The Netherlands Cancer Institute, Antoni van Leeuwenhoekhuis, Division of Cell Biology, 1066 CX Amsterdam, The Netherlands
Rho-like GTPases, including Cdc42, Rac, and Rho, regulate signaling pathways that control actin cytoskeletal structures and transcriptional activation. The Tiam1 gene encodes an activator of Rac1, and similarly to constitutively activated (V12)Rac1, overexpression of Tiam1 in fibroblasts induces the formation of membrane ruffles. Tiam1 contains a Dbl homology (DH) domain and adjacent pleckstrin homology (PH) domain, hallmarks for activators of Rho-like GTPases. Unique for Tiam1 are an additional PH domain and a Discs-large homology region in the NH2-terminal part of the protein. Here we show that both in fibroblasts and COS cells, membrane localization of Tiam1 is required for the induction of membrane ruffling. A detailed mutational analysis, in combination with confocal laser scanning microscopy and immunoelectron microscopy, demonstrates that the NH2-terminal PH domain of Tiam1, but not the DH-adjacent PH domain, is essential for membrane association. This NH2-terminal PH domain of Tiam1 can be functionally replaced by the myristoylated membrane localization domain of c-Src, indicating that the primary function of this PH domain is to localize the protein at the membrane. After serum starvation, both membrane association of Tiam1 and ruffling can be induced by serum, suggesting that receptor stimulation induces membrane translocation of Tiam1. Similar to V12Rac1, Tiam1 stimulates the activity of the c-Jun NH2-terminal kinase (JNK). This Rac-dependent stimulation of JNK also requires membrane association of Tiam1. We conclude that the regulated membrane localization of Tiam1 through its NH2-terminal PH domain determines the activation of distinct Rac-mediated signaling pathways.
Rho-like GTPases, which include Cdc42, Rac1, and
RhoA, control distinct signal transduction pathways that determine the organization of the actin
cytoskeleton in response to various extracellular stimuli.
In fibroblast cells, activation of Cdc42 and Rac results in
the formation of focal complexes and filopodia or lamellipodia, respectively, while Rho is involved in the formation
of stress fibers and focal contacts (Ridley and Hall, 1992 Apart from their effects on the cytoskeleton, both Cdc42
and Rac1 can activate the c-Jun NH2-terminal kinase (JNK)1
pathway, while all three Rho-like GTPases activate serum
response factor (Coso et al., 1995 Rho-like GTPases are activated by guanine nucleotide
dissociation stimulators (GDSs), also known as guanine
nucleotide exchange factors. Inactivation occurs by hydrolysis of bound GTP, which is stimulated by interaction
with GTPase activating proteins, while guanine nucleotide
dissociation inhibitors (GDIs) lock the GTPases either in
the active or in the inactive state (Boguski and McCormick, 1993 Tiam1 was originally identified in T-lymphoma cells as
an invasion- and metastasis-inducing gene (Habets et al.,
1994 Apart from the DH domain, Tiam1 contains several
other conserved motifs, including a consensus myristoylation sequence at the NH2 terminus, a Discs-large homology region (DHR), and two pleckstrin homology (PH) domains (Habets et al., 1994
Since little is known of the intracellular localization and requirements of GDS proteins to stimulate Rho-like GTPases,
we analyzed the conditions for Tiam1 to stimulate Racdependent membrane ruffling and JNK activity. For this,
truncated Tiam1 proteins, or proteins that contain small deletions in each of the conserved domains, were expressed
in fibroblast cells and COS cells, and their intracellular localization and biological activity were analyzed by confocal
microscopy, immunoelectron microscopy, and analysis of
JNK activity. We demonstrate that membrane association of Tiam1 is absolutely required for Rac-dependent induction of membrane ruffling and stimulation of JNK activity.
Membrane association of Tiam1 is accomplished through
the NH2-terminal PH domain, but not the COOH-terminal
PH domain or the DHR domain. Moreover, PH domain-
dependent membrane association of Tiam1 could be induced by serum. These results suggest that the controlled
membrane association of Tiam1 is an important factor in
the activation of distinct Rac-mediated signaling pathways.
Transfection Constructs
Full-length mouse Tiam1 (FL1591) and the NH2-terminally truncated
C1199 and C580 Tiam1 constructs (nomenclature refers to the number of
COOH-terminal amino acids [aa] of the encoded Tiam1 proteins) contain
artificial SpeI and SalI restriction sites in front of the first ATG and a hemagglutinin tag (HA-tag), followed by a SalI restriction site, at their 3 Cells and Transfection Experiments
NIH3T3 cells, stably expressing the C1199 Tiam1 protein and C682 Tiam1
protein, have been described previously (Van Leeuwen et al., 1995). NIH3T3
cells and COS-7 cells were cultured in a humidified CO2 incubator in
DME supplemented with 10% newborn calf serum or 10% fetal calf serum, respectively. For transient expression assays, NIH3T3 cells were
seeded in dishes with or without coverslips (8,000 cells/cm2). 18 h after
seeding, cells were transfected with lipofectamin (Life Technologies, Inc.,
Grand Island, NY). COS-7 cells were seeded in dishes with or without
coverslips and transfected using DEAE dextran (Seed and Aruffo, 1987 Immunocytochemistry
Cells on coverslips were washed and fixed in 3.7% formaldehyde in PBS.
Cells were subsequently stained as described (Michiels et al., 1995 Immunoelectron Microscopy
48 h after transfection, cells were fixed for 1 h on the dish with a mixture
of 4% paraformaldehyde and 0.5% glutaraldehyde in 0.1 M phosphate
buffer. Fixed cells were scraped with a rubber policeman, washed, embedded in 10% gelatin, and infiltrated overnight with 1 M sucrose. After a further infiltration (2 h) with 1.8 M sucrose with 20% polyvinylpyrolidone,
the samples were frozen in liquid nitrogen. Frozen thin sections were collected on formvar/carbon-coated grids, preincubated with 0.15% glycine
in PBS, followed by immunoincubations with antibodies for 45 min at
25°C. Subsequently, the sections were incubated with 10-nm gold-labeled
secondary antibodies for 30 min at 25°C. Antibody dilutions were made in
phosphate buffer with 1% BSA and sections were washed in the same
buffer, before and in between the incubation steps. Finally, sections were
embedded and stained with 1.5% methylcellulose containing 0.3% uranyl
acetate and examined in an electron microscope (model CM10; Philips
Electronic Instrs., Mahwah, NJ). Where appropriate, the distribution of
immunogold particles in at least 40 cross sections of cells was analyzed.
