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Molecular and Cellular Biology, June 2001, p. 3750-3762, Vol. 21, No. 11
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.11.3750-3762.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Activation of the Ral and Phosphatidylinositol 3'
Kinase Signaling Pathways by the Ras-Related Protein TC21
Marta
Rosário,
Hugh
F.
Paterson, and
Christopher J.
Marshall*
CRC Centre for Cell and Molecular Biology,
Chester Beatty Laboratories, Institute of Cancer Research, London
SW3 6JB, United Kingdom
Received 6 November 2000/Returned for modification 12 December
2000/Accepted 9 March 2001
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ABSTRACT |
TC21 is a member of the Ras superfamily of small GTP-binding
proteins that, like Ras, has been implicated in the regulation of
growth-stimulating pathways. We have previously identified the
Raf/mitogen-activated protein kinase pathway as a direct TC21 effector
pathway required for TC21-induced transformation (M. Rosário,
H. F. Paterson, and C. J. Marshall, EMBO J. 18:1270-1279, 1999). In this study we have identified two further effector pathways for TC21, which contribute to TC21-stimulated transformation: the
phosphatidylinositol 3' kinase (PI-3K) and Ral signaling pathways. Expression of constitutively active TC21 leads to the activation of Ral
A and the PI-3K-dependent activation of Akt/protein kinase B. Strong
activation of the PI-3K/Akt pathway is seen even with very low levels
of TC21 expression, suggesting that TC21 may be a key small
GTPase-regulator of PI-3K. TC21-induced alterations in cellular
morphology in NIH 3T3 and PC12 cells are also PI-3K dependent. On the
other hand, activation of the Ral pathway by TC21 is required for
TC21-stimulated DNA synthesis but not transformed morphology. We show
that inhibition of Ral signaling blocks DNA synthesis in human tumor
cell lines containing activating mutations in TC21, demonstrating for
the first time that this pathway is required for the proliferation of
human tumor cells. Finally, we provide mechanisms for the activation of
these pathways, namely, the direct in vivo interaction of TC21 with
guanine nucleotide exchange factors for Ral, resulting in their
translocation to the plasma membrane, and the direct interaction of
TC21 with PI-3K. In both cases, the effector domain region of TC21 is
required since point mutations in this region can interfere with
activation of downstream signaling.
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INTRODUCTION |
The regulation of several cellular
processes including proliferation, differentiation, and the modulation
of the cytoskeleton has been ascribed to the Ras subfamily of small
GTP-binding proteins. This family includes the classical Ras proteins
(H-Ras, N-Ras, K4A-Ras, and K4B-Ras), the R-Ras-like proteins (R-Ras,
TC21, and M-Ras/R-Ras 3), the Rap proteins (Rap 1A, Rap 1B, Rap 2A, and Rap 2B) and the Ral proteins (Ral A and Ral B) (3). While
the role of the classical Ras proteins is becoming clearer, the role of
other members of the subfamily is still unclear. The classical Ras
proteins have been of particular interest, given the high incidence of
mutation of these genes in human cancers. Apart from the classical Ras
proteins, the only other member of the Ras subfamily of GTPases
found to be mutated in human cancers is TC21 (also called R-Ras 2)
(2, 5, 19). Constitutively active TC21 will transform a
wide variety of fibroblast and epithelial cell lines, and injection of
TC21-transformed fibroblasts into nude mice results in the formation of
highly aggressive tumors (5, 6, 14, 19). In addition,
up-regulation of the wild-type protein has been observed in seven of
nine breast tumor cell lines (6). These observations have
suggested that TC21, like the classical Ras proteins, may be involved
in the regulation of growth.
TC21 has 55% amino acid identity to the classical Ras proteins but has
an absolutely conserved core effector domain (residues 32 to 40 in
H-Ras and 43 to 51 in TC21), a region that is required for the
interaction of all identified Ras effectors (10). However, other family members such as R-Ras, which is 70% identical to TC21,
also share conserved effector domains but do not activate the same
effector pathways and are not transforming (8, 17, 29,
35).
We have previously demonstrated that TC21 will directly interact with
the Raf serine/threonine kinases and therefore lead to the activation
of the Raf/mitogen-activated protein (MAP) kinase pathway, a step that
is crucial to TC21-stimulated cellular transformation (39). Here we identify two further effector pathways of
TC21: the phosphatidylinositol 3' kinase (PI-3K) and Ral signaling
pathways. We demonstrate that TC21 leads to a strong PI-3K-dependent
activation of the serine/threonine kinase Akt/protein kinase B (PKB).
Activation of this pathway is probably due to the direct interaction of
TC21 with PI-3K and is required for TC21-induced transformation of NIH
3T3 cells and for TC21-induced morphological alteration of PC12 cells.
In addition, we show that TC21 interacts directly with several exchange
factors for Ral, leading to their membrane localization and to the
subsequent activation of Ral A. Activation of this pathway by TC21 is
crucial for TC21-induced DNA synthesis both in human tumor cell lines
harboring activating mutations in TC21 and in fibroblasts expressing
active TC21, but it does not appear to be involved in the morphological
alterations stimulated by TC21 expression. These results define a novel
role for the Ral pathway in the stimulation of DNA synthesis in human
tumor cell lines. The effector domain of TC21 is shown to be required for the activation of both PI-3K and Ral signaling pathways by TC21,
since point mutations in this region specifically interfere with signaling.
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MATERIALS AND METHODS |
Plasmids.
