling, and Cdc42
evokes filopodia on the plasma membrane of fibroblasts (2-5).
Evidence has accumulated that the Rho family proteins have a variety of
roles in distinct types of cells and tissues. For instance, they are
involved in extension and collapse of neurites (6, 7), chemotaxis of
macrophages when stimulated by colony-stimulating factor-1 (8), and the
generation of tissue polarity in Drosophila (9). However, it
is not clear how the activities of Rho family proteins are spatially
and temporally regulated to express their functions in
vivo.
There are three kinds of proteins that regulate the activities of Rho
family proteins by controlling the ratio of the GTP-bound active form
to the GDP-bound inactive form.
GEFs1 activate the G proteins
by converting the GDP-bound inactive state to the GTP-bound active
state, whereas GTPase-activating proteins and guanine nucleotide
dissociation inhibitors inactivate Rho family proteins (10). Recently,
more than 20 putative GEFs for Rho family GTPases have been identified
and are characterized by the presence of the conserved amino acid
sequence, Dbl homology (DH) domain, which catalyzes the guanine
nucleotide exchange reaction. These proteins have been shown to
regulate various cellular events including morphological changes of
cells, oncogenesis, or activation of transcription factors (11).
Genetic analyses also revealed that GEFs play important roles in
development; DRhoGEF2 mediates cell shape changes in gastrulation of
Drosophila embryos (12), and UNC-73A is required for cell
and growth cone migrations in Caenorhabtidis elegans (13).
Although the Rho family GTPases appear to be widely distributed in a
variety of tissues, several GEFs are preferentially expressed in a
limited number of tissues at certain developmental stages. Therefore,
it is hypothesized that each of the GEFs has a distinct role in the
regulation of various cellular events by activating the GTPases during development.
One of the GEFs whose tissue and subcellular localization has been
clearly shown is SIF protein, which is encoded by the still life (sif) gene of Drosophila, identified by
our behavioral mutant screen (14). SIF protein is predominantly
expressed in the nervous system and confined to the synaptic terminals.
At the ultrastructural level, it is found in lateral regions of the
active zones for neurotransmission. A loss-of-function sif
mutation causes reduced motor activities, which can be rescued by
expression of a sif minigene in the nervous system.
Moreover, expression of a truncated SIF protein induced membrane
ruffling with altered actin localization in human KB cells. These data
suggested that SIF protein regulates the formation or maintenance of
synapses, possibly by organizing the actin cytoskeleton through the
activation of Rho family GTPases. SIF protein contains a DH domain, two
pleckstrin homology (PH) domains (15), and a PDZ domain (16, 17). The
organization and amino acid sequences of these domains are highly
related to those of mouse Tiam1.
The Tiam1 gene was originally identified by its ability to
induce invasion of T-lymphoma cells (18). Tiam1 protein functions as a
GEF that specifically reacts to Rac1 and Cdc42 in vitro and induces membrane ruffling in NIH 3T3 cells by activating Rac1 (19).
Tiam1 affects the morphology of neuroblastoma cells, including neurite
outgrowth (20), and E-cadherin-mediated cell-cell adhesion of
epithelial cells (21). The transcripts are mainly expressed in the
brain and testis, and the developmental distribution patterns in the
brain suggest that Tiam1 contributes to cytoskeletal reorganization required during cell migration and neurite extension in a specified population of neurons (22).
To further understand the molecular mechanisms underlying the
activation of Rho family GTPases, particularly Rac1, and the biological
phenomena that the related signaling cascade may regulate, we have made
efforts to identify a new GEF. Here we report a novel mouse protein,
STEF (SIF and Tiam1-like exchange
factor), which was isolated on the basis of sequence
similarity to both SIF and Tiam1. Our biochemical and cell biological
analyses showed that STEF protein functions as a GEF specific for Rac1
in vitro and in vivo. These data together with
the similar domain organization of STEF and Tiam1 suggest that these
two proteins execute their functions in similar molecular environments
to activate Rac1 cascades in cells. However, in situ
hybridization revealed that the stef transcript is
preferentially localized in narrower regions when compared with the
distribution pattern of the Tiam1 transcript, suggesting a
distinct or more limited role of STEF in mouse development.