JNK Assay
Semiconfluent COS-7 cells or NIH3T3 cells were transfected with pCMVFlag-JNK1 and pcDNA-neo-Rac1- or Tiam1-expression vectors as described above. 48 h after transfection, cells were washed with ice-cold PBS
and lysed on ice in a buffer containing 50 mM Tris-HCl, pH 7.5, 1% NP40, 100 mM NaCl, 5 mM EDTA, 100 µM sodium orthovanadate, 1 mM sodium fluoride, 20 µM aprotonin, 20 µM leupeptin, and 1 mM Pefabloc.
After centrifugation (10 min, 10,000 g), flag-tagged c-Jun kinase was immunoprecipitated using 2 µg/ml of M2 monoclonal antibody (Kodak,
Rochester, NY) for 1 h (4°C). Immune complexes were captured using
protein A-Sepharose beads (Pharmacia LKB Biotechnology Inc.) coated
with rabbit anti-mouse immunoglobulins (Nordic Immunological Labs,
Capistrano Beach, CA). Beads were washed three times in lysis buffer and
once in kinase buffer (20 mM Hepes, 10 mM MgCl2). Dried beads were incubated for 20 min at 30°C with 5 µg bacterially expressed glutathione
S-transferase (GST)-c-Jun (amino acids 1-135) in 40 µl kinase buffer containing 1 mM DTT, 20 µM ATP, 5 µCi [ Membrane Association of Tiam1 Is Required for
Membrane Ruffling
We have shown previously that full-length Tiam1 (FL1591)
or a large COOH-terminal fragment of the Tiam1 protein
(C1199) causes Rac1-dependent induction of membrane
ruffling in NIH3T3 fibroblast cells (Michiels et al., 1995
The NH2-terminal PH Domain Is Essential for
Membrane Localization of Tiam1
To investigate which of the conserved domains in Tiam1
determines the intracellular localization, small deletions
were made in each of these domains within the C1199 construct (see Fig. 3 and Materials and Methods). These mutant proteins were transiently expressed in COS-7 cells to
analyze their intracellular localization and ability to induce
membrane ruffling. Transfection of these mutant Tiam1
constructs in NIH3T3 cells resulted in the same phenotypic changes (not shown). Although Tiam1 is endogenously expressed in COS-7 cells and NIH3T3 cells, the levels are too low to be visualized by immunocytochemistry
or Western blotting. If phenotypic changes were induced
by the transfection of Tiam1 mutants, they were found in
>80% of the transfected cells.
As shown in Fig. 3 B, the C1199 Tiam1 protein carrying
a small internal deletion in the conserved COOH-terminal
part of the DH-adjacent PH domain (C1199- Immuno-EM analyses of transfected COS cells showed
that all mutant proteins were equally present in the cytoplasm and at the plasma membrane (Fig. 5), except for
mutant C1199 Tiam1, which contains a deletion in the NH2terminal PH domain (C1199-
The NH2-terminal PH Domain of Tiam1 Can
Be Functionally Replaced by the c-Src Membrane
Localization Domain, but Not by Other
PH Domains
To determine whether membrane localization is the only
function of the NH2-terminal PH domain, we fused the NH2terminal 20 amino acids of c-Src, containing the myristoylation site and a basic region, in front of a C580 Tiam1
protein, resulting in MS-C580 Tiam1 (see Materials and
Methods). C580 Tiam1 encompasses the COOH-terminal
580 amino acids and encodes the DH domain and adjacent
PH domain (Fig. 6 A). Similar to the C682 Tiam1 protein (see Fig. 2 B), the C580 Tiam1 protein did not induce
membrane ruffling and was not localized at the plasma
membrane (Figs. 6 A and 7 A). In contrast, the MS-C580
Tiam1 protein induced ruffling in both NIH3T3 cells and COS-7 cells (Fig. 6 B), albeit less extensively than C1199
Tiam1 (Fig. 3 A). No differences were observed in the expression levels of these proteins (Fig. 4, lanes 10-12). Immuno-EM showed that MS-C580 Tiam1 was able to associate with the plasma membrane (Fig. 7 B), although this
protein seemed to be more interiorly located than the mutant C1199 Tiam1 proteins (compare Figs. 5 and 7 B). Furthermore, part of the expressed MS-C580 Tiam1 protein
was present around vesicle-like structures in the cytoplasm, similar to other MS-containing Tiam1 fusion proteins (see for example Fig. 6 E, inset). A fusion protein
containing the NH2-terminal Src domain in front of the
C1199-
To test whether the NH2-terminal PH domain of Tiam1
is sufficient for membrane localization, we fused a region
containing this PH domain to the C580 Tiam1 protein (see
Materials and Methods). The resulting protein, however,
did not induce membrane ruffling and was not associated
with the plasma membrane (Michiels, F., and J.C. Stam,
unpublished results). This might argue that additional sequences are required to ensure membrane localization.