The Ral binding domain of RalBP1 (residues 397 to
518) (51) was subcloned by PCR into the pEF Plink HA.6
vector that incorporates a HA tag at the N terminus of the protein. The
Ral binding domain of RalBP1 and full-length wild-type RalGDS were
subcloned into the pEGFP-C1 mammalian expression vector (Clontech) that
fuses the green fluorescent protein (GFP) at the N terminus of RalGDS. Mutations in the effector domain of Myc-tagged V23 TC21 were generated using PCR-directed mutagenesis. All other constructs have been described previously (39).
Cell culture, transfection, and microinjection.
Untransformed and transformed NIH 3T3 cell lines were cultured in
Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 or 5%
calf serum (GibcoBRL Life Technologies), respectively. Madin-Darby
canine kidney (MDCK) cells and all human tumor cell lines were
maintained in DMEM supplemented with 10% fetal calf serum. PC12 cells
were grown in DMEM supplemented with 10% horse serum and 5% fetal
calf serum. DNA transfections were performed using LipofectAMINE
(GibcoBRL Life Technologies), and microinjections were performed on a
Zeiss Microinjection Workstation (Carl Zeiss, Oberkochen), both as
previously described (39). Cells undergoing DNA synthesis
were identified by incorporation into newly synthesized DNA of
bromodeoxyuridine (BrdU; Amersham Life Sciences) present at 10 mM in
the culture medium. Microinjected samples were fixed and stained as
previously described (39) using the A14 rabbit polyclonal
anti-Myc antibody (Santa Cruz), 9E10 mouse monoclonal antibody, anti-HA
antibody (3F10, Boehringer Mannheim), rat anti-BrdU monoclonal
antibody, or Texas red-conjugated phalloidin (Molecular Probes) to
detect polymerized actin. All immunofluorescence samples were analyzed
with a Bio-Rad MRC 1024 confocal imaging system equipped with a Nikon
Eclipse 400 microscope.
Preparation of cell lysates for immunoprecipitation, pull-down,
and Western blotting.
Samples for detection of phosphorylated Akt
or extracellular signal-regulated kinase (ERK) or for Ral pull-down
assays were prepared in NP-40 buffer (50 mM Tris [pH 7.4], 1% NP-40,
15% glycerol, 200 mM NaCl, 5 mM MgCl2) containing protease
inhibitors (10 µg of leupeptin per ml, 10 µg of aprotinin per ml, 1 mM phenylmethylsulfonyl fluoride [PMSF], 10 µg of pepstatin A per
ml) on ice. Lysates were cleared by centrifugation, and protein
concentrations were determined. A 100-µg sample of cleared whole-cell
lysate was analyzed by Western blotting with anti-phosphorylated Ser
473 or Thr 308 Akt polyclonal antibodies (NEB), anti-Akt (NEB),
anti-ERK2 122 rabbit polyclonal antibody (7), and
anti-phosphorylated ERK monoclonal antibody (Sigma).
For analysis of Ral-GTP levels, equal levels of protein were incubated
for 1 h at 4°C with glutathione-Sepharose beads that had been
precoupled to recombinant glutathione S-transferase
(GST)-RalBP1 RalBD as described by Wolthuis et al. (51).
The beads were washed three times in large volumes of NP-40 buffer, and
precipitated endogenous Ral A was analyzed by Western blotting with an
anti-Ral A antibody (Transduction Laboratories).
Cell lysates for the analysis of coimmunoprecipitated Ras-related
proteins and endogenous p110
were prepared in modified
Ras lysis
buffer (20 mM Tris [pH 7.5], 1% [vol/vol] Triton X-100,
10%
glycerol, 100 mM KCl, 5 mM MgCl
2, 5 mM NaF, 1 mM EGTA)
supplemented
with inhibitors (1 mM Na
3VO
4, 10 µg of leupeptin per ml 10 µg
of aprotinin per ml, 10 µg of
pepstatin A per ml, 10 mM benzamidine,
0.5 µg of microcystin LR per
ml). Cleared lysates equilibrated
for levels of protein were used
immediately after preparation
in immunoprecipitations with anti-Myc
antibodies (mouse 9E10 monoclonal
antibody and A14 rabbit anti-Myc
polyclonal antibody; Santa Cruz).
Coimmunoprecipitates were washed
three times in modified Ras lysis
buffer before being analyzed for
coimmunoprecipitating endogenous
p110
by Western blotting with
anti-p110
antibody (sc-602; Santa
Cruz). Cell lysates for the
analysis of coimmunoprecipitated Rlf
and TC21 proteins were prepared in
Ras lysis buffer as previously
described (
39). TC21
proteins were immunoprecipitated with the
9E10 anti-Myc monoclonal
antibody, and coimmunoprecipitated Rlf
was detected by Western blotting
with an anti-HA monoclonal antibody
(3F10; Boehringer
Mannheim).
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RESULTS |
Constitutive activation of the ERK/MAP kinase and Akt/PI-3K
signaling pathways in TC21-transformed cells.
Activating mutations
in TC21 transform a variety of different cell types and appear to
transform NIH 3T3 cells with an efficiency as high as or even higher
than that of H-Ras (5, 6, 14, 19). Other R-Ras-like
GTPases have a very low transforming efficiency (8, 25,
33).
As we have previously reported, NIH 3T3 fibroblasts stably transformed
with constitutively active TC21 or H-Ras show elevated
levels of
phosphorylated active ERK1 and ERK2 as compared to the
parental
untransformed NIH 3T3 cells (
39) (Fig.
1A). This activity
is completely inhibited by the MEK inhibitor UO126. Since activation
of
the PI-3K pathway is associated with signaling by Ras, we have
investigated whether this pathway is also involved in TC21-stimulated
transformation. TC21 transformed cells showed elevated levels
of
phosphorylated active Akt/PKB, a downstream effector of PI-3K
(Fig.
