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INTRODUCTION |
Ras proteins are monomeric GTPases that play a pivotal role in the
control of cell proliferation; they function as binary switches by
cycling between an inactive form bound to GDP and the active GTP-bound
state (1). Activation, through the dissociation of bound GDP and
subsequent binding of GTP, is catalyzed by
GEFs,1 such as CDC25/Ras-GRF
and Sos (2-4). Return to the inactive state is ensured via stimulation
of the low intrinsic GTPase activity of Ras by GTPase-activating
proteins, such as p120-GTPase-activating protein, neurofibromin, and
GapIP4BP (2, 5). In the active GTP-bound state, Ras exerts
its biological effects by turning on several effectors that activate
downstream pathways. Soluble serine/threonine kinases B-Raf and c-Raf,
once activated by Ras-GTP through a mechanism that is not fully
understood, trigger a cascade of protein kinases that results in the
activation of mitogen-activated protein kinases extracellular
signal-regulated kinases 1 and 2 (6). Another target of Ras is the
catalytic subunit of phosphatidylinositol 3-OH kinase (PI3K) (7); this pathway leads to the activation of