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INTRODUCTION |
The c-Myc gene was discovered as the cellular homologue of the
retroviral v-Myc oncogene and has been proven to have a crucial role in
various pathological and physiological events in cellular biology. In
human cancers, alterations of the c-Myc gene have in fact been
reported, which have included chromosomal translocation, point
mutation, gene amplification, and heterotopic expression (1). On the
other hand, the levels of c-Myc oncoprotein increase and remain
elevated in normal cells that have been induced to divide, indicating
it is required throughout the cell cycle for proliferation. Deregulated
c-Myc expression is asserted to be sufficient to drive quiescent cells
into the S phase to prevent cell cycle exit (2-4).
Previous studies have revealed one of these actions is through the
modulation of transcription of various genes, i.e. c-Myc recognizes the E-box sequence in the genome together with Max as a
partner and activates transcription. In this case the C-terminal basic/helix-loop-helix/leucine zipper
(B-HLH-LZ)1 domain of c Myc
mediates binding to a specific area of DNA. c-Myc also interacts
directly with a wide variety of other proteins through this C-terminal
B-HLH-LZ domain. For example, dimerization with a protein known as Max
is essential for the binding of Myc to DNA (5). In addition, the
C-terminal domain of c-Myc binds to other transcription factors,
TFII-I, YY1, and AP-2, which regulate the DNA binding and
transcriptional activities of c-Myc (reviewed in Ref. 6). On the other
hand, the N-terminal domain of c-Myc (amino acids (aa) 1-144) has both
transcriptional activation and repression activities. This domain
contains two evolutionarily conserved regions termed Myc box (MB) I (aa
47-62) and MB II (aa 106-143). A c-Myc mutant deleted within MB I has
been shown to diminish Myc-mediated transactivation (7). MB II has been
reported to mediate several functions, including transformation,
induction of apoptosis, blocking differentiation, and transcriptional
repression (8-13). These studies show MB II is a crucial domain for
diverse c-Myc functions. Along these lines, there is a growing interest in c-Myc-binding proteins to realize the mechanisms of multiple c-Myc functions.
In this paper, we report on Tiam1, which has been already reported as a
specific guanine nucleotide exchange factor (GEF) for Rac1, a Rho
family p21 low molecular weight G protein, which also binds to c-Myc
(14). Tiam1 can modify actin cytoskeleton and cell migration, and can
activate the mitogen-activated protein kinase cascade through
activating Rac1. Although some papers have recently reported that Tiam1
or Rac1 is also related to apoptosis, both these proteins show various
effects for apoptosis depending on the cell type (15-26). As far as we
know, no previous paper has ever addressed a potential interaction
between Tiam1 and c-Myc. We here report the direct interaction between
c-Myc and Tiam1, and the negative regulation of Tiam1 of c-Myc
transactivation and its apoptosis activity. This interaction with c-Myc
may provide a new facet of Tiam1, demonstrating various unexpected functions.
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MATERIALS AND METHODS |
Plasmids and Constructs--
A full-length human c-Myc was
cloned in pGEX-4T3 (Amersham Biosciences), and that with a flag
epitope at the C terminus was cloned in pCS2+ (provided by D. Turner,
Hutchinson Cancer Research Center, Seattle, WA) for transfection into
293T cells. Full-length human Tiam1 cDNA (a gift from K. Nakaya,
Showa University, Tokyo, Japan) was generated as described (15) and
cloned into pCS2+. Deletion mutants of c-Myc (1-142, 1-128, 129-439,
and 143-355; these numbers refer to the amino acids included in the
encoded c-Myc protein) and those of Tiam1 (C1199, C682, and N392;
nomenclature refers to the number of C-terminal or N-terminal amino
acids of the encoded Tiam1 proteins) were generated by two-step PCR
mutagenesis, as previously described (27). Tiam1(N392) was tagged by
hemagglutinin (HA) at the C terminus. Plasmids encoding c-DNAs of human
-Pix CH domain deleted mutant and human Vav1 were gifts from T. Nagase (Kazusa DNA Research Institute, Kisarazu, Japan) and M. Shibuya (Institute of Medical Science, University of Tokyo, Japan),
respectively. Oncogenic Vav1 was generated by two-step PCR mutagenesis
(28). The dominant negative mutant of Rac1 (N17Rac1), tagged with HA and cloned into pCS2+, was provided by B. J. Mayer (University of
Connecticut Health Center, Farmington, CT) (29).
Antibodies--
The monoclonal antibodies for the HA epitope
tag, c-Myc, and nucleoporin p62 were from Berkeley Antibody (Richmond,
CA), Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and Transduction
Laboratories (Lexington, KY), respectively. The monoclonal antibodies
for the flag tag and
-tubulin were from Sigma. The polyclonal
antibodies for Tiam1(C-16) and Tiam1(N-15) (Tiam1C and Tiam1N,
respectively) were from Santa Cruz Biotechnology, Inc. The polyclonal
antibody anti-c-Myc was from Upstate Biotechnology, Inc. (Lake Placid, NY) Anti-mouse or anti-rabbit immunoglobulin conjugated or unconjugated with alkaline phosphatase and TRITC (tetramethylrhodamine
isothiocyanate)-conjugated secondary antibodies were from DAKO
(Copenhagen, Denmark).