1B). Activation of Akt/PKB in these cells
is absolutely
dependent on the activation of PI-3K, as indicated by the
strong
inhibitory effect of the PI-3K inhibitor LY294002.
H-Ras-transformed
NIH 3T3 cells had elevated levels of Akt/PKB
phosphorylation,
in agreement with previous work (
9,
36,
38); however, we
consistently found that activation of the
Akt/PI-3K pathway in
H-Ras-transformed cell lines was significantly
lower than that
observed in TC21-transformed cell lines (Fig.
1).
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FIG. 1.
Activation of the ERK/MAP kinase and Akt/PI-3K pathways
in TC21-transformed cells. (A) NIH 3T3 cell lines stably expressing
oncogenic TC21, H-Ras, or TC21 proteins containing mutations in the
effector domain and untransformed NIH 3T3 were transferred to
serum-free medium for 4 h. TC21-transformed cells were also
exposed either to 25 µM LY294002, 20 µM UO126 (a MEK inhibitor), or
the equivalent volume of ethanol carrier for 2 h prior harvesting.
Whole-cell lysates were separated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and specific
antibodies were used to detect phosphorylated ERK1 and ERK2 as well as
total ERK2. Two independently isolated clones of TC21-transformed cells
(cl.1 and cl.2) are shown. (B) The transformed NIH 3T3 cell lines were
also analyzed for levels of phosphorylated Akt/PKB and total Akt/PKB in
the presence of 50 µM LY294002, 20 µM UO126, or the equivalent
volume of dimethyl sulfoxide (DMSO) carrier added for 2 h prior to
harvesting as described above. Antibodies against the phosphorylated
Ser 473 or Thr 308 sites in Akt/PKB gave similar results.
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We generated point mutations in the effector domain of TC21 based on
effector mutations in H-Ras that preferentially affect
the binding and
activation of specific effectors (
39,
47,
50). Like the
cognate mutations in H-Ras, the point mutations
in the effector domain
of TC21 E48G and T46S but not Y51C interfere
with the constitutive
activation of Akt in stably expressing cells,
further suggesting that
TC21-directed signaling is responsible
for Akt activation (Fig.
1B). We
have also observed elevated levels
of activated Akt in the human tumor
cell lines that contain activating
mutations in TC21 (data not
shown).
We confirmed that activation of the Akt/PI-3K pathway in the
transformed NIH 3T3 cells was due to the expression of the TC21
oncogene, rather than being acquired as a result of clonal selection
procedures, by determining the effect of transient transfection
of
activated forms of H-Ras, TC21, or R-Ras on the phosphorylation
state
of endogenous Akt/PKB. A 5-min stimulation with platelet-derived
growth
factor (PDGF) was included for comparison (Fig.
2). In
accordance with our observations
in stable cell lines (Fig.
1),
Fig.
2 shows that expression of
constitutively active TC21 results
in strong activation of the
Akt/PI-3K pathway. Point mutations
in the effector domain of TC21
partially or completely inhibit
this activation, indicating that direct
signaling by TC21 is required.
The Y51C mutation partially interferes
with the activation of
Akt/PKB, since higher expression levels are
required for activation
to be observed (Fig.
2B). The equivalent V12
Y40C H-Ras mutant
has been reported to induce a three- to
fourfold-lower activation
of the Akt/PI-3K pathway than does the
wild-type oncogenic H-Ras
(
21,
37,
50). Longer-term
expression of the Y51C mutant
does, however, allow activation of the
Akt/PKB pathway comparable
to that seen by V23 TC21 (Fig.
1B and data
not shown), indicating
that several mechanisms may be responsible for
Akt/PKB activation
by TC21.
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FIG. 2.
Activation of endogenous Akt/PKB by TC21. NIH 3T3 cells
were transiently transfected with fixed or increasing amounts of
expression vectors for Myc-tagged constitutively active forms of H-Ras,
TC21, the TC21 effector mutants, R-Ras, or an empty-vector control
(EV). The cells were harvested 20 h posttransfection following a
4-h serum starvation. One of the empty-vector transfections was
stimulated with 25 ng of platelet-derived growth factor (PDGF) per ml
for 5 min prior to harvesting. Whole-cell lysates equilibrated for
protein levels were separated by SDS-PAGE. Specific anti-phosphorylated
Akt/PKB and anti-Myc antibodies were used to detect the active Akt/PKB
and the Ras-like proteins, respectively.
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R-Ras has also been proposed to lead to a strong activation of the
Akt/PI-3K pathway (
29). However, side-by-side titrations
of oncogenic TC21 and R-Ras indicate that activation of this pathway
by
TC21 is considerably stronger than activation by R-Ras (Fig.
2C).
The notion that the effect of TC21 expression on the phosphorylation of
Akt/PKB is due to the stimulation of 3'-phosphorylated
inositide
production was confirmed using the pleckstrin homology
(PH) domain of
Akt/PKB as a specific probe for these lipids (
45)
(Fig.
3). Expression of the GFP:Akt PH domain
fusion protein in
MDCK epithelial cells resulted in a diffuse
cytoplasmic and nuclear
staining pattern, with some protein also
visible at cell-cell
junctions as previously reported (
45)
(Fig.
3). Coexpression
of active TC21 resulted in a strong
translocation of the GFP:Akt
PH domain fusion protein to the plasma
membrane and internal vesicular
membranes, presumably as a result of
3'-phosphoinositide generation,
since incubation with LY294002 prevents
this translocation, with
the fusion protein again accumulating in the
nucleus and cytoplasm
(Fig.