Isolation of the STEF Gene--
To isolate a murine gene
homologous to the Drosophila sif gene, we designed
degenerated oligonucleotide PCR primers corresponding to the conserved
amino acid residues between SIF and Tiam-1. The first set of primers
(PF1, PR1, and PR3) was designed in the PHn-TSS domain. The second set
(DF1, DR1, and DR2) was designed in the DH domain. The primer
sequences are as follows: PF1,
5'-GG(A/C/G/T)(A/G)C(A/C/G/T)GT(A/C/G/T)(A/C)G(A/C/G/T)AA(A/G)AC-3' (amino acids G(A/T)VRKA); PR1,
5'-GC(A/C/G/T)GC(A/G)CA(A/C/G/T)GC(A/C/G/T)(C/G)(A/T)(A/G)TG(A/T/G)AT-3' (complementary to amino acids IHSACAA); PR3,
5'-CCA(A/G)TT(C/T)TC(A/C/G/T)A(A/G)(C/T)TC(A/C/G/T)(A/G)(C/T)(C/T)TG-3' (complementary to amino acids Q(V/T)ELENW); DF1,
5'-AC(A/C/G/T)GA(A/G)(A/C)G(A/C/G/T)AC(A/C/G/T)TA(C/T)GT(A/C/G/T)AA-3' (amino acids TERTYVK); DR1,
5'-(C/T)(G/T)(C/T)TGCAT(C/T)TC(A/G)TT(A/G/T)AT(A/G)TG-3' (complementary to amino acids HINEMQ(R/K)); and DR2,
5'-TC(C/T)TC(A/G)TG(A/T/G)AT(A/C/G/T)(C/T)(G/T)(C/T)TGCAT-3' (complementary to amino acids MQ(R/K)IHEE).
Poly(A)+ RNA was isolated from the brains of ICR mice with
the QuickPrep mRNA Purification kit (Amersham Pharmacia Biotech). 4 µg of RNA was used as a template for first strand cDNA synthesis using the First-strand cDNA Synthesis kit (Amersham Pharmacia Biotech), and the entire product was subsequently used as a template for one PCR reaction. PCR was performed in the 100-µl scale with the
primer set PF1 and PR1 or the primer set DF1 and DR2, employing the
following protocol: 30 cycles of 94 °C for 1 min, 45 °C for 2 min, and 72 °C for 2 min. Then 1 µl of each of the products was
used as a template for a second PCR reaction with the nested primer set
PF1 and PR3 or nested primer set DF1 and DR1. The DNA fragments of
predicted sizes were recovered from agarose gels, cloned into
pBluescriptII SK Expression and Purification of Recombinant Small G Proteins and
GEFs--
Bacterially expressed GST-Dbl, GST-RhoA, GST-Rac1, and
GST-Cdc42 fusion proteins were purified as described previously (23, 24). The 1.2-kb fragment encoding the DH-PHc domain of STEF (amino
acids 1096-1502) was obtained by PCR amplification from the STEF
cDNA clones, digested with SmaI and XhoI, and
inserted into the SmaI and XhoI sites of pGEX4T-1
(Amersham Pharmacia Biotech) to construct pGEX-STEF. Escherichia
coli strain BL21(DE3) transformed with pGEX-STEF was cultured at
37 °C and treated for 4 h at 25 °C with 0.1 mM
isopropyl GEF Assays--
Effects of STEF on the dissociation of
[3H]GDP from and the binding of
[35S]GTP In Situ Hybridization of Sections--
Wax sections were
obtained from Novagen (Hybrid-Ready Tissues). After treatment with
xylene and rehydration through an ethanol series and PBS, and the
specimens were fixed with 4% paraformaldehyde in PBS for 15 min and
then washed with PBS for 2 min. The sections were treated with 7.5 µg/ml proteinase K in PBS at 37 °C for 1 h, washed with PBS
for 2 min, refixed with 4% paraformaldehyde in PBS, again washed with
PBS for 2 min, and placed in 0.2 M HCl for 10 min. After
washing with PBS for 1 min, the specimens were acetylated by incubation
in 0.1 M triethanolamine-HCl, pH 8.0, for 1 min and further
in 0.1 M triethanolamine-HCl, 0.25% acetic anhydride for
10 min. After washing with PBS for 2 min, the samples were dehydrated
through a series of ethanols. Hybridization was performed with probes
at concentrations of 200-500 ng/ml in a hybridization solution (50%
formamide, 5× SSC, 1% SDS, 50 µg/ml tRNA, and 50 µg/ml heparin)
at 55 °C for 16 h. After hybridization, the specimens were
washed in 5× SSC at 55 °C for 15 min and then in 50% formamide,
2× SSC at 55 °C for 15 min, followed by RNase treatment in 50 µg/ml RNase A in 10 mM Tris-HCl, pH 8.0, 1 M
NaCl and 1 mM EDTA. Then the sections were washed twice
with 2× SSC at 50 °C for 15 min, twice with 0.2× SSC at 50 °C
for 15 min, and once with TBST (0.1% Tween 20 in TBS) for 5 min. After
treatment with 0.5% blocking reagent (Roche Molecular Biochemicals) in
TBST for 1 h, the samples were incubated with anti-DIG AP
conjugate (Roche Molecular Biochemicals) diluted 1:2000 with TBST for
1 h. The sections were washed twice with TBST containing 2 mM levamisole and then incubated in 100 mM
NaCl, 50 mM MgCl2, 0.1% Tween 20, 100 mM Tris-HCl, pH 9.5, and 2 mM levamisole.