Cell Culture, Transfection, and Immunoprecipitation--
Rat1/CM
cells were kindly supplied by Y. Kuchino (National Cancer Center
Research Institute, Tokyo, Japan) (30). 293T human embryonal kidney
cells and Rat1 fibroblasts were cultured in Dulbecco's modified
Eagle's medium (Invitrogen) supplemented with 10% fetal bovine
serum. Rat-1/CM cells were maintained as previously described (30). For
transient expression assays, 293T cells were transfected with a maximum
of 5 µg of plasmid DNA/6-cm diameter dish by a calcium phosphate
coprecipitation method with concurrent treatment with 25 µM chloroquine, essentially as described previously (27). 293T cells and Rat1 cells were also transfected using LipofectAMINE 2000 (Invitrogen) with a maximum of 3.5 µg of plasmid DNA/3.5-cm diameter dish. Cells were harvested 48 h after transfection. To monitor the effect of fibronectin on the interaction of the following molecules, we harvested Rat1 and Rat1/CM after 20 or 50 min after their
placement on fibronectin-coated dishes with a diameter of 6.0 cm. Cell
lysates were prepared using protease inhibitors in TXB buffer (10 mM Tris (pH 7.6), 150 mM NaCl, 5 mM
EDTA (pH 8.0), 10% glycerol, 1 mM
Na3VO4, and 1% Triton X-100). Whole brain
tissue from 5-week-old ICR mice was also lysed in TXB buffer with a
Dounce homogenizer. Lysates were precleared by incubation with protein G-agarose (Roche Molecular Biochemicals) for 3 h at 4 °C. To
purify the protein, 1 µg of monoclonal or affinity-purified
polyclonal antibody was incubated with 500 µl of cell lysate for
2 h at 4 °C and precipitated with protein G-agarose for 4 h at 4 °C. Immunoprecipitates were extensively washed with TXB
buffer, separated by SDS-PAGE (27).
In Vitro Binding Assay--
293T cells lysates transiently
expressing Tiam1 and its deletion mutants were prepared as described
above. Glutathione S-transferase (GST)-c-Myc, GST deletion
mutants of c-Myc, and GST were purified from Escherichia
coli transformed with wild-type and deletion mutants of c-Myc
cloned into pGEX-4T3 and pGEX-4T3 alone, incubated, and analyzed as
described in Ref. 31.
Direct Binding Assay--
The proteins of Tiam1(FL) and
Tiam1(N392) were produced in in vitro reticulocyte lysate,
by TNT quick-coupled translation/transcription systems
(Promega, Madison, WI), according to the instructions from the
manufacturer, and incubated with wild-type and deletion mutants of
GST-c-Myc, and GST alone as described above. The solution was purified
by glutathione-Sepharose beads (Roche Molecular Biochemicals), separated by SDS-PAGE. The results were visualized and quantitated with
a Bio-Imaging Analyzer (BAS1000, Fuji).
Subcellular Fractionation of Mouse Brain--
The purification
of the nuclei and cytoplasm from Rat1 cells and Rat1/CM cells was
carried out as described (32).
Immunohistochemistry of Mouse Brain--
Whole brain tissue from
5-week-old ICR mice was fixed with PBS containing 4% paraformaldehyde
for 24 h and embedded in paraffin. Sections (4 µm thickness)
were cut and stained with hematoxylin and eosin stain. An
immunohistochemical study was with the Histofine Simple Stain PO
(multi) kit (Nichirei, Tokyo, Japan), according to the instructions
from the manufacturer. Antigen retrieval was achieved with trypsin
digestion. The primary antibodies used are described under
"Results."
Cell Staining--
For immunohistochemical analysis, cells were
plated on uncoated slides or fibronectin-coated slides (Roche Molecular
Biochemicals) for 30, 50, 70, or 90 min, and fixed with PBS containing
4% paraformaldehyde for 20 min at 25 °C, and then permeabilized
with PBS containing 0.5% Triton X-100 for 5 min. The cells were
preincubated in 1% bovine serum albumin for 10 min and incubated with
an anti-Tiam1 (Tiam1N) antibody for 1 h at 25 °C. The cells
were then rinsed in PBS containing 1% bovine serum albumin. The cells
were further incubated with a TRITC-conjugated secondary antibody for
1 h at 25 °C. Cells were examined with an Axiophot microscope
(Carl Zeiss, Inc., Thornwood, NY).