Alternatively, the juxtaposition of the PH domain to the
DH domain might interfere with the proper folding of
these domains. A construct expressing solely the region
containing the NH2-terminal PH domain did not function
as a dominant-negative Tiam1 mutant. However, C1199- To analyze the specificity of the NH2-terminal PH domain of Tiam1, a region containing this PH domain was exchanged for the corresponding regions containing the PH
domain of DbL, the PH domain of The NH2-terminal PH Domain Is Required for the
Induction of Membrane Ruffles by the Full-Length
Tiam1 Protein
The full-length Tiam1 protein (FL1591) carries a consensus myristoylation sequence at the NH2 terminus. By labeling with [3H]myristate, we have confirmed that the NH2
terminus of Tiam1 is myristoylated (not shown). To test
whether the myristoylation is sufficient for the membrane
association of full-length Tiam1, a small deletion was also
made in the NH2-terminal PH domain of full-length FL1591
Tiam1. However, similar to C1199- Controlled Cellular Localization of Tiam1
PH domains have been identified in other signaling molecules as protein-protein and/or protein-phospholipid interaction motifs that are required for the controlled targeting of these proteins to the plasma membrane (Lemmon
et al., 1996
Unexpectedly, neither PDGF nor insulin could substitute for serum in the induction of membrane ruffling (data
not shown). Both growth factors induce lamellipodia formation in NIH3T3 cells within 5 min, which can easily be
discriminated from the Tiam1-induced ruffling. This might
indicate that Tiam1 is not activated by PDGF or insulin.
Membrane-associated Tiam1 Activates JNK
To investigate whether membrane association of Tiam1 is
also required for the induction of other Rac-mediated signaling pathways besides membrane ruffling, the activity of
JNK was determined after cotransfection with different
Tiam1 mutants (see Materials and Methods). Both in
COS-7 cells and NIH3T3 cells, cotransfection of fulllength Tiam1 led to an almost sevenfold stimulation of JNK activity, similar to constitutively activated (V12)Rac1
(Fig. 9 A). Cotransfection of tagged mitogen-activated protein kinase (MAPK) with full-length or mutant Tiam1 or
(V12)Rac did not result in activation of MAPK (not
shown). The stimulation of JNK by Tiam1 was dependent
on the activation of Rac, since cotransfection of dominantnegative (N17)Rac1 partly blocked the activation of JNK by Tiam1 (Fig. 9 A). Higher amounts of (N17)Rac interfered with the expression of JNK. Furthermore, a deletion
in the catalytic DH domain of Tiam1 prevented JNK activation (not shown). Cotransfection of dominant-negative
(N17)Cdc42 did not interfere with the activation of JNK by
Tiam1 (data not shown). Transfection of C1199 Tiam1 stimulated JNK activity to a similar extent as FL1591
(Fig. 9 B). However, shorter COOH-terminal Tiam1 fragments such as C682 and C580 Tiam1, which did not localize to the plasma membrane, were not able to activate
JNK above background levels. Also, mutant C1199-
The data presented here demonstrate that Tiam1 is a cytoplasmic protein that is capable of associating with the
plasma membrane. The NH2-terminal PH domain is essential for membrane association of Tiam1, and this membrane association is required for Rac-mediated induction
of membrane ruffling and stimulation of JNK activity.
A short COOH-terminal fragment of Tiam1, C682 Tiam1,
which lacks the NH2-terminal PH domain, did not associate with the plasma membrane in NIH3T3 cells or COS-7
cells and was unable to induce membrane ruffling (see
Figs. 1 and 2). Similar to C1199 Tiam1 and (V12)Rac1, the
C682 Tiam1 protein induces a tumorigenic phenotype in
NIH3T3 cells (Van Leeuwen et al., 1995). This indicates that the Tiam1-induced formation of membrane ruffles and induction of a tumorigenic phenotype are triggered by two
distinct signaling pathways. Since JNK activity could not
be stimulated by C682 Tiam1 and none of the truncated
or mutant Tiam1 proteins activated MAPK, it is unlikely
that the induction of a tumorigenic phenotype is due to activation of JNK or MAPK. An explanation could be that tumorigenicity is caused by activation of cytoplasmic Rac,
although we cannot exclude that cytoplasmic Tiam1 activates another, yet unknown GTPase. Both the C1199 and
C682 Tiam1 proteins were also able to induce an invasive
phenotype in T-lymphoma cells (Habets et al., 1994 A detailed structural analysis showed that a small deletion in the NH2-terminal PH domain, but not the DHadjacent COOH-terminal PH domain, abolished the membrane localization of Tiam1 and the subsequent induction
of membrane ruffling. In all DH domain-containing GDS
proteins analyzed thus far, the DH domain is flanked by a PH domain (Cerione and Zheng, 1996 Several reports have demonstrated that isolated PH domains can bind in vitro to G
Since Rac-dependent membrane ruffling and activation
of JNK are induced by membrane-associated Tiam1, the
question of how Rac is activated arises. The current hypothesis is that inactive Rac is complexed to RhoGDI and
localized in the cytoplasm, as suggested for endogenous
Rac in neutrophils (Bokoch, 1994 Given the importance of PH domains in Tiam1 and
other GDS proteins, it is conceivable that phospholipids
and/or G;
Ridley et al., 1992
; Kozma et al., 1995
; Nobes and Hall,
1995a
). Each of these GTPases can be activated by specific
growth factors, and they may also constitute a hierarchical
signaling cascade (Kozma et al., 1995
; Nobes and Hall,
1995a
). The resulting cytoskeletal rearrangements influence both adhesion and motility of cells, implicating Rholike GTPases as key regulators of these processes (Nobes
and Hall, 1995b
).
; Hill et al., 1995
; Minden
et al., 1995
; Olson et al., 1995
). Rho-like proteins thus may
also play a role in transcriptional activation of genes.
Moreover, both Rac1 and RhoA are involved in the transformation of cells by Ras (Khosravi-Far et al., 1995
; Qiu
et al., 1995a
,b), and constitutively active mutants of Rac1
(V12Rac1) and RhoA (V14RhoA) can induce an oncogenic phenotype in NIH3T3 cells (Perona et al., 1993
; Qiu et al.,
1995a
; Van Leeuwen et al., 1995).
). The role of these regulators in the diverse signaling pathways mediated by Rho-like proteins is largely
unknown.