Coloring reactions were performed with BM purple substrate (Roche
Molecular Biochemicals) overnight and then washed with PBS. The samples
were dehydrated and mounted with HSR (Kokusai-Shiyaku).
Transfection of Expression Plasmids into KB Cells--
The
plasmid DNA, pFCMVstef To isolate a mouse gene homologous to the Drosophila
sif gene, we performed a RT-PCR experiment using mRNA
extracted from the mouse adult brain because the sif
transcript in Drosophila is abundant in the adult
brain.2 Two sets of
degenerated oligonucleotide PCR primers were designed from conserved
amino acid sequences found in SIF and Tiam1 to amplify two
corresponding DNA fragments expected to contain the first PH (PHn)
domain or the DH domain (Fig.
1B). The first primer set
produced an unidentified DNA sequence that encodes a PH domain with
high similarity to the PHn domains of SIF and Tiam-1 proteins. Using
the second primer set, we obtained another new DNA sequence that
encodes a DH domain, which is highly related to the DH domains of SIF
and Tiam-1. To test whether these two DNA fragments were derived from
one novel gene, we again performed the RT-PCR experiment with a new set
of primers, one in the sequence of PHn domain and the other in DH
domain. A 2.0-kb DNA fragment was successfully amplified, and the
sequencing analysis revealed that this fragment contained nucleotide
sequences encoding a PDZ domain as well as the PH and DH domains
described above. The overall amino acid sequences and the organization
of the domains were highly conserved among the novel protein, SIF and
Tiam1. These data suggested that the DNA fragment amplified in our
procedure was derived from a novel gene that may be a mouse homologue
of the Drosophila sif gene. Screening a cDNA library
prepared from the mouse newborn brain RNA using the 2.0-kb DNA fragment
as a probe identified several cDNA clones. We further obtained the
corresponding cDNA fragments expected to extend to the 5' or 3'
ends of the full-length transcript by 5' or 3' rapid amplification of
cDNA ends. Sequencing several overlapping cDNAs revealed the
structure of the stef transcript, a stretch of 6157-base
pair sequence that contains the 5'-untranslated region, a long open
reading frame, the 3'-untranslated region, and
poly(A)+.
The sequence of the 6157-base pair cDNA contains a long open
reading frame that predicts a protein of 1715 amino acids (Fig. 1A). As found in Tiam1 and SIF proteins, this protein has a
potential myristoylation site at the N terminus, two PH domains, one
PDZ domain, and one DH domain (Fig. 1B). Because these
motifs are present in many intracellular signaling molecules and STEF
protein does not contain a signal sequence or a transmembrane region, STEF is likely to be a cytoplasmic protein. Several regions of STEF
protein are highly related in amino acid sequence to Tiam1 and SIF
proteins (Fig. 1, B-E); the PHn domain together with its C-terminally flanking region is 66.9% identical to Tiam1 and 52.5% identical to SIF, and the DH domain with the following second PH domain
(PHc) is 63.6% identical to Tiam1 and 47.8% to SIF. However, the PDZ
domain of STEF is moderately conserved, being 27.6% identical to Tiam1
and 30.0% identical to SIF. We hereafter refer to the conserved region
flanking the PHn domain as the TSS domain
(Tiam1-STEF-SIF homologous domain).