Luciferase Assay--
293T cells in a 3.5-cm dish were
transiently transfected with 0.02 µg of pRL-TK (Toyo Ink, Tokyo,
Japan), 1 µg of p4x(WT)E-SVP-Luc (a gift from H. Ariga, Graduate
School of Pharmaceutical Sciences, Hokkaido University, Sapporo,
Japan), and various plasmids described under "Results" using
LipofectAMINE 2000 (Invitrogen). Two days after transfection, whole
cell extracts were prepared and the luciferase activity caused by the
reporter plasmid was determined by using the Pica Gene dual kit (Toyo Ink).
Apoptosis Assay--
To induce apoptosis in Rat-1 cells and
their transfectants, 2 days after transfection, the cells were seeded
24 h before serum deprivation and then transferred to medium
containing 0.3% FBS unless otherwise indicated, after treatment or no
treatment of transient transfection. The cells were judged to be
apoptotic when they were nonadherent and showed typical nuclear changes (definite chromatin condensation and/or nuclear fragmentation) (30).
After photography, the cells were harvested by trypsinization and fixed
in PBS containing 4% paraformaldehyde for 20 min at 25 °C. The
cells were stained with 4',6-diamidino-2-phenylindole and observed as
described in Ref. 33.
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RESULTS |
c-Myc Specifically Associates with Guanine Nucleotide Exchange
Factor Tiam1--
We explored the binding between c-Myc and a GEF of
Rac1, Tiam1. First, to determine the location of the Tiam1 binding site in the c-Myc protein, we made truncated c-Myc cDNA constructs (Fig.
1a). Bacterially extracted GST
fusioned wild-type (WT) and the truncated form of c-Myc, which were
purified by glutathione-Sepharose beads, were incubated with the cell
lysate of Tiam1(WT)-transfected 293T cells. In this in vitro
condition, Tiam1(WT) could associate with some of these c-Myc
constructs that contain the intact N-terminal MB II domain (Fig.
2a). We also
prepared truncated Tiam1 cDNA constructs to determine the
c-Myc-binding region in Tiam1 (Fig. 1b). GST-c-Myc(WT)
extracted from bacteria could associate only to the WT and N392 (aa
1-392) truncated mutants of Tiam1, but not to the C1199 and C682
mutants in the in vitro condition described above (Fig.
2b). These observations imply that MB II in the N-terminal domain of c-Myc and N-terminal 1-392 amino acids of Tiam1 may be the
principal regions for binding in vitro with each other.

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Fig. 1.
Schematic representation of the wild-type and
truncated c-Myc (A) and Tiam1 (B)
constructs used in this study. Proteins are depicted to scale.
NLS, nuclear localization signal; M,
myristoylation signal; P, region rich in proline, glutamate,
serine, and threonine; PHn and PHc, N-terminal
and C-terminal pleckstrin homology domain; wt,
wild-type.
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Fig. 2.
c-Myc specifically associates with the
guanine nucleotide exchange factor Tiam1. A, 293T cells
were transiently transfected with Tiam1(WT) construct. Bacterially
purified GST-fusioned wild-type and truncated c-Mycs were incubated
with the 293T cell lysates and affinity-precipitated (AP) by
glutathione beads, according to the in vitro binding assay.
Bound proteins were immunoblotted (IB) with anti-Tiam1C
(lanes 1-6), which reacted with epitopes located
at the C terminus of Tiam1. GST fusion proteins are shown at the
bottom. 293T cell total lysates are shown in lane
7. B, 293T cells were transiently transfected
with Tiam1(WT), Tiam1(C1199), Tiam1(C682), and HA-tagged Tiam1(N392)
constructs. Bacterially purified GST-fusioned wild-type c-Myc was
incubated with the 293T cell lysates, according to the in
vitro binding assay. Bound proteins were immunoblotted with
anti-Tiam1C (lanes 1-3) and anti-HA
(lane 4). GST-c-Myc is shown at the
right. Expressions of Tiam1s are shown at the bottom. C, 293T cells were
transiently transfected with plasmid encoding flag-tagged c-Myc
together with Tiam1(WT) construct. Cell lysates were immunoprecipitated
(IP) with anti-Tiam1C and anti-flag, and immunoblotted with
anti-flag and anti-Tiam1C, respectively. Expression of c-Myc and Tiam1
is shown at the bottom. D, 293T cells were
transiently transfected with Tiam1(WT) and -(C1199), and plasmid
encoding HA-tagged Vav1 and -Pix Pix CH domain deleted mutant
together with c-Myc(WT) construct. Cell lysates were immunoprecipitated
(IP) with anti-Myc, and immunoblotted with anti-HA and
Tiam1C. Expression of c-Myc and Tiam1 are shown at the
bottom. E, bacterially purified GST-fusioned
wild-type and truncated c-Mycs were incubated with Tiam1(N392)
translated and transcribed in vitro. Bound proteins
were separated by SDS-PAGE and visualized by imaging analyzer (BAS
1000; Fuji) (lanes 1-3). Lane
4 was the expression of Tiam1s. GST fusion proteins are
shown at the bottom. F, whole brains of ICR mice
were lysed and immunoprecipitated with anti-Tiam1C antibody, preimmune
rabbit immunoglobulin, and anti-Myc (polyclonal) antibody. Precipitants
were subjected to immunoblotting with the indicated antibodies. These
experiments were performed at least three times.