). The predicted Tiam1 protein consists of 1,591 amino
acids and contains a Dbl homology (DH) domain. This domain is present in GDS proteins for Rho-like GTPases
and is considered to be the catalytic domain (Hart et al.,
1994
). DH domains are found in a rapidly expanding group of proteins, many of which are encoded by transforming genes that were identified after transfection of genomic DNA or cDNA libraries into NIH3T3 cells (Cerione and Zheng, 1996
; Collard, 1996
). A number of these
proteins, like Dbl, Ost, Cdc24, Lbc, and Vav were shown
to activate Rho-like GTPases (Hart et al., 1991
; Horii et al.,
1994
; Zheng et al., 1994
, 1995; Olson et al., 1996
). Tiam1 is
the only GDS protein identified that specifically activates
Rac in vitro as well as in vivo (Michiels et al., 1995
). In fibroblasts, Tiam1 induces the formation of membrane ruffles, similarly to constitutively activated V12Rac1 (Michiels
et al., 1995
). The Tiam1-induced phenotype is inhibited by
coexpression of dominant-negative mutant (N17)Rac1, but
not by coexpression of (N17)Cdc42 or by inactivation of
RhoA with C3 transferase (Michiels et al., 1995
; Collard,
1996
). This indicates that the Tiam1-induced membrane
ruffling is primarily caused by activation of Rac and that
other Rho-like GTPases are not involved. Moreover,
V12Rac1, but not V14RhoA, induces an invasive phenotype in T-lymphoma cells, similar to Tiam1, suggesting that
Tiam1-induced invasiveness is also caused by activation of
Rac (Michiels et al., 1995
).
; Collard, 1996
; see Fig. 1 A).
One of the PH domains is present COOH-terminally adjacent to the DH domain, as found in all other GDS proteins for Rho-like GTPases (Collard, 1996
), while a second PH
domain is located in the NH2-terminal part of Tiam1.
DHR domains have been implicated as protein-protein
interaction motifs (Ponting and Phillips, 1995
). PH domains, primarily found in signaling molecules, are thought
to determine the cellular localization and/or activity of
these proteins by interacting with proteins or phospholipids (for review see Lemmon et al., 1996
).
Fig. 1.
Confocal images of NIH3T3 cells expressing truncated
Tiam1 proteins. A depicts the Tiam1 constructs encoding the
COOH-terminal 1,199 amino acids (C1199) or 682 amino acids
(C682) that were used for transfection of NIH3T3 cells, in comparison to the full-length Tiam1 protein (FL1591). Established
cell lines that express the C1199 Tiam1 protein (B) or the C682
Tiam1 protein (C) were fixed and stained with anti-Tiam1 antibodies (Habets et al., 1994), followed by FITC-coupled secondary antibodies (left) and by TRITC-labeled phalloidin for F-actin
(right). Note the prominent membrane ruffling in cells that express the C1199 Tiam1 protein. Stress fibers are hardly visible in
these cells, since the optical sections were made through the upper half of the cells to reveal the presence or absence of membrane ruffling. No significant differences were found in the
amount or appearance of stress fibers between Tiam1-expressing cells and controls. M, myristoylation signal; P, PEST regions; PHn and PHc, NH2-terminal and COOH-terminal pleckstrin homology domains. Magnification is 250×. Bar, 40 µm.
[View Larger Version of this Image (132K GIF file)]
Materials and Methods
ends.
They were cloned as SpeI-SalI fragments into the eukaryotic expression
vector pUTSV1 (Eurogentec S.A., Belgium), which was linearized by SpeIXhoI. C1199-
PHc carries a small internal PstI (aa 1377) to BsmI (aa 1390) deletion in the conserved COOH-terminal part of the DH-adjacent PH domain. C1199-
DH1 carries a BamHI (aa 1103) to XbaI (aa 1130) deletion in a nonconserved part of the DH domain. C1199-
DHR carries a large NotI (aa 853) to Asp718 (aa 905) deletion in the DHR region that
eliminates most of the DHR region. C1199-
PHn carries a small internal
Asp700 (aa 513) to EarI (aa 528) deletion in the conserved COOH-terminal part of the NH2-terminal PH domain, which is quite similar to the deletion made in the COOH-terminal PH domain (C1199-
PHc). Overhangs were blunted by either Klenow end filling reactions (Boehringer
Mannheim Corp., Indianapolis, IN) or with T4 DNA polymerase (Boehringer Mannheim Corp.) before ligation. The myristoylation sequence of
c-Src, encoding the NH2-terminal 20 amino acids, was amplified with SpeISalI adaptors by PCR with Pfu polymerase (Stratagene, La Jolla, CA) and
cloned between the SpeI and SalI sites at the 5
ends of the FL1591-
PHn,
C1191-
PHn, and C580 Tiam1 constructs. Fragments containing the COOHterminal PH domain of Tiam1, the PH domain of Dbl, or the PH domain
of
-Ark were amplified by PCR with SmaI and PvuII adapters and used
to replace the region containing the NH2-terminal PH domain of Tiam1 in
the C1199 construct. The exchanged regions contain 17 additional aa at
the NH2-terminal end and 36 additional aa (33 aa for
-Ark) at the COOHterminal end, in comparison to the consensus PH domain (Gibson et al., 1994
). To analyze whether the NH2-terminal PH domain is sufficient for
membrane targeting, a region containing the NH2-terminal PH domain,
from SalI (aa 393) to BstX1 (aa 647), was fused to the Bsu36I site (aa
1022) at the NH2 terminus of the C580 Tiam1 protein. All amplified fragments were sequenced with the T7 polymerase sequencing kit (Pharmacia
LKB Biotechnology Inc., Uppsala, Sweden).
).
16 h after transfection, the medium was either replaced and cells were incubated for an additional 30 h before washing and fixation, or cells were
incubated for 24 h in serum-free medium, followed by the addition of serum for 2 h. Western blot analyses were performed to monitor the size and expression levels of the mutant proteins, using either polyclonal antibodies directed against the COOH-terminal part of Tiam1 (Habets et al.,
1994
) or monoclonal antibodies against the HA-tag (12CA5; Boehringer
Mannheim Corp.) at the COOH terminus of all mutant Tiam1 proteins.