Several DH domain-containing proteins including Tiam1 have been shown
to be GEFs that specifically function for the Rho family proteins
in vitro (11). We therefore examined whether STEF has any
influence on the dissociation of GDP from and the association of GTP to
Rho family proteins. Because the truncated GEFs comprising only the DH
domain and the adjacent PH domain have been shown to sufficiently
exhibit the GDP/GTP exchange activity for the specific Rho family
proteins (30, 31, 39), we employed a recombinant GST fusion protein
carrying the DH-PHc domain of STEF to assay its exchange activity. In
addition, the GST-Dbl protein was used as a positive control, which is
known to exhibit GEF activity for all RhoA, Rac1, and Cdc42 (25).
Because it has been reported that Tiam1 is a GEF for Rac1 (19), we
first analyzed whether GST-STEF enhanced the dissociation of GDP from
Rac1. The results obtained in this assay indicated that GST-STEF as
well as GST-Dbl stimulated the dissociation of [3H]GDP
from Rac1 in a time-dependent manner (Fig.
2A). To confirm these
findings, we investigated the influence of STEF on the exchange of
preloaded GDP for [35S]GTP Department of Molecular Genetics,
Division of Signal Transduction,
Inheritance and Variation
Group,
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(Stratagene), and sequenced. The PHn-TSS
domain primer pair yielded mr5-23, and the DH domain primer pair
produced mr1-5. To test whether these fragments were derived from the
same gene, RT-PCR was again executed using the LA-PCR kit version 2 (Takara) with a primer pair to amplify a fragment spanning the two
fragments. The primers used were: mrF1,
5'-GGAAAGAATTCCACAGAGCAGAATAGTGCC-3', and mrR3,
5'-AGCTTAAAGTGATCCGCATAGTAGAGGAAAG-3'. The PCR protocol was as follows:
40 cycles of 98 °C for 20 s and 68 °C for 10 min, and
finally 72 °C for 10 min. A 2.0-kb DNA fragment was successfully amplified.
-D-thiogalactoside to induce expression of
GST-STEF. The fusion protein was purified through a
glutathione-Sepharose 4B column.
S to the Rho family proteins were assayed as
described previously (24-27). Regarding the dissociation assay, in
short, the [3H]GDP-bound form of small G proteins were
obtained by incubating 20 pmol of each small G protein with 1 µM [3H]GDP (1000-2000 cpm/pmol) for 20 min
at 30 °C in the reaction mixture I (20 mM Tris-HCl, pH
7.5, 1 mM dithiothreitol, 5 mM
MgCl2, and 10 mM EDTA). To prevent the
dissociation of [3H]GDP from the G proteins, 0.375 M MgCl2 was added to a final concentration of
20 mM, and the mixtures were immediately cooled on ice. The
dissociation of [3H]GDP was performed at 25 °C by
adding a 200-fold excess of unlabeled GTP and the indicated amounts of
GST-Dbl or GST-STEF to the reaction mixture II (50 mM
Tris-HCl, pH 8.0, 1 mM dithiothreitol, 10 mM MgCl2, and 2.9 mM EDTA). As to the association
assay, the GDP-bound form of GST-Rac1 was obtained by incubating 10 pmol of GST-Rac1 with 2 µM unlabeled GDP for 20 min at
30 °C in the reaction mixture I and by successive addition of 0.375 M MgCl2 to a final concentration of 20 mM. The association of [35S]GTP
S to the
GDP-bound form of GST-Rac1 was carried out at 25 °C by adding 10 µM [35S]GTP
S (2000 cpm/pmol) and
indicated amounts of GST-Dbl or GST-STEF to the reaction mixture II. In
both dissociation and association assays, the reaction was stopped at
the indicated time by adding 2 ml of an ice-cold solution (20 mM Tris-HCl, pH 7.5, 20 mM MgCl2, and 100 mM NaCl). The diluted mixtures were filtrated
through nitrocellulose filters, and then the filters were washed a few times with the same solution. The radioactivity trapped on the filters
was counted. Protein concentrations were determined with bovine serum
albumin as a standard protein.
N, was constructed by inserting DNA fragments
encoding portions of STEF (amino acids 411-1715) into pFLAG-CMV-2
(Kodak). pFCMVsif
N, pEF-BOS-HA-Rac1val12, and pEF-BOS-HA-Cdc42val12
were prepared as described (14, 28). KB cells were cultured and seeded
onto a polylysine-coated dish (28). The expression plasmids were
transfected into the KB cells (3 × 106/well dish)
using LipofectAMINE (Life Technologies, Inc.) according to the
manufacturer's recommendations. pGFP (CLONTECH)
was cotransfected only when pFCMVsif
N was used for transfection.