3).
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FIG. 3.
Translocation of the PH domain of Akt/PKB to the plasma
membrane by TC21. Confluent MDCK cells were microinjected with 10 µg
of an expression vector for the GFP:Akt PH domain fusion protein per ml
alone or in conjunction with 50 µg of an expression vector for the
Myc-tagged V23 TC21 per ml. LY294002 (25 µM) or the equivalent volume
of ethanol was added to the medium just prior to microinjection. Cells
were fixed 8 h after microinjection. GFP fluorescence is shown.
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Direct interaction of PI-3K with TC21.
H-Ras interacts
directly with the catalytic subunit (p110) of PI-3K 
, PI-3K , and
PI-3K 
through the Ras effector domain (36, 38, 40).
This region is completely conserved in TC21, although it is likely that
other regions outside the effector domain are also involved in binding
or in conferring specificity (44). Therefore it is
possible that the strong activation of this pathway by TC21 is mediated
through the direct interaction of TC21 with PI-3K.
To demonstrate the interaction between TC21 and PI-3K, we sought to
coimmunoprecipitate endogenous PI-3K with the Ras-related
proteins from
NIH 3T3 stable cell lines that express Myc-tagged
versions of these
proteins (Fig.
4). Endogenous p110 could
be
coimmunoprecipitated with TC21 and H-Ras but not with R-Ras,
consistent
with the observed activation of Akt/PKB by H-Ras and TC21
but
not by R-Ras (Fig.
4A). Point mutations in the effector domain
of
TC21, all of which affect activation of Akt/PKB by TC21, interfered
with binding to p110, indicating that this region is required
for the
interaction (Fig.
4B). Despite a weak activation of Akt
by the Y51C
mutant in transient transfections, only very little
or no p110 was
found associated with this protein by coimmunoprecipitation.
This
failure to coimmunoprecipitate p110 with Y51C may be a consequence
of
low levels of expression of this mutant in NIH 3T3 cells, making
it
technically difficult to immunoprecipitate sufficient Y51C
protein to
reveal the interaction.
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FIG. 4.
Interaction of TC21 with PI-3K. Myc-tagged oncogenic
TC21, R-Ras, and H-Ras (A) or Myc-tagged constitutively active TC21
proteins containing specific mutations in the effector domain (B) were
immunoprecipitated from NIH 3T3 cell lines stably expressing these
proteins and grown in medium supplemented with 5% serum.
Coimmunoprecipitating endogenous p110 was detected after SDS-PAGE by
immunoblotting with specific antibodies.
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The role of TC21-induced PI-3K activation in TC21-stimulated
signaling.
Several biological functions have been ascribed to the
PI-3K pathway, including effects on cell proliferation (11,
12), actin polymerization and thus cell morphology (27,
32, 34, 37, 46), and cell survival after exposure to a number of
apoptotic stimuli (21, 23, 52).
Treatment of TC21-transformed NIH 3T3 with LY294002 resulted in
inhibition of DNA synthesis and morphological reversion of
these cells,
as seen by an inhibition in the incorporation of
bromodeoxyuridine
(BrdU) into newly synthesized DNA and the loss
of spindle shape and
cellular flattening (Fig.
5A).
LY294002 affected
the ability of
parental NIH 3T3 cells to incorporate BrdU without
affecting their
cellular morphology, indicating that basal PI-3K
levels are required
for cell cycling of untransformed NIH 3T3
cells. The PI-3K pathway is
thus crucial to TC21-induced morphological
transformation.
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FIG. 5.
TC21-induced transformation of NIH 3T3 cells (A) and
morphological alteration of PC12 cells (B) are dependent on PI-3K
activation. (A) V23 TC21-transformed NIH 3T3 or parental untransformed
NIH 3T3 cells were seeded on several plastic dishes in DMEM
supplemented with 5% serum. The cells were exposed to various
concentrations of LY294002 or to the equivalent volume of the ethanol
carrier for 42 h and allowed to incorporate BrdU during the last
15 h. Samples were then fixed and stained for the incorporated
BrdU (shown in green) and for polymerized actin (shown in red). (B)
PC12 cells were seeded on laminin-coated plates in medium containing
5% fetal calf serum and 10% horse serum. The medium was diluted 1:4
with serum-free medium 24 h after seeding. At 48 h after seeding,
the cells were microinjected with 25 µg of an expression vector for
Myc-tagged oncogenic TC21 or H-Ras per ml. LY294002 (25 µM) was added
to a second duplicate plate of cells after injection. The cells were
fixed 15 h after injection and stained for TC21 or H-Ras
expression using anti-Myc antibodies. Levels of phosphorylated Akt/PKB
in duplicate plates exposed to 25 µM LY294002, 20 µM UO126, or the
equivalent volume of dimethyl sulfoxide carrier for 4 h prior to
stimulation with 100 ng of NGF per ml for 5 min are shown.
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Nerve growth factor (NGF) stimulation or the expression of active H-Ras
results in a two-stage differentiation process of
PC12 neuronal
precursor cells, involving transient rapid spreading
of lamellipodia
followed by cell cycle arrest and neurite outgrowth
(
1,
16). Expression of constitutively active forms of the
MAP kinase
kinase MEK1 is sufficient to induce neurite outgrowth
in these cells
with little or no initial cellular flattening (7;
H. Paterson,
unpublished data). Expression of active TC21 in PC12
cells resulted in
a distinctive morphology (Fig.
5B). The cells
exhibited intense
lamellipodia formation (reminiscent of the first
stages of NGF-induced
differentiation) with peripheral filopodia.