Proteins were visualized using the enhanced chemiluminescence protocol
(Amersham Corp., Arlingtin Heights, IL).
) with a
polyclonal rabbit antiserum against Tiam1 (Habets et al., 1994
). Antibodies were visualized with FITC-labeled secondary antibodies (Zymed Labs
Inc., S. San Fransisco, CA). Cells were stained simultaneously for F-actin
with TRITC-labeled phalloidin (Molecular Probes, Eugene, OR) at a concentration of 2 U/ml. At least 100 positive cells were analyzed by fluorescence microscopy and confocal laser scanning microscopy. Optical sections were taken through the apical part of the cells to show the presence or absence of membrane ruffles. Confocal images were recorded separately and subsequently merged. Colocalization of green (FITC) and red
(TRITC) fluorescence appears in yellow in the merged images.
-32P]ATP (5,000 Ci/mmol), and
0.5 mM para-nitrophenylphosphate. Reactions were stopped with SDSsample buffer, and the amount of radioactive GST-c-Jun was quantified
after SDS-PAGE by phosphorimaging. The level of expression of Tiam1,
mutant Rac, and tagged JNK in the NIH3T3 or COS-7 cells was monitored by Western blotting.
Results
).
Established NIH3T3 cell lines expressing either of these
proteins are flat and epithelial-like and contain many membrane ruffles (Fig. 1 B), a phenotype that is also induced by transfection or microinjection of constitutively activated (V12)Rac1 (Ridley et al., 1992
; Michiels et al., 1995
; Van
Leeuwen et al., 1995). In contrast, a smaller COOH-terminal part of the Tiam1 protein (C682), which contains only
the DH domain and the adjacent PH domain, did not induce this phenotype (Fig. 1 C). As shown by confocal laser
scanning immunofluorescence microscopy, the truncated
large C1199 Tiam1 protein was present in the cytoplasm and colocalized with F-actin in membrane ruffles (Fig. 1
B). In contrast, the short C682 Tiam1 protein seemed to be
restricted to the cytoplasm (Fig. 1 C). Western blot analyses
(see Fig. 4, lanes 1-3) indicated that both proteins were intact and equally expressed. This suggested that the difference in phenotypes induced by these truncated proteins is
probably caused by a different intracellular localization,
and not by differences in stability. Immunoelectron microscopy (immuno-EM) indeed confirmed that the C1199
Tiam1 protein is present in the cytoplasm as well as at the
cell membrane and particularly in the membrane ruffles,
whereas the C682 Tiam1 protein is almost exclusively located in the cytoplasm (Fig. 2). We hypothesized, therefore, that membrane localization of Tiam1 is required for
morphological transformation of NIH3T3 cells, including
the formation of membrane ruffles.
Fig. 4.
Immunoblotting of mutant Tiam1 proteins. Cell lysates
were separated on a 7.5% SDS-polyacrylamide gel and blotted to
nitrocellulose. All mutant C1199 and C682 Tiam1 proteins were
detected with a polyclonal anti-Tiam1 antibody (Habets et al.,
1994). Mutant C580 Tiam1 proteins were detected with a monoclonal antibody against the HA-tag (12CA5). Proteins were visualized using enhanced chemiluminescence (Amersham Corp.). Molecular mass markers (in kD) are shown. The arrow indicates the
position of the mutant C1199 Tiam1 proteins.
[View Larger Version of this Image (31K GIF file)]
Fig. 2.
Immunoelectron microscopic localization of truncated
Tiam1 proteins. Frozen sections of established NIH3T3 cell lines
expressing either C1199 Tiam1 (A) or C682 Tiam1 (B) were stained
with anti-Tiam1 antibodies followed by gold-conjugated secondary antibodies. The C682 Tiam1 protein is almost exclusively located in the cytoplasm, whereas the C1199 Tiam1 protein is also
present at the plasma membrane and in the membrane ruffles.
Bar, 400 nm.
[View Larger Version of this Image (121K GIF file)]
Fig. 3.
Confocal images of COS-7 cells expressing mutated C1199 Tiam1 proteins. In this figure, the first column depicts the mutant Tiam1 proteins that were used. The second column shows the FITC-staining for Tiam1 expression. The third column shows the TRITClabeled phalloidin staining for F-actin, while the merged images of Tiam1 (green) and F-actin (red) are shown in the fourth column.
Colocalization appears in yellow. Deletions within the conserved domains are indicated by arrowheads. See Materials and Methods section for a description of the mutations. For abbreviations, see legend to Fig. 1. Magnification is 200×. Bar, 40 µm.
[View Larger Versions of these Images (8 + 79 + 26K GIF file)]
PHc) was
still able to induce membrane ruffling. This was an unexpected finding since deletions in the DH-adjacent PH domain of other GDS proteins interfered with their proper
functioning (Hart et al., 1994
; Whitehead et al., 1995b
), and mutations in this region of the PH domain of
-Ark
abolished interactions with both G
proteins and phospholipids (Touhara et al., 1995
). In contrast, a C1199
Tiam1 protein with a deletion in the DH domain (C1199
DH) did not induce membrane ruffling (Fig. 3 C), corroborating a previously proposed catalytic function for the
DH domain (Hart et al., 1994
). Deletion of most of the
DHR region (C1199-
DHR) did not affect ruffling (Fig. 3 D), indicating that the DHR domain is not required for
the induction of membrane ruffles. However, a small deletion in the NH2-terminal PH domain (C1199-
PHn), comparable to the deletion made in the COOH-terminal PH domain, completely abolished membrane ruffling (Fig. 3 E).
Western blot analyses showed that all mutant proteins were of the predicted size and expressed at similar levels (see Fig. 4, lanes 4-9), although somewhat less than the C1199
Tiam1 protein. This excludes the possibility that these results
were due to major differences in protein stability. We conclude that both the catalytic DH domain and the NH2-terminal PH domain are essential for Tiam1-induced membrane ruffling.