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We next examined the Tiam1 association with c-Myc under another
condition. Coexpression and coprecipitation analyses in 293T cells
revealed that immunoprecipitated Tiam1(WT) could associate to c-Myc(WT)
tightly in vivo, but not to the aa 143-355 truncated mutant
of c-Myc, which could not associate to Tiam1(WT) in vitro (Fig. 2c). Conversely, immunoprecipitated c-Myc(WT) could
also associate to Tiam1(WT), but c-Myc(143-355) could not (Fig.
2c).
Then we were interested in whether the other Rac1-GEFs, including
-Pix and Vav1, could associate to c-Myc in vivo.
Immunoprecipitated c-Myc(WT) overexpressed in 293T cells
could specifically associate to Tiam1(WT), but not to the
C1199 truncated mutant of Tiam1, other Rac1-GEFs and their mutants; the
wild-type Vav1 and CH domain truncated
-Pix (Fig.
2d).
Next we tested the direct binding between Tiam1 and c-Myc by the
in vitro translation and transcription reticulocyte lysate system (described under "Materials and Methods"). GST-fusioned c-Myc(WT) could directly bind to in vitro transcribed
Tiam1(N392) constructs, but GST fusion mutated c-Myc(143-355) and GST
alone could not directly bind them (Fig. 2e).
Among native mouse tissues, Tiam1 has been reported to be highly
expressed in the brain and the testis (14). The expression of c-Myc is
also reported to be detected in the mouse brain and to be enhanced by
methamphetamine-induced apoptotic processes in neurons (34). In the
protein extracted from mouse brain, c-Myc was coprecipitated with
immunoprecipitated Tiam1 by the specific Tiam1 antibody but was not
coprecipitated by preimmune rabbit immunoglobulins (Fig.
2f). Despite the weak expression of c-Myc in the brain, we
believe we could prove the considerable interaction between c-Myc and
Tiam1 in the mouse brain.
Nuclear Localization of Tiam1--
We next attempted to
investigate the intracellular location of both proteins. A
transcription factor c-Myc is known to be present in the nucleus (34).
We tested whether Tiam1 is mainly present in the same location as
c-Myc. First, we applied the immunohistochemical technique for the
mouse brain tissue in which c-Myc and Tiam1 associated with each other
(Fig. 2f). As shown in Fig. 3,
the expression of c-Myc was mainly and diffusely located at the nucleus of the cerebral neuronal cells in the cortex and the basal ganglia by
the c-Myc-specific antibody. The immunoreactivity of Tiam1 was also
demonstrated in the nuclei of cerebral neuronal cells, but only in
those of the thalamus, hypothalamus, and dentate nucleus (Fig. 3). That
immunoreactivity disappeared by absorption with peptide used as the
immunogen for the Tiam1-specific antibody (Fig. 3).

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Fig. 3.
The nuclear localization of Tiam1. The
same regions of the basal ganglia of ICR mice brain were stained
with hematoxylin and eosin stain and immunohistochemically. For the
immunohistochemistry, anti-c-Myc, anti-Tiam1N, and peptide for
neutralization to anti-Tiam1N antibodies were used as shown. These
experiments were performed at least three times, and evaluation of
immunoreactivity was performed by two independent pathologists.
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Previously, several investigators including us reported that an
F-actin-containing membrane ruffle was observed at 15 min after
stimulation (adherence of Rat-1 fibroblasts to the extracellular matrix, fibronectin) and a Tiam1 localization on that membrane ruffle
was detected (27, 35). When Rat-1 cells cultured on uncoated slides
were analyzed, we found that endogenous Tiam1 was demonstrated in the
nucleus, but endogenous c-Myc was barely detectable by
immunofluorescence with the specific antibody (Fig. 4, a and b). On the
other hand, both endogenous Tiam1 and stably transfected c-Myc were
detected clearly and abundantly in the nucleus, when Rat-1/CM (Rat-1
cells stably transfected with c-Myc), were used in the same conditions
as Rat-1 cells (Fig. 4, d and e). A negative
control against anti-Tiam1 antibody using preimmune rabbit
immunoglobulins as the primary antibody revealed no immunoreactivity in
both Rat-1 and Rat-1/CM (Fig. 4, c and f).

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Fig. 4.
Nuclear localization of Tiam1.
a-f, Rat1 cells (a-c) and Rat1 cells stably
expressing c-Myc (Rat1/CM) (d-f) were stained for Tiam1N
(a and d), c-Myc (b and e),
and preimmune rabbit immunoglobulin (c and f) on
uncoated slides, respectively. g-n, Rat1 cells
(g-j) and Rat1/CM cells (k-n) were replated on
fibronectin-coated slides for 30 (g and k), 50 (h and l), 70 (i and m),
and 90 min (j and n) and stained with Tiam1N.
o, total cell lysates and purified nuclei and cytoplasm from
Rat1 cells (lanes 1-3) and Rat1/CM cells
(lanes 4-6) were immunoblotted against various
antibodies, as indicated. Ten micrograms of protein from each fraction
was applied to SDS-PAGE. p, Rat1 and Rat1/CM cells were
plated on fibronectin-coated dishes and cultured for 20 or 50 min, as
indicated. The cell lysates were immunoprecipitated (IP)
with anti-c-Myc and Western blotted (WB) with anti-Tiam1C.