There was no inhibition of
DNA synthesis (data not shown) or induction
of neurites. Unlike Movilla
et al. (
31) and Graham et al. (
15),
we did
not observe neurite outgrowth on TC21 expression (Fig.
5B). However, in
their experiments, cells were analyzed 14 to
20 days after introduction
of activated TC21, whereas NGF stimulation
or introduction of activated
Ras or MEK1 induces neurite outgrowth
within 24 h (
7,
16).
Since PI-3K activation is required for the differentiation of the
neuronal precursor PC12 rat pheochromocytoma cell line in
response to
NGF (
20), we tested whether the TC21-induced morphology
was a result of the activation of the PI-3K pathway by using LY294002.
Inhibition of PI-3K did not cause these cells to revert to the
original
morphology but did partly inhibit flattening and resulted
in the
formation of elongated processes (Fig.
5B). Surrounding
uninjected PC12
cells did not exhibit any significant morphological
alterations on
exposure to LY294002 (data not
shown).
Interaction of TC21 with RalGEFs.
Guanine nucleotide exchange
factors for Ral proteins (RalGEFs) have been proposed to be effectors
of TC21, based on the identification of one of the members of this
family, RalGDS, in yeast two-hybrid screens with activated TC21
(28).
RalGEFs are cytoplasmic proteins, and several lines of evidence suggest
that translocation of these proteins to the plasma
membrane is a
crucial step in activation of the membrane-bound
Ral proteins
(
18,
26,
50). Like other Ras proteins, TC21
is localized
to the plasma membrane through C-terminal posttranslation
lipid
modifications (
4). We examined whether activated TC21
could recruit RalGEFs to the plasma
membrane.
Figure
6
shows that two RalGEF family members,
RalGDS and Rlf, translocate from the cytoplasm to the plasma membrane
on coexpression
of active TC21. The translocation of Rlf by TC21 is
particularly
complete. Further confirmation of the interaction of TC21
with
Rlf was obtained by coimmunoprecipitation of transiently
coexpressed
wild-type Rlf with oncogenic TC21 (Fig.
7). Insertion of the T46S
and Y51C point
mutations in the effector domain region of TC21
completely abolished
both TC21-induced plasma membrane translocation
and
coimmunoprecipitation of the RalGEFs with TC21. However, like
the
cognate E37G mutation in H-Ras, the E48G mutation in TC21
did not
inhibit the interaction with the RalGEFs (Fig.
6 and
7)
(
37,
50).
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FIG. 6.
Plasma membrane translocation of RalGDS and Rlf by V23
and V23 E48G TC21. Confluent MDCK cells were microinjected with 20 µg
of an expression vector for a GFP:wild-type RalGDS fusion protein per
ml (A) or 50 µg of an expression vector for HA-tagged wild-type Rlf
per ml (B) alone or in conjunction with expression vectors for
Myc-tagged V23 TC21 wild-type or effector mutant proteins (25 and 50 µg/ml, respectively). Cells were fixed and stained 14 h after
microinjection for Myc-tagged TC21 or HA-tagged Rlf using specific
antibodies. GFP fluorescence is shown for the GFP:RalGDS fusion
protein. Samples were analyzed by confocal microscopy.
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FIG. 7.
Coimmunoprecipitation of Rlf with V23 and V23 E48G TC21.
NIH 3T3 cells were transiently transfected with expression vectors for
HA-tagged Rlf alone or in conjunction with expression vectors for
Myc-tagged V23, V23 T46S, V23 E48G, or V23 Y51C TC21. Myc-tagged TC21
proteins were immunoprecipitated from whole-cell lysates, and
coimmunoprecipitated Rlf was detected after SDS-PAGE by immunoblotting
with anti-HA antibodies.
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Activation of Ral A by TC21.
Several members of the Ras family
of GTPases interact with RalGEFs (28, 41, 43, 48, 49).
However, interaction has not always correlated with the ability of
these GTPases to activate the Ral pathway. Indeed, R-Ras and Rap have
been reported to bind RalGDS in vitro but do not activate this pathway
in vivo (17, 26, 41, 43, 49). Therefore we tested whether
the interaction of TC21 with the RalGEFs results in the activation of
Ral A in vivo, using the Ral binding domain of the Ral effector RalBP1 (RalBP1 RalBD) to selectively pull down GTP-bound Ral
(51). First we determined the constitutive levels of
Ral-GTP in stably transformed cell lines (Fig.
8). TC21-transformed NIH 3T3 clones consistently exhibited elevated levels of Ral-GTP compared to parental
untransformed NIH 3T3 cells. A smaller elevation of Ral-GTP levels was
also seen in Ras-transformed cells. Figure 8B shows that total levels
of endogenous Ral A are equivalent in the transformed and untransformed
cell lines.
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FIG. 8.
Constitutive activation of Ral A in TC21-transformed
cells. H-Ras-transformed, TC21-transformed, and untransformed NIH 3T3
cells were transferred to serum-free medium for 4 h and then
lysed. GST-RalBP1-RalBD-conjugated glutathione-Sepharose beads were
used to pull down endogenous Ral-GTP from whole cell lysates.
Precipitated Ral A-GTP (A) and total Ral A and Glu-tagged and
Myc-tagged H-Ras and TC21 proteins in whole-cell lysates (B) were
detected after SDS-PAGE by immunoblotting with specific antibodies. Two
independently isolated TC21 clones (cl.1. and cl.2.) are shown. Fold
increases in the levels of Ral A-GTP are indicated below panel A.
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We confirmed these results by investigating the activation state of
endogenous RalA on transient expression of TC21 and H-Ras
in NIH 3T3
cells (Fig.