PHn). This protein was almost exclusively localized in the cytoplasm (Fig. 5 D), similar to the C682 Tiam1 protein (Fig. 2 B). The C1199-
DH
Tiam1 protein, which carries a deletion in the catalytic DH
domain and does not induce membrane ruffling, was still
capable of associating with the plasma membrane (Fig. 5 B).
This clearly shows that membrane localization of Tiam1 is
not a consequence of the induction of membrane ruffling.
Therefore, it can be concluded that the NH2-terminal PH
domain is essential for the membrane localization of
C1199 Tiam1 and that this membrane localization is required for the formation of membrane ruffles.
Fig. 5.
Ultrastructural localization of mutant C1199 Tiam1 proteins in COS-7 cells. Immunogold labeling of mutant C1199 Tiam1
proteins with a deletion in the COOH-terminal PH domain (A), a deletion in the DH domain (B), a deletion in the DHR domain (C),
and a deletion in the NH2-terminal PH domain (D). C1199-PHn Tiam1 is almost exclusively localized in the cytoplasm, while other
mutant Tiam1 proteins also associate with the plasma membrane. Bar, 400 nm.
[View Larger Version of this Image (113K GIF file)]
PHn Tiam1 protein was also able to localize at the plasma membrane and to cause ruffling (data not shown).
The Src membrane localization domain thus enables C580
Tiam1 and C1199-
PHn to induce membrane ruffling by
tethering these proteins to the plasma membrane.
Fig. 6.
Confocal images of COS-7 cells expressing mutant Tiam1 proteins. Confocal laser scanning microscopic images of COS-7 cells expressing C580 Tiam1 (A), MS-C580 Tiam1 (B), full-length Tiam1 (FL1591) (C), full-length Tiam1 with a deletion in the NH2-terminal PH
domain (FL-PHn) (D), and FL-
PHn containing a Src myristoylation signal at the NH2 terminus (MS-FL-
PHn) (E). The insert in E is
a higher magnification of the framed region showing the vesicle-like structures that are surrounded by the MS-FL-
PHn Tiam1 protein.
Indicated in blue is the myristoylation signal from c-Src (MS), containing the NH2-terminal 20 amino acids. In the merged images, Tiam1
is shown in green and F-actin in red. Magnification is 200×. Bar, 40 µm.
[View Larger Versions of these Images (8 + 83 + 27K GIF file)]
Fig. 7.
Immunoelectron microscopic localization of mutant
C580 Tiam1 proteins. Immunogold labeling of transiently transfected COS cells expressing C580 Tiam1 (A) or MS-C580 Tiam1
(B). Note that the MS-C580 Tiam1 protein is located at and underneath the plasma membrane. Bar, 400 nm.
[View Larger Version of this Image (125K GIF file)]
DH, which contains a deletion in the catalytic DH domain, also
does not interfere with the induction of membrane ruffling
by Tiam1. The reason for this is not clear at the moment.
-ARK, or the COOHterminal PH domain of Tiam1 (see Materials and Methods). However, none of these mutant Tiam1 proteins localized at the plasma membrane and induced membrane
ruffling (data not shown). This means either that additional sequences are required for correct functioning of
these PH domains or that the primary function of the NH2terminal PH domain, namely to localize Tiam1 through specific interactions with components of the plasma membrane, cannot be substituted for by other PH domains.
PHn, mutant fulllength Tiam1 (FL1591-
PHn) was unable to induce membrane ruffling in NIH3T3 cells or COS cells (Fig. 6, C
and D), and immuno-EM showed that the protein was not
localized at the plasma membrane (data not shown). Apparently, the NH2-terminal myristoylation of full-length
Tiam1 alone is not sufficient for membrane localization. To further substantiate this, the NH2-terminal c-Src sequences were fused in front of the FL1591-
PHn1 Tiam1
protein. This region of Src contains a basic region that is
required for optimal membrane translocation in addition
to the myristoylation sequence, whereas other Src-like tyrosine kinases contain a palmitoylated cysteine in this region (Superti-Furga and Courtneidge, 1995
). Both additional sequences are absent in Tiam1. Cells expressing the
MS-FL1591-
PHn1 Tiam1 protein again showed membrane ruffling, although less extensive than cells expressing FL1591 Tiam1 (Fig. 6, C-E). Again, no main differences in expression levels were observed between these
proteins (data not shown). Immuno-EM confirmed that the MS-FL1591-
PHn1 Tiam1 protein was present at and
close to the plasma membrane, as found for MS-C580 Tiam1
(data not shown). Apparently, the accessory basic region
of c-Src also functions to properly localize MS-FL1591
PHn1 Tiam1. Similar to MS-C580, the expression of MSFL1591-
PHn Tiam1 led to the formation of small cytoplasmic vesicle-like structures, and the protein was also
located around these structures (Fig. 6 E). These observations indicate that the NH2-terminal PH domain also
serves as an essential membrane tag, or perhaps membrane anchor, for full-length Tiam1.
). In tissue sections of skin and certain carcinomas, endogenous Tiam1 is predominantly present in the
cytoplasm. Also in some cell lines, where we can envision
endogenous Tiam1 by Western blotting, including T-lymphoma cells and neuronal cells, it is mainly localized in the
cytoplasmic fraction (data not shown), suggesting that Tiam1
may translocate to the plasma membrane after receptor
stimulation. So far, however, we have not been able to
identify a receptor-mediated signaling pathway involving
Tiam1 activation. To investigate whether membrane translocation of exogenous Tiam1 could be visualized in NIH3T3 cells, we analyzed whether serum could affect the localization and capacity of Tiam1 to induce membrane ruffling.