The expression of c-Myc and Tiam1 are shown at the bottom of
the figure. These experiments were performed at least three
times.
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We next attempted to record the time-course profile of Tiam1
localization in Rat-1 and Rat-1/CM placed on fibronectin-coated slides.
In this experiment, we observed immunofluorescence with a specific
antibody in these cells after various periods from plating, as
described under "Materials and Methods." In Rat1 cells, endogenous
Tiam1 was localized both in the nucleus and in the cell membrane ruffle
at 30, 50, and 70 min after cells were placed on fibronectin (Fig. 4,
g-i). However, by 90 min, endogenous Tiam1 gradually
localized in the nucleus and disappeared from the membrane ruffle (Fig.
4j). In the case of Rat-1/CM, endogenous Tiam1 was also
localized both in the nucleus and in the cytoplasmic membrane ruffle at
50 and 70 min, and mainly in the nucleus but not in the membrane ruffle
at 90 min after plating (Fig. 4, l-n). In Rat-1/CM,
granular expression of endogenous Tiam1 in cytoplasm was detected but
was weak in nucleus for the first 30 min (Fig. 4k). During
the course of these experiments, c-Myc expression was constantly
detected in the nucleus in Rat-1/CM (data not shown).
The influence of fibronectin stimulation on the interaction between
c-Myc and Tiam1 was examined using a time-course experiment as
described under "Materials and Methods." The in vivo
binding between Tiam1 and c-Myc in Rat1/CM cells was detected at 50 min (Fig. 4p, lane 2) but not at 20 min
(Fig. 4p, lane 1) after the plating on fibronectin.
To confirm these results, we examined and checked the intracellular
localization of both Tiam1 and c-Myc in Rat-1 and Rat-1/CM by
subcellular fractionation and Western blotting. We could also detect
Tiam1 expression in the nuclear fraction, but weakly in the cytoplasmic
fraction in both Rat-1 and Rat-1/CM (Fig. 4o). In contrast,
c-Myc expression of Rat-1/CM was detected only in the nuclear fraction
(Fig. 4o, lane 1). In Rat-1 cells,
endogenous c-Myc was not detected by this method (Fig. 4o,
lanes 4-6). In addition, stably overexpressed
c-Myc did not affect the location of endogenous Tiam1 in Rat-1 cells in
this subcellular fractionation assay.
Tiam1 represses the transcriptional activity of c-Myc. To determine
whether the association with Tiam1 affects the transcription properties
of c-Myc, 293T cells were transiently transfected with the expression
vectors harboring c-Myc and Tiam1 together with the reporter plasmid
containing the luciferase gene linked to the tetramerized E-box
sequence (4XE) followed by the SV40 promoter, using a cationic lipid
(36). First, we examined the single luciferase activity of c-Myc or
Tiam1. As shown in Fig. 5a,
c-Myc stimulated the E-box-dependent luciferase activity.
The relative luciferase activity of c-Myc is ~3 times that of pCS2
mock control vector (Fig. 5a, lane 3).
On the other hand, Tiam1 showed no effect on the luciferase activity by
transfection of Tiam1 with the reporter plasmid (Fig. 5a,
lane 4). We next checked the influence of Tiam1 toward c-Myc luciferase activity. When 293T cells were transfected with
c Myc, pCS2 mock control vector and reporter plasmid, the luciferase
activity was stimulated (Fig. 5b, lane
2), whereas the mock vector alone showed no effect on that
activity (Fig. 5b, lane 1). The
luciferase activity was enhanced by c-Myc, and was repressed by
co-transfection of Tiam1 with c-Myc and the reporter plasmid, in a
dose-dependent manner (Fig. 5b, lanes
4 and 5). Co-expression of N17Rac1, a dominant
negative mutant of Rac1, under the same conditions as in Fig.
5b (lane 4) had no Tiam1-mediated suppression of the transcriptional activity of c-Myc (Fig.
5b, lane 3). In the case of
Tiam1(C682), Vav1, and oncogenic Vav1, which is constitutive active
mutant of Vav1, none of these constructs showed any effect on the
c-Myc-enhanced luciferase activity (Fig. 5b,
lanes 6-8). Because the Tiam1 deletion mutant
(C682), which could not associate with c-Myc, did not affect the
luciferase activity in this experimental system, and because another
Rac1-GEF (here Vav1) did not, either, the repression activity of Tiam1 that we observed here is dependent on its binding capability to c-Myc
and may be specific to Tiam1.