9). Similar to our
observations in stably
transformed cells, expression of active TC21
resulted in an increase
in the level of GTP-bound Ral A, as did
expression of H-Ras (Fig.
9A). Insertion of the T46S point mutation in
the effector domain
of TC21 interfered with RalGEF binding to TC21 and
also inhibited
the activation of Ral by TC21, while the E48G mutation,
which
has no effect on binding, also does not prevent the activation
of
Ral by TC21 (Fig.
9B). This indicates that the ability of TC21
to bind
RalGEFs is crucial to the activation of Ral by TC21.
View larger version (31K):
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[in a new window]
|
FIG. 9.
Activation of endogenous Ral A by V23 and V23 E48G TC21.
Expression vectors for Myc-tagged V12 H-Ras and V23 TC21 (A) as well as
for V23 T46S and V23 E48G TC21 (B) were transiently transfected into
NIH 3T3 cells. The cells were transferred to medium containing 1.5%
serum after transfection and harvested 20 h later. Ral A-GTP was
specifically precipitated using GST-RalBP1-RalBD-conjugated
glutathione-Sepharose beads. Ral A and Myc-tagged H-Ras and TC21
proteins were detected by immunoblotting with specific antibodies as
described in Materials and Methods.
|
|
Requirement for Ral activation for TC21-stimulated DNA
synthesis.
The direct interaction of TC21 with RalGEFs and the
activation of endogenous Ral by constitutively active TC21 strongly
suggest that the Ral pathway is a TC21 effector pathway. It has been
previously reported that this pathway cooperates with other Ras
effector pathways, such as the Raf/MAP kinase pathway, in cellular
transformation (43, 47). To assess the function of the Ral
pathway in TC21-induced transformation, we used overexpression of the
Ral-GTP binding domain of the Ral effector RalBP1 (RalBP1 RalBD) as a
way of selectively inhibiting Ral signaling (Fig.
10). Overexpression of this domain prevents the signaling of Ral to its effectors by forming nonproductive complexes with GTP-bound Ral.
View larger version (67K):
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[in a new window]
|
FIG. 10.
Requirement of Ral activation for TC21-stimulated DNA
synthesis. (A) TC21-transformed NIH 3T3 and the human tumor cell lines
CAL51 and SK-UT-1 were seeded on collagen-coated dishes for
microinjection. The cells were transferred to serum-free medium
containing ITS 10 h after seeding, with the exception of SK-UT-1
cells, which were kept in low-serum medium. The cells were
comicroinjected the next day with 6 mg of Y13-259 or whole
immunoglobulin G (IgG) per ml alone or in conjunction with 0.2 mg of
recombinant GST-RalBP1-RalBD per ml or 0.2 mg of GST protein per ml.
The culture medium of injected TC21-transformed NIH 3T3, CAL51, and
SK-UT-1 cells was supplemented with 10 mM BrdU 8 or 20 h (for the
human tumor cell lines) after injection, and the cells were fixed 21, 26, or 44 h after injection, respectively, and stained for the
microinjected proteins and for incorporated BrdU using specific
antibodies. SK-UT-1 cells were also transfected with an expression
vector for GFP or with an expression vector for the GFP:RalBP1 RalBD
fusion protein and then changed to serum-free medium. Transfected
SK-UT-1 cells were allowed to incorporate BrdU for 7 h at 17 h after transfection and were then fixed and stained as described
above. The percentage of BrdU-positive cells is shown. Under these
conditions, untransformed NIH 3T3 cells do not synthesize DNA. The
experiments were repeated at least three times with similar results.
Similar results were obtained using the dominant negative N28 Ral
mutant protein. (B) The morphology of injected TC21-transformed NIH 3T3
cells injected with IgG alone or together with GST-RalBP1-RalBD is
shown after staining for the rabbit IgG injection marker. (C) SK-UT 1 cells were transfected with low levels (10 ng) of an expression vector
for GFP, for the GFP:RalBP1 RalBD fusion protein, or for N28 Ral in the
absence or presence of low levels (5 ng) of an expression vector for
the constitutively active form of Ral A, Q72L Ral. The cells were kept
in 1% serum and allowed to incorporate BrdU for 7 h at 24 h
after transfection and were then fixed and analyzed as before. The
percentage of cells in DNA synthesis, the percent inhibition in DNA
synthesis after expressing the RalBD or N28 Ral, and the percent rescue
after coexpressing Q72L Ral are shown. The experiment was repeated
three times with similar results. (D) Swiss 3T3 cells were seeded on
glass coverslips and allowed to reach contact inhibition. They were
then transferred to serum-free medium for 24 h. The cells were
microinjected with 12.5 µg of an expression vector for Myc-tagged V23
TC21 per ml and 25 µg of an expression vector for HA-tagged RalBP1
RalBD per ml in the combinations indicated. The culture medium was
supplemented with 10 mM BrdU 20 h postinjection, and the cells
were fixed 44 h postinjection and stained for Myc-tagged TC21,
HA-tagged RalBP1 RalBD, and incorporated BrdU using specific
antibodies. NA, not applicable.
|
|
Expression of the RalBD in serum-starved TC21-transformed NIH 3T3
resulted in a strong inhibition of DNA synthesis with no
significant
effect on cellular morphology (Fig.
10A and B). Significantly,
we also
found that inhibition of Ral signaling in the breast
adenocarcinoma-derived
CAL51 cell line and the SK-UT-1 uterine
leiomyosarcoma cell line,
which are human tumor cell lines that harbour
activating mutations
in TC21 (
2,
13,
19), inhibited DNA
synthesis (Fig.
10A).