After the cells were cultured in the growth medium for 24 h, they
were incubated in the serum-starved medium for 24 h, fixed, and
doubly stained with rhodamine-labeled phalloidin and with either
anti-Flag antibody or anti-HA monoclonal antibody (12CA5) (29).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Amino acid sequence and domain structure of
STEF protein. A, deduced amino acid sequence of STEF
protein. PH domains are single-underlined, the TSS domain is
double-underlined, the PDZ domain is underlined
by a wavy line, and the DH domain is underlined
by a dotted line. B, schematic domain structures
of STEF, SIF and Tiam1 proteins. The PH (PHn and PHc), TSS, PDZ, and DH
domains are represented by yellow, green,
light blue, and red boxes, respectively. The
potential myristoylation site is indicated by the letter M
at the N terminus of each protein. The scores of the amino acid
identity between the corresponding domains are also shown.
C-E, sequence similarities among STEF, SIF, and Tiam1
proteins. Alignment of the PHn-TSS domains (C), PDZ domains
(D), and DH-PHc domains (E) of STEF, Tiam1,
and SIF proteins. Amino acid residues conserved among two or more
proteins are highlighted by a black background.
S on Rac1. GST-STEF and
GST-Dbl enhanced the incorporation of [35S]GTP
S to
Rac1 (Fig. 2B). We then examined the kinetic properties of
GST-STEF and GST-Dbl for Rac1. Both stimulated the dissociation of
[3H]GDP from Rac1 in a dose-dependent manner
(Fig. 2C). However, measurement of the kinetic parameters
(e.g. kcat and Km) of GST-STEF was unsuccessful because the reaction was not saturated under our experimental conditions (data not shown). We subsequently performed the dissociation assay for RhoA and Cdc42 under the same
conditions. GST-STEF did not show the dissociation activity for RhoA or
Cdc42, while GST-Dbl clearly increased the release of
[3H]GDP from RhoA and Cdc42 (Fig. 2C).
Consistently, GST-STEF did not prompt the exchange of preloaded GDP for
[35S]GTP
S on RhoA or Cdc42 (data not shown). From the
data obtained in these experiments, we conclude that STEF is a GDP/GTP
exchange factor that specifically reacts to Rac1.
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Fig. 2.
GEF activity of STEF protein. The
dissociation of [3H] GDP from Rho family GTPases and the
association of [35S]GTP S to Rac1 were assayed. In
these experiments, the DH-PHc region of STEF conjugated with GST was
used instead of the full-length STEF. Dbl protein showed GEF activity
for all RhoA, Rac1, and Cdc42 and was therefore employed as a positive
control. Time courses of the effects of STEF (1 µM) and
Dbl (0.5 µM) proteins on the dissociation of
[3H] GDP from Rac1 (A) and on the association
of [35S]GTP
S to Rac1 (B). In each
experiment, both proteins showed GEF activity for Rac1 in a
time-dependent manner. C, substrate specificity
of STEF protein. The dissociation of [3H] GDP from Rac1,
Cdc42, and RhoA was assayed in the presence of various doses of STEF or
Dbl. STEF catalyzed the exchange reaction specifically for Rac1 in a
dose-dependent manner.
We next examined whether STEF also activates Rac1 in cultured cells.
When KB cells were transfected with the cDNA of constitutively activated Rac1 (V12Rac1), the cells exhibited membrane ruffling accompanied by the accumulation of actin filaments along the altered plasma membrane as previously reported (Fig.
3, g-i) (2), whereas constitutively activated Cdc42 (V12Cdc42) induced filopodia (Fig. 3,
j-l). Because many DH domain-containing proteins express
their oncogenic or invasive activities when their N-terminal portions are truncated (18, 31, 32), we constructed the N-terminally truncated
SIF (SIFN) or STEF (STEF
N) and made them overexpressed in KB
cells to examine their effects on cytoskeleton and cellular morphology.
SIF
N, as previously reported (14), was found to induce membrane
ruffling in KB cells (Fig. 3, d-f). When STEF
N was
introduced into KB cells (Fig. 3, a-c) and NIH3T3 cells
(data not shown), membrane ruffles were also evoked, where STEF
N
colocalized with F-actin along the periphery. However, STEF
N induced
neither filopodia nor stress fibers in KB cells or NIH3T3 cells. These data implied that STEF activated Rac1 but not Cdc42 or RhoA in vivo, which was consistent with the results of the GEF assay
in vitro.
|
To determine when the stef gene is expressed during
embryogenesis, we performed a Northern blot analysis for the RNA
samples prepared from embryos at various stages (Fig.