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Fig. 5.
Tiam1 represses the transcriptional activity
of c-Myc. A, 293T cells were transiently transfected
with pRL-TK, reporter plasmid, 1 µg of c-Myc(WT), and 1 µg of
Tiam1(WT) by the LipofectAMINE procedure as indicated. Relative c-Myc
luciferase activity compared with that of lane 2 is shown at the bottom. B, 293T cells were
transiently transfected with pRL-TK reporter plasmid, 1 µg of
c-Myc(WT) as indicated. Lanes show 1 µg of Tiam1(WT)
(lanes 3 and 4), 0.5 µg of Tiam1(WT)
(lane 5), 1 µg of Tiam1(C682) (lane
6), 1 µg of HA-tagged Vav1(WT) (lane
7), 1 µg of HA-tagged oncogenic-Vav1 (lane
8), and 1 µg of HA-tagged N17Rac1 (lane
3). Relative c-Myc luciferase activity compared with
lane 1 was shown in the bottom. These
experiments were performed at least three times.
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Tiam1 Interfered with c-Myc-mediated Apoptosis in Rat-1
Cells--
As mentioned above, c-Myc has been implicated in the
regulation of apoptosis in certain settings. To clarify the effect of Tiam1 on the apoptotic activity of c-Myc, we used Rat-1 cells and
Rat-1/CM cells, which were stably expressed with c-Myc. In this
experimental system, Rat-1/CM causes c-Myc-related apoptosis in a
serum-deprived cultured medium (30). Beforehand, we checked the ratio
of apoptotic cells in Rat-1 and Rat-1/CM. As previously reported, both
Rat-1 and Rat-1/CM were cultured in 10 or 0.3% FBS-containing medium
for 24 h, respectively (30), and we counted cells with apoptotic
phenotypes. When Rat 1/CM were harvested in 10 and 0.3% FBS,
approximately 8 and 46% cells showed apoptotic phenotypes,
respectively (Fig.
6a,
lanes 3 and 4). Rat-1 in 10% FBS grew
normally and showed the low apoptotic ratio (Fig. 6a, lane 1). Rat-1 in 0.3% FBS exhibited a ratio of
~14% apoptotic cells (Fig. 6a, lane
2). These results showed c-Myc-induced apoptosis is related
to the status of the serum deprivation.

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Fig. 6.
Tiam1 interfered with c-Myc-mediated
apoptosis in Rat1 cells. A, induction of c-Myc-mediated
apoptosis by serum deprivation. Rat1 cells and Rat1/CM cells were
seeded at a density of 1 × 105 cells in 60-mm dishes
and cultured for 12 h in medium containing 10% FBS. The cells
were transferred to the medium containing 10 or 0.3% FBS. The
percentage of apoptotic cells was determined 24 h later as
described under "Materials and Methods." B-E, Rat1/CM
cells were transiently transfected with 2 µg of GEFs and 1 µg of
HA-tagged N17Rac1 or 1 µg of flag-tagged c-Myc(1-142), as indicated. Twenty-four hours after
transfection, the cells were transferred to a medium containing 0.3%
FBS. The apoptotic cells were photographed 24 h later
(C and E), and the percentage of apoptotic cells
was determined, as described under "Materials and Methods"
(B and D). The lane numbers
in B correspond to the numbers in C,
and the lane numbers in D correspond
to the numbers in E. These experiments were
performed at least three times.
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Next, we examined whether Tiam1 interfered with the apoptosis
inducing activity of c-Myc in this experimental system. Constructs of
Tiam1(WT), Tiam1(C1199), Tiam1(C682), Vav1, or mock control vector were
transiently transfected with or without N17Rac1 in Rat-1/CM by using
cationic lipid (Fig. 6, b and c). We had already monitored the transfection efficiency and obtained the results that
more than 90% of cells showed expression of the construct when
Rat-1/CM cells were transfected by this transfection protocol (data not
shown). These transfected cells after serum deprivation for 24 h
showed that the exogenous expression of Tiam1(WT) reduced the number of
apoptotic cells (Fig. 6, b and c, lane
3), whereas Tiam1(C682) and Vav1, which could not bind to
c-Myc, did not act on apoptosis compared with the mock control
(Fig. 6, b and c, lanes 5 and 6). However, the expression of Tiam1(C1199) increased the apoptotic cell ratio compared with the mock control. In the case of
transfection by Tiam1 together with N17Rac1, which is a dominant
negative Rac1, the number of apoptotic cells also reduced (Fig. 6,
b and c, lane 2). These
results showed that Tiam1 inhibited the apoptotic activity of c-Myc
that was dependent on the binding capacity of Tiam1 to c-Myc, whereas
N17Rac1 showed no effect on this inhibition. On the other hand,
Tiam1(C1199), which is known to be an active form for GEF activity and
does not bind with c-Myc, had no effect on this inhibition (Fig. 6,
b and c, lane 4). This observation probably reflects the induction of apoptosis by
Tiam1(C1199) through the activated mitogen-activated protein kinase
cascade by its dominant GEF activity for Rac1, not through c-Myc interaction.