This is the first demonstration to our knowledge
of the involvement
of the Ral pathway in human tumor cell line growth.
CAL51 and
SK-UT-1 cells have activating mutations in TC21 and are
independent
of Ras function, as demonstrated by their insensitivity to
the
Ras-neutralizing antibody, Y13-259 (Fig.
10A).
Inhibition of Ral activation by expression of the dominant negative Ral
form, N28 Ral, also leads to an inhibition in DNA
synthesis in these
cells (Fig.
10C and data not shown). Furthermore,
inhibition of DNA
synthesis in SK-UT-1 cells due to expression
of either the RalBD or
N28Ral can be partially reversed by coexpression
of constitutively
active Ral, demonstrating that these proteins
act by having specific
effects on signaling by endogenous Ral
proteins (Fig.
10C).
To confirm that activation of the Ral pathway was required for
TC21-induced DNA synthesis, we coinjected an expression vector
for
RalBP1 RalBD with an expression vector for constitutively
active TC21
into quiescent Swiss 3T3 fibroblasts. Expression of
active TC21 in
these cells resulted in a strong stimulation of
DNA synthesis that was
partially inhibited by expression of the
RalBD (Fig.
10D). Again, no
effect of the RalBD on TC21-induced
cellular morphology was observed
(data not
shown).
| |
DISCUSSION |
Constitutively active TC21 will transform NIH 3T3 and other
fibroblast and epithelial cell lines with high efficiency (5, 6,
14, 19, 24, 33, 39). We have previously shown that the high
transformation potential of TC21 in NIH 3T3 cells is mediated by direct
activation of the Raf/MAP kinase pathway by TC21 (39).
Despite the aggressive transformation and strong stimulation of DNA
synthesis by TC21, activation of the Raf/MAP kinase pathway by TC21 is
less potent than by the classical Ras proteins (39),
suggesting that the activation of other signaling pathways is involved
in TC21-induced transformation. We now show that TC21-transformed cells
exhibit constitutive activation not only of the Raf/MAP kinase pathway
but also of the PI-3K and the Ral pathways, both of which contribute to
several aspects of TC21-induced transformation. Unlike the Raf/MAP
kinase pathway, constitutive activation of these pathways in
TC21-transformed NIH 3T3 cells appears to be higher than in
H-Ras-transformed cells. We have also found that the human tumor cell
lines which have activating mutations in TC21 and do not require
classical Ras function have very high levels of constitutively active
Akt/PKB and require Ral activation for DNA synthesis.
Activation of the Akt/PI-3K pathway by TC21.
Not only do
TC21-transformed NIH 3T3 and tumor cell lines containing activating
mutations in TC21 show a strong constitutive PI-3K-dependent activation
of Akt/PKB, but TC21 expression in transiently transfected NIH 3T3
cells also leads to a parallel increase in Akt/PKB activation.
Microinjection of oncogenic TC21 together with a GFP:Akt PH domain
fusion protein expression vector results in PI-3K-dependent recruitment
of the PH domain to the plasma membrane, demonstrating that TC21 can
stimulate the generation of 3'-phosphorylated lipids. Furthermore,
microinjection of the Y13-259 Ras-neutralizing antibody into
TC21-transformed cells does not inhibit the activation of Akt/PKB,
suggesting that TC21-stimulated activation of PI-3K is not mediated by
classical Ras proteins (data not shown). In transient-transfection
assays and in assays with stably transformed cell lines, activation of
Akt/PKB by TC21 is comparable to or higher than that by other Ras
family members, suggesting that TC21 may be a key regulator of PI-3K.
H-Ras has been previously found to interact directly with PI-3K,
suggesting a mechanism for the activation of this pathway
by H-Ras
(
36). From the crystal structure of p110
, it has been
predicted that Glu 37, Asp 38, Tyr 40, and Tyr 64 in H-Ras lie
at the
PI-3K:Ras interface (
44). Residues equivalent to Glu
37, Asp 38, and Tyr 40 are conserved in TC21. The equivalent residue
to Tyr
64 in H-Ras is a phenylalanine in TC21, which would interfere
with the
proposed hydrogen bond formation at this site (
44).
However, endogenous p110 is readily coimmunoprecipitated with
oncogenic
TC21. Other Ras-related proteins such as R-Ras and M-Ras/R-Ras
3 also
have a Phe residue at this site and have been reported
to activate the
Akt/PI-3K pathway (
25,
29).
We have observed that in vivo activation of the PI-3K pathway by TC21
can be inhibited by mutations in the effector domain
of TC21. The T46S
and E48G point mutations in the effector domain
of TC21 interfere with
binding of TC21 to PI-3K, the generation
of 3' phosphoinositides, and
the activation of Akt/PKB in vivo,
further indicating the requirement
for the direct interaction
of TC21 with PI-3K for the activation of
this pathway by TC21.
Equivalent mutations in H-Ras also interfere with
activation of
the PI-3K pathway by this oncogene (
37).
Activation of the PI-3K pathway appears to be central to various facets
of cellular transformation by TC21 or H-Ras oncogenes
in NIH 3T3 cells,
including DNA synthesis, cellular morphology,
and protection from cell
death resulting from loss of adherence,
known as anoikis (Fig.
5A and
data not shown). The TC21-induced
morphological alteration of PC12
pheochromocytoma cells is also
dependent on activation of PI-3K (Fig.
5B). This unique phenotype
does not appear to be dependent on
activation of the Ral or the
MAP kinase pathway since inhibition of
either pathway (by expression
of the RalBD or by exposure to the MEK
inhibitor UO126) has no
effect on the TC21-induced morphology (data not
shown). Previous
work has demonstrated that activation of the MAP
kinase pathway
is central to the induction of neurites in these cells
(
7).