As shown in Fig. 8 A, NIH3T3 cells transiently expressing
the C1199 Tiam1 protein showed no membrane ruffling
after serum starvation for 24 h. Almost no Tiam1 protein
was present at the plasma membrane, and F-actin was
mostly concentrated in lamellipodia in the Tiam1-expressing cells. Since these optical sections were taken at the
basal site of the cells to illustrate the lamellipodia, stress
fibers are also visible. Note that after serum starvation for
24 h, NIH3T3 cells still contain some stress fibers, in
contrast to Swiss 3T3 cells. Immuno-EM confirmed that
most of the cells contained significantly less Tiam1 at the
plasma membrane after serum starvation (Fig. 8, D and F).
The residual membrane-associated Tiam1 might be sufficient for the presence of lamellipodia under these conditions. Addition of serum induced membrane localization
and subsequent ruffling of C1199 Tiam1-expressing cells
after 2 h (Fig. 8, B and E), at which time point seruminduced stress fibers have already decreased. A quantification of these results is presented in Fig. 8 F. Similar results
were obtained with FL1591 Tiam1, C1199-
PHc, and C1199
DHR Tiam1 (data not shown). In contrast, after 24 h of
serum starvation the MS-C580 Tiam1 protein remained at
the plasma membrane and still induced membrane ruffling
(Fig. 8 C). This suggests that the serum-induced membrane translocation of Tiam1 is mediated by the NH2-terminal PH domain.
Fig. 8.
Serum-dependent localization of Tiam1. Confocal images of NIH3T3 cells expressing C1199 Tiam1 (A and B) or MS-C580
Tiam1 (C) proteins. Cells were serum starved for 24 h (A and C), followed by stimulation with serum for 2 h (B). In the merged pictures,
Tiam1 proteins are shown in green and F-actin in red. These optical sections were taken at the basal sites of the cells and reveal the presence of stress fibers in serum-starved NIH3T3 cells. Magnification is 300×. (D and E) Immuno-EM localization of C1199 Tiam1 after
serum starvation (D) or after readdition of serum for 2 h (E). In F, the distribution of gold particles in at least 40 cross sections of cells
were scored and presented as the percentage of cross sections with gold particles being predominantly at the membrane (M) or in the
cytoplasm (C). Bars: (A-C) 40 µm; (D and E) 400 nm.
[View Larger Versions of these Images (95 + 11 + 27 + 14K GIF file)]
PHn
Tiam1, which did not localize to the plasma membrane because of the deletion in the NH2-terminal PH domain, was
unable to activate JNK (Fig. 9 B). MS-C580 Tiam1 caused
no stimulation of JNK activity (Fig. 9 B), and neither did
MS-C1199-
PHn or MS-FL1591-
PHn Tiam1 proteins (data
not shown). These proteins, however, induced membrane
ruffling and were present at the plasma membrane (Fig.
6). A possible explanation for this somewhat unexpected result is that activation of endogenous Rac by MS-containing
Tiam1 proteins is sufficient for induction of membrane ruffling but not for stimulation of JNK. Alternatively, the
lack of induction of JNK activity is caused by the different
ultrastructural localization of the MS-C580 Tiam1 protein
(compare Figs. 5 and 7) or may reflect the need for an intact NH2-terminal PH domain. Interestingly, similar results were obtained with membrane-targeted p110 phosphoinositide-3-kinase which was able to stimulate membrane
ruffling but not gene transcription from the Jun-inducible
Fos promoter (Reif et al., 1996
). Whatever the mechanism,
these results indicate that the localization of Tiam1 at the
plasma membrane is also required for Rac-mediated stimulation of JNK activity.
Fig. 9.
Stimulation of JNK activity by Tiam1. (A) COS-7 cells
were transfected with pCMV-Flag-JNK1 and empty vector
(pcDNA-neo), full-length Tiam1 (FL1591), or constitutively
activated (V12)Rac1. Cotransfection with dominant-negative
(N17)Rac1 was performed as indicated. The activity of immunoprecipitated Flag-JNK was determined 48 h after transfection by
an in vitro kinase assay using bacterially expressed GST-Jun as a
substrate. The expression of Tiam1, Myc-tagged (N17)Rac, and
Flag-tagged JNK was analyzed by Western blotting and is shown underneath the graph. (B) NIH3T3 cells were transfected with
pCMV-Flag-JNK1 and mutant Tiam1 constructs. JNK activity
was determined as described. All data are presented as fold stimulation of empty vector controls. Equal amounts of expressed
Flag-JNK1 and Tiam1 proteins, as determined by Western blotting, were used. Data represent mean ± standard deviation of at
least two independent experiments.
[View Larger Version of this Image (24K GIF file)]
Discussion
). As
the localization of Tiam1 might be differently regulated in
lymphoid cells compared to adherent COS and fibroblast cells, we are currently testing whether or not JNK is involved in the Tiam1/Rac-mediated induction of an invasive phenotype in T-lymphoma cells.
; Collard, 1996
),
suggesting that the DH-PH combination is a functional
unit. Indeed, deletions in either the DH domain or the
flanking PH domain of the protooncogenes Dbl, Ost, Dbs,
and Lfc result in loss of transforming capacity (Ron et al.,
1991
; Horii et al., 1994
; Whitehead et al., 1995a
,b; Zheng
et al., 1996
). The transforming capacity of a mutant of Lfc,
which carried a deletion in the PH domain, could be restored by the addition of a COOH-terminal Ras CAAX
motif, suggesting that the localization of Lfc at the plasma
membrane is required for its transforming capacity (Whitehead et al., 1995b
). In contrast, the Ras-CAAX motif was
not sufficient to replace the PH domain of Dbl, which is
required for the presence of Dbl in the particulate fraction
of cells (Zheng et al., 1996
). Corresponding results were
obtained for the NH2-terminal PH domain of Ras-guanine
nucleotide release factor (GRF), suggesting that the association of Ras-GRF and Dbl in the particulate fraction is a
prerequisite for their activity (Buchsbaum et al., 1996
;
Zheng et al., 1996
). Together with pleckstrin and RasGRF, Tiam1 is one of a few proteins that contains two PH
domains (Habets et al., 1994
). Similarly to Ras-GRF, only
the NH2-terminal PH domain of Tiam1 is involved in the
localization of the protein. The COOH-terminal PH domain of Tiam1 has only moderate homology with other
PH domains and lacks a highly conserved tryptophan residue in the COOH-terminal
-helix (Habets et al., 1994
).