Next, we examined whether the ectopic expression of the MBII domain of
c-Myc, which binds Tiam1, could reverse the inhibition of c-Myc
apoptosis observed upon the co-expression of Tiam1. We used a deletion
mutant of c-Myc, c-Myc(1-142), which contains the MB II domain but not
the nuclear localization signal, basic, HLH, and LZ domains. We
expected that c-Myc(1-142) would bind to Tiam1, as shown in Fig.
2a, but would not be translocated to the nucleus. We
transiently transfected both Tiam1(WT) and c-Myc(1-142) in a Rat1/CM
cell line and performed an apoptosis assay. When both Tiam1 and the
mock control vector were transfected, the apoptotic activity of c-Myc
was inhibited (by the interaction of Tiam1; Fig. 6, d and
e, lane 1), but when both Tiam1 and
c-Myc(1-142) were transfected, the apoptotic activity of c-Myc was
only partially inhibited (Fig. 6, d and e,
lane 2). We interpreted this finding as implying
that the coexpression of excess c-Myc(1-142) partially rescued the
Tiam1(WT)-mediated inhibition of c-Myc-induced apoptosis.
 |
DISCUSSION |
The mechanisms through which the effects of c-Myc mediate on the
fate of cells (for example cell transformation or apoptosis) have
been reviewed (37, 38). A broad body of work argues convincingly that
c-Myc is a transcription factor, which activates and represses many
target genes (39). However, a large number of studies have recently
identified cellular factors that, through their interaction with c-Myc
in cells, have provided a deeper insight into other possible functions.
Many proteins that interact with the c-Myc N-terminal domain have, in
particular, been identified, and their respective effects on various
c-Myc functions have been analyzed, including transactivation, cell
transformation, and apoptosis. As described in the Introduction, MB II
seems to be the most crucial domain in N-terminal domain for
several c-Myc functions, and thus identification of MB II-binding
proteins has been thought to significantly advance our understanding of
the mechanisms of c-Myc. Several MB II-binding proteins, which are
indispensable to control several c-Myc functions, have in fact been
identified, such as Bin1, TRRAP, Pam, and so on (reviewed in Ref. 6).
Tiam1, on which we report here, is a new N-terminal domain and MB
II-binding protein. As shown in the results, the direct interaction of
Tiam1 and c-Myc induces repression of the transactivation and apoptotic
activities of the latter.
The question may be raised whether Tiam1 can directly associate with
c-Myc. Recently it was reported that Cdc24, a GEF for Cdc42 that is one
of the Rho family GTPases (40, 41), was sequestered in the cell nucleus
by the adapter protein Far1. It was then relocated to the cytoplasm by
degradation of Far1 by the G1 cyclin-dependent
kinase Cdc28-Cln at the actin cytoskeletal change, and by the importin
-family member Msn5, which is required for nuclear export (40, 41).
These reports demonstrated an important process; a GEF for the
Rho-family GTPases could be sequestrated in the nucleus and relocated
in the cytoplasm by other proteins (41). Our observations of the Tiam1
localization we have presented in our study here remind us of the
situation of Cdc24, whereby Tiam1 was mobilized to the cytoplasm in
Rat-1 cells at the first response to contact with the extracellular
matrix fibronectin, was recruited to both the membrane ruffle and
nucleus during the next phase, and then was finally relocated to the
nucleus (Fig. 4, g-n). There was no difference between
Rat-1 and Rat-1/CM in that Tiam1 was present in the nucleus when plated
on the uncoated slides (Fig. 4, b and e). On the
contrary, the membrane ruffle localization of Tiam1 was detected as
early as 30 min later in Rat-1 cells, whereas it takes 50 min in Rat
1/CM (Fig. 4, g-n). These phenomena may show that
overexpression of c-Myc itself has no effect on the stable localization
of Tiam1, but it affects the Tiam1 mobilization between the cytoplasm
and the nucleus. c-Myc seems to slow down the Tiam1 mobility to the
membrane ruffle and the cytoplasm from the nucleus, but we could not
conclude at what point in this nuclear import system the c-Myc had its effect. At this moment, information about the nuclear localization of
Rho-GTPases-GEFs is only available about Tiam1 and Cdc24. We could not
find other endogenous Rac1 GEFs, Vav1 and
-Pix, expressed in the
Rat-1 and Rat-1/CM nucleus by immunofluorescence when we examined them
under the same experimental conditions as Tiam1 (data not shown). The
nuclear localization of Tiam1 may be important in terms of enabling
direct interaction with c-Myc. Specific interaction of c-Myc with
Tiam1, but not with Vav1 and
-Pix, was validated by coexpression and
coprecipitation in our in vivo association analysis (Fig.