However, the failure of TC21-expressing cells to
produce neurites
is not due to a lower activation of the Raf/MAP kinase
pathway
(
39), since coexpression of activated MEK1 with
V23 TC21 in
PC12 cells does not induce neurites (data not
shown).
Association of TC21 with RalGEFs and the role of TC21-induced Ral
activation in transformation.
Apart from the Akt/PI-3K pathway, we
also observed the constitutive activation of the Ral pathway in
TC21-transformed cells and the activation of endogenous Ral A upon
transient transfection of TC21. This activation requires an intact
effector domain since certain mutations in this region of TC21 abolish
activation of Ral A by TC21.
A possible mechanism for this activation was uncovered after
identification in a yeast two-hybrid screen of RalGDS as a
GTP-dependent
binding partner for TC21 (
28). We have
expanded these initial
observations to show that TC21 will interact not
only with the
prototypical member, RalGDS, but also with another Ral
nucleotide
exchange factor, Rlf. Rlf has only approximately 30%
homology
to other RalGEFs, with the homology lying for the most part in
the catalytic and Ras binding domains (
49). We show that
interaction
with TC21 results in the translocation of these cytosolic
exchange
factors to the plasma membrane. Consistent with these
observations,
previous work has shown that the posttranslational
modification,
and therefore plasma membrane localization of Ras and Ral
proteins,
is essential for the Ras-dependent activation of Ral
(
18,
26).
Targeting RalGEFs to the plasma membrane by
fusion to a Ras lipid
modification CAAX box is sufficient for
constitutive activation
of this pathway (
50). These
observations suggest that the recruitment
of cytosolic RalGEFs to the
plasma membrane by Ras GTPases is
a key event in Ral activation.
Interaction with RalGEFs and plasma
membrane translocation of RalGEFs
are both inhibited by the T46S
and Y51C mutations in the effector
domain of TC21, mutations that
also interfere with the ability of TC21
to activate endogenous
Ral A, again arguing that activation of Ral by
TC21 is achieved
through the direct recruitment of RalGEFs to the
plasma membrane.
A further mutation in the effector domain, E48G, has
no effect
on the interaction of TC21 with RalGEFs, on the
TC21-stimulated
plasma membrane translocation of RalGEFs, or on the
activation
of Ral A by TC21. Equivalent mutations in H-Ras have similar
effects
on the ability of Ras to interact with RalGEFs (
22,
37,
50),
suggesting that TC21 and H-Ras may interact with RalGEFs in
a
similar
fashion.
We have found that activation of the Ral pathway is required for
TC21-induced stimulation of DNA synthesis but not for morphological
alterations in human tumor cell lines harboring activating mutations
in
TC21 or in TC21-transformed NIH 3T3 cells. Earlier suggestions
that the
Ral pathway may be involved in the stimulation of DNA
synthesis came
from the observation that the RalGEF-binding H-Ras
effector mutant
(E37G) could stimulate DNA synthesis when expressed
in thyrocytes
(
30). However, we found that activation of the
Ral pathway
is not sufficient for the induction of DNA synthesis
or for
transformation, since the E48G effector mutant of TC21
does not
stimulate DNA synthesis in quiescent fibroblasts and
alone is unable to
transform cells (reference
39 and data not
shown).
These studies, together with previous work (
14,
15,
28,
31,
39), demonstrate that TC21 activates the same signal
transduction pathways as the classical Ras proteins, although
there may
be quantitative differences in the magnitude of activation
that could
give rise to different biological outcomes. This is
clearly seen in the
PC12 cell system, where activated Ras induces
neurite differentiation
through a MAP kinase-dependent pathway
whereas TC21 induces cell
flattening through a PI-3K-dependent
pathway. On the other hand, the
ability of TC21 to activate similar
pathways to those activated by
classical Ras proteins also suggests
that TC21 can perform many of the
same functions as Ras. This
conclusion is supported by the observation
that while microinjection
of the Ras-neutralizing antibody, Y13-259,
into 14 of the 15 human
tumor cell lines so far tested, blocks DNA
synthesis (
42; R.
Wilson, H. Paterson, and C. J. Marshall, unpublished observations),
microinjection of Y13-259 into two
tumor cell lines containing
activated TC21 has no effect (Fig.
10).
Furthermore, Ras mutations
are rare in the tumor types in which
oncogenic mutations in TC21
have so far been
found.
| |
ACKNOWLEDGMENTS |
We thank Mark Crompton for the CAL51 cell line and Andrew
Chan (Mount Sinai) for the SK-UT-1 cell line. The PH domain Akt pEGFP
C1 plasmid was a kind gift of Matilda Katan. We also thank Johannes Bos
for the kind gifts of the full-length RalGDS cDNA, RalBP1 RalBD
pGEX4T3, N28 Ral A pMT2-HA and full-length Rlf pMT2-HA plasmids, as
well as for advice on the Ral pull-down assays.
This work was funded jointly by a Wellcome Prize Fellowship (M.R.) and
a CRC project grant (C.J.M. and M.R.). C.J.M. is a Gibb life fellow of
the Cancer Research Campaign.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: CRC Centre for
Cell and Molecular Biology, Chester Beatty Laboratories, Institute of Cancer Research, 237 Fulham Rd., London SW3 6JB, United Kingdom. Phone:
44-20 7352 9772. Fax: 44-20 7352 5630. E-mail:
chrism{at}icr.ac.uk.
| |
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Molecular and Cellular Biology, June 2001, p. 3750-3762, Vol. 21, No. 11
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.11.3750-3762.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