Although a complete deletion of the DH-adjacent PH domain of Tiam1 abolished membrane ruffling, it did not interfere with the membrane localization of the protein
(data not shown). What other functions can be attributed
to this PH domain remains to be elucidated.
subunits and phospholipids like phosphatidylinositol (4,5)bisphosphate (PIP2) and inositol (3,4,5)trisphosphate (IP3) (Harlan et al., 1994
; Ferguson et al., 1995
; Mahadevan et al., 1995
; Touhara et al.,
1995
). Phosphoinositide-3-kinase, which stimulates the synthesis of phosphatidylinositol (3,4,5)trisphosphate (PIP3)
from PIP2 (Hiles et al., 1992
), has been implicated in the
activation of Rac by PDGF and insulin (Wennstrom et al.,
1994
; Hawkins et al., 1995
) and might be required for the
activation of Rac by Ras (Rodriguez-Viciana et al., 1994
).
Although we have shown that the association of Tiam1 with
the plasma membrane is induced by serum, neither PDGF
nor insulin could substitute for serum in NIH3T3 cells. G
subunits and phospholipids represent potential downstream effectors of G-protein-coupled receptors and/or
activated tyrosine kinase receptors. Based on the in vitro
binding data of PH domains, it is tempting to speculate that G
subunits and/or specific phospholipids regulate the association of Tiam1 with the plasma membrane, as
depicted in Fig. 10. The fact that we were not able to functionally exchange the NH2-terminal PH domain of Tiam1
for other PH domains argues for a specific targeting function for this PH domain, and similar results have been reported for PH domains in other signaling proteins (Buchsbaum et al., 1996
; Kolanus et al., 1996
). However, at the moment we cannot determine whether PH domains can
function as independent modules or whether additional
sequences are required for their activity.
Fig. 10.
Model for activation of Rac by Tiam1. Activation of
Rac requires membrane translocation of Tiam1 and might be
analogous to activation of Ras by receptor-induced translocation
of SOS. Membrane association of Tiam1 could be mediated by
interactions between the NH2-terminal PH domain and putative
effectors of activated G-protein coupled receptors (GPCR), tyrosine kinase receptors (TKR), or Ras, which may include G
subunits, PIP2, PIP3, IP3, and perhaps G
subunits. Activation of
Rac by membrane-associated Tiam1 leads to membrane ruffling
and JNK activation. It is unlikely that JNK is involved in the induction of an oncogenic phenotype in NIH3T3 cells, since mutant
C682 Tiam1 induces an oncogenic phenotype without activation
of JNK. Which Tiam1/Rac-mediated signaling pathways are involved in the induction of an invasive phenotype in T-lymphoma
cells remains to be established.
[View Larger Version of this Image (36K GIF file)]
; Hawkins et al., 1995
)
and for exogenous Rac and Rho proteins in other cell types
(Adamson et al., 1992
; Ridley et al., 1992
; Takaishi et al.,
1995
; Robertson et al., 1995
). According to this model, activation involves the dissociation of the Rac-GDI complex,
the translocation of GDP-bound Rac to the plasma membrane, followed by the exchange of GDP for GTP by interaction with a membrane-bound GDS (Bokoch, 1994
; Hawkins et al., 1995
). Our findings, however, also indicate that
Tiam1 translocates from the cytoplasm to the plasma
membrane after induction by serum. The most straightforward explanation for the fact that membrane-associated
C1199 Tiam1, but not cytoplasmic C580 Tiam1, induces
ruffling and activates JNK would be that GDP-bound Rac
is partly present at the plasma membrane in NIH3T3 and
COS-7 cells. The activation of this pool of membranebound, inactive Rac by Tiam1 is then sufficient for the induction of membrane ruffles and activation of JNK. Alternative explanations would be that the activity of Tiam1 is
enhanced at the plasma membrane, or that Tiam1 is also
involved in the translocation of Rac from the cytoplasm to
the plasma membrane. In all these models, the controlled
membrane association of Tiam1 is the only determinant
for the induction of membrane ruffling by Rac. Since MSC580 Tiam1, but not C580 Tiam1, induces membrane ruffling, it is unlikely that the activation of cytoplasmic Rac
by Tiam1 leads to the membrane translocation of GTPbound Rac. As discussed before, however, cytoplasmic GTPbound Rac might be responsible for the induction of a tumorigenic phenotype by C682 Tiam1. Further exploration of these models will require the generation of antibodies
that efficiently detect endogenous Tiam1 and Rac and tools
to discriminate between active and inactive endogenous Rac.
subunits are involved in their intracellular localization and/or activation. The next challenge will be to
identify the upstream signaling pathways and molecules
that translocate and activate these GDS proteins.
Received for publication 8 October 1996 and in revised form 23 January 1997.
1. Abbreviations used in this paper: aa, amino acids; DH, Dbl homology; DHR, Discs-large homology region; GDI, guanine nucleotide dissociation inhibitors; GDS, guanine nucleotide dissociation stimulator; GRF, guanine nucleotide release factor; GST, glutathione S-transferase; HA, hemagglutinin; IP3, inositol (3,4,5)trisphosphate; JNK, c-Jun NH2-terminal kinase; MAPK, mitogen-activated protein kinase; PH, pleckstrin homology; PIP2 and PIP3, phosphatidylinositol (4,5)bisphosphate and (3,4,5)trisphosphate.We thank Drs. M. Karin, A. Eva, and T. van Biezen for their gift of constructs; G. Habets and F. van Leeuwen for their help in the initial studies; L. Oomen for his assistance with the confocal microscope; L. Oomen and N. Ong for preparation of some of the figures; and F. van Leeuwen, E. Roos, and R. Engers for critically reading the manuscript.
This work was supported by grants from the Netherlands Organization for Scientific Research and the Dutch Cancer Society to J.G. Collard.