2d). On the other hand, Tiam1 has recently been reported to
interact with other proteins related to cell migration and cytoskeletal
modification (27). However, until now, there has been no report that
has proved any interaction between Tiam1 and any nuclear protein. We
have been able to show for the first time the direct interaction of
Tiam1 with the c-Myc nuclear protein. As shown in Fig. 4p,
the attachment of the cells to the fibronectin changed the degree of
binding between Tiam1 and c-Myc, probably by altering the localization
of Tiam1. We think that these data are also consistent with the idea
that the direct binding of these proteins takes place in the nucleus.
Naturally, further investigation regarding the influences of cell
attachment on apoptosis and Myc function is needed. We delineated the
c-Myc-binding locus in Tiam1 as the N terminus of Tiam1 (aa 1-392).
Although Tiam1 (aa 1-392) contains two PEST domains (aa 58-92 and
100-132), which are considered to be predictors of protein instability
(14, 42) (Fig. 2, b and e), the presence of a
c-Myc binding motif in Tiam1 should be elucidated in further experiments.
c-Myc is known to act as a transcription factor in its classical
function. Many genes, in which c-Myc activates or represses expression,
have been identified (reviewed in Refs. 37 and 43). As mentioned under
"Results," the Tiam1 mutant and Vav1, which lacked any binding
activity to c-Myc, could not modify the transactivation of c-Myc (Fig.
5b), indicating that Tiam1 interaction may repress transcription of some genes via c-Myc. However, we have so far been
unable to find any kind of gene that Tiam1 could ultimately down-regulate. The dominant negative Rac1(N17Rac1) failed to relieve Tiam1-mediated inhibition of the transcriptional and apoptosis inducing
activities of c-Myc (Fig. 5b, lane 3),
suggesting that Rac1, a known effector of Tiam1, may not be involved in
the regulation of c-Myc functions by Tiam1.
In terms of the ultimate effector of the apoptotic function of c-Myc,
many transactivated target genes have also been reported, including
p53, ornithine decarboxylase, and cyclin A (44-47). Recently several
genes indirectly induced by c-Myc during apoptosis have also been
reported. The CD95/Fas ligand is additionally known as an indirect
target gene of c-Myc. c-Myc controls apoptosis through regulation of
CD95/Fas ligand expression in some cells, but not in the others (48).
Along these lines, we monitored the expression of CD95/Fas ligand in
both Rat-1 and Rat 1/CM by reverse transcription-PCR, but we did not
detect any change in expression (data not shown). Soucie et
al. (49) also reported that c-Myc activates Bax and elicits
cytochrome c release from mitochondria into the cytoplasm
during apoptosis, and this effect is inhibited by Bcl-2. It is not,
however, clear what kind of proteins were directly transactivated by
c-Myc in this situation. In our results, Tiam1(WT) inhibited
c-Myc-related apoptosis in Rat-1/CM cells, but the Tiam1 mutant and
Vav1, which have no binding activity to c-Myc, could not (Fig. 6,
b and c). As shown in Fig. 6 (d and
e), the coexpression of excess c-Myc(1-142) partially rescued the Tiam1(WT)-mediated inhibition of c-Myc-induced apoptosis. These results further verify the inhibitory role of Tiam1 in the c-Myc
apoptosis mechanism, based on its association with aa 1-142 of
c-Myc.
The roles of GTPases in the apoptosis pathway have recently drawn
considerable attentions. For example, in fibroblasts and some
hematopoietic cells, constitutively active Rac1 protects cells from
apoptosis induced by Ras or serum withdrawal (17, 19). In contrast,
activated Rac and Cdc42 can induce apoptosis in Jurkat T lymphocytes,
thymocytes, peripheral T cells, and neuronal cell lines (20-26). Tiam1
has also been shown to promote apoptosis in certain cell types.
Treatment of promyelocytic HL-60 or myeloid U937 cells with the
apoptotic agent bufalin induced a modest increase in endogenous Tiam1
mRNA and protein levels (15). Furthermore, overexpression of Tiam1
enhanced bufalin-induced apoptosis, whereas expression of antisense
Tiam1 RNA inhibited apoptosis. Another paper (16), however, reported
that Tiam1 is cleaved by caspase during apoptosis. Considering the
argument in the papers mentioned above, Rac1 and its GEF Tiam1 have a
somewhat dichotomous effect on apoptosis. On the other hand in our
results, an active form of Tiam1, Tiam1(C1199), induced apoptosis in
the Rat-1/CM cell system (Fig. 6, b and c,
lane 4). This may imply that Tiam1 activates the
mitogen-activated protein kinase cascade through Rac1 and induces
apoptosis. However, this observation may be the case only in a
particular system, namely the Rat-1/CM, and further studies are
necessary to clarify the relationship between c-Myc apoptotic activity
and the effects of Rac1 and Tiam1 on apoptosis already reported. We
showed here only a descriptive phenomenon that Tiam1 is localized in
the nucleus and negatively regulates the transactivation and
apoptosis induced by c-Myc, and future studies will be aimed at
addressing the functional role of Tiam1 in the nucleus.