4A). A weak signal of the
stef RNA was detected at 6.3 kb in the E7 embryo, and the signal intensity increased as development proceeded up to E17 during
embryogenesis.
|
To reveal the tissue distribution of the stef transcript, we performed further Northern blot analysis for the RNA samples extracted from various tissues of adult mice (Fig. 4B). A strong signal was detected in the brain, and much weaker signals were found in the heart, lung, liver, skeletal muscle, kidney, and testis. This expression pattern was different from the Tiam1 profile, which showed strong signals in the brain and testis (18).
We also performed in situ hybridization for mouse embryos to
reveal the spatial distribution of the stef transcript.
During late embryonic stages, stef was mainly expressed in
the cartilaginous tissues and in restricted parts of the central
nervous system (Fig. 4C). In E14.5 embryos, signals were
detected in cartilaginous tissues including Meckel's, costal,
vertebral, and tracheal cartilage. In the central nervous system at the
same stage, stef was very strongly expressed in the cortical
plate, moderately expressed in a part of the striatum, and very weakly
expressed at the floor of the fourth ventricle. In E16.5 embryos, the
distribution pattern of the stef transcript was similar to
that in E14.5 embryos (data not shown).
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DISCUSSION |
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Utilizing RT-PCR, we cloned DNA fragments that represent a novel gene, stef, which is highly related to Drosophila sif and mouse Tiam1 genes. From a cDNA library prepared from the brain of a newborn mouse and by means of 5' and 3' rapid amplification of cDNA ends experiments, we succeeded in obtaining overlapping cDNA clones that nearly covered the full-length transcript of the stef gene. The longest open reading frame predicts an amino acid sequence of the STEF protein that consists of 1715 amino acid residues. This protein contains a potential myristoylation site at its N terminus, a PH (PHn) domain, a PDZ domain in the middle part, and a DH domain followed by a second PH (PHc) domain in the C-terminal part. All these motifs are also found in SIF and Tiam1 in the same order, and their sequences are remarkably conserved. In addition, in the C-terminal flanking region of PHn, the TSS domain was defined on the basis of sequence similarity among these proteins. This conservation found in amino acid sequence and domain organization indicates that both stef and Tiam1 are orthologues of the Drosophila sif gene. Computer analyses revealed that the similarity between STEF and Tiam1 is greater than that between SIF and STEF or Tiam1. From an evolutional point of view, these relationships suggest that at the time of the divergence of arthropods and chordates, a common ancestral gene of these three genes separated into sif and a second ancestral gene, and the latter further split into stef and Tiam1 afterward.
Rho-like GTPases become activated when bound GDP is exchanged for GTP, a process catalyzed by GEFs. More than 20 putative GEFs specific for Rho family GTPases have been identified, which contain a conserved catalytic domain, the DH domain (11). The substrate specificity for the exchange reaction differs among members of the DH family. For example, Lbc, Lfc, and Lsc only catalyze the exchange for RhoA (33, 34), whereas Ost and Dbs react for Cdc42 and RhoA (11, 35). We found that the STEF protein is a GEF that specifically functions for Rac1 in vitro and in cultured cells.
We initially examined whether the DH-PHc domain of STEF conjugated with
GST activated the dissociation of GDP from each member of the Rho
family. Although the DH-PH domain of Dbl showed activity with Rac1,
Cdc42, and RhoA (25), the DH-PHc of STEF only enhanced GDP release from
Rac1, not from the other molecules. This substrate specificity of STEF
revealed in vitro was further examined in cultured cells. It
has been shown that each of the activated Rho family GTPases induces a
distinct cellular morphology; Cdc42 induces filopodia, Rac1 causes
lamellipodia or membrane ruffling, and RhoA exhibits stress fibers in
fibroblasts or partly in KB cells (2-5). Our experiment showed that
N-terminally truncated STEF (STEFN) induced membrane ruffling but
did not induce either filopodia or stress fibers in KB or NIH3T3 cells.
In addition, STEF
N colocalized with F-actin at the edge of the
ruffled membrane, suggesting local activation of Rac1 in the altered
structure. Thus, STEF appears to specifically activate Rac1 in culture
cells as well as in vitro.
Many Rho family proteins are believed to be widely distributed in various tissues, each having multiple functions during development. For example, Rac1 is involved in the fusion of myoblasts (6) and migration of neuronal cells (36). Furthermore, each member is potentially able to regulate several developmental processes even in the same type of cells; in Purkinje cells, the activated Rac1 appears to affect the formation of dendritic spines and also perturbs axonal extension (37). These facts imply that a temporally and spatially organized regulation of the Rho family proteins is required for each cell to properly differentiate, possibly through activation by GEFs localized at limited subcellular sites during a specific developmental time window. Indeed, SIF protein, a Drosophila putative GEF, is localized in synaptic terminals of mature synapses but is hardly detected during axonogenesis (14). Expression of each GEF may therefore correspond to the spatiotemporally programmed activation of Rho family members and thereby induce a subset of cellular events during development.
We reported that the stef transcript is preferentially distributed in a limited number of tissues during embryogenesis and at the adult stage. In E14.5 and E16.5 embryos, stef is expressed in several tissues including the developing cerebral cortex and cartilage. Notably, in the cortex during these stages, there are numerous neurons migrating through the intermediate zones toward the cortical plates and some extending neurites. It is therefore possible that STEF may regulate neuronal migration or neurite extension by locally activating Rac1. This possibility is supported by the previous findings that Rac1 is involved in cellular migration and neurite extension (6, 19, 36). At the same embryonic stage, Tiam1 expression is also observed in the brain and cartilage (22) and spatially overlaps stef expression. The similarities in the expression pattern as well as in protein structure suggest that the two proteins may essentially have overlapping functions in similar molecular environments and affect the same cellular events. Alternatively, these proteins may be localized at distinct subcellular sites to differentially activate Rac1 and regulate distinct processes of neuronal differentiation. To clarify these possibilities, the subcellular localization of both gene products should be investigated.
Although stef and Tiam1 transcripts are distributed in several overlapping tissues, we are aware that stef expression is confined to narrower areas compared with Tiam1 expression. At the adult stage, Tiam1 is strongly expressed in testis (18), where stef is detected at a much lower level. In E14.5 embryos, there are a number of tissues in which Tiam1 is expressed, but stef expression is hardly detected; among them are the roof of the midbrain and the olfactory epithelium (22). These distinct expression patterns suggest that stef and Tiam1 have differential functions, and stef may play a more limited role in the regulation of tissue development.
We have shown that STEF, Tiam1 and SIF share several conserved domains.
PDZ is a domain for protein interaction and is found in many proteins
associated with specialized junctions (16, 17), and the PH domain is
implicated in the binding to membranes and is frequently present in
many signaling molecules (15). The TSS domain also appears to mediate
protein interaction (38). These conserved domain structures suggest
that the three proteins interact with similar molecules to constitute a
protein complex at the plasma membrane and activates Rac1 in a similar
mode, possibly responding to extracellular cues. Through this possible
signaling cascade, STEF may control neuronal migration or neurite
extension and also synaptic events, as suggested for SIF. To understand what biological events STEF regulates during and after development, genetic analyses will be necessary after the gene has been disrupted.
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ACKNOWLEDGEMENTS |
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We are grateful to M. Nakagawa, N. Ito, N. Matsuo, M. Washida, and M. Yoshizawa for help with enzyme assay, culture experiment, and in situ hybridization, F. Michiels and J. G. Collard for a comment of Tiam1 and for a gift of Tiam1 cDNA, and T. Terashima for the invaluable discussion on mouse histology.
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FOOTNOTES |
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* This work was supported by CREST of Japan Science and Technology Corporation, by research grants from the Ministry of Education, Science, Sports, and Culture, and by a Research Grant for Nervous and Mental Disorders from the Ministry of Health and Welfare of Japan.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/EMBL Data Bank with accession number(s) AB022915.
2 M. Sone and C. Hama, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are:
GEF, guanine
nucleotide exchange factor;
DH, Dbl homology;
PH, pleckstrin homology;
PCR, polymerase chain reaction;
RT, reverse transcription;
kb, kilobase pair(s);
GTPS, guanosine 5'-3-O-(thio)triphosphate;
GST, glutathione S-transferase;
PBS, phosphate-buffered saline;
HA, hemagglutinin;
En, embryonic day n.
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REFERENCES |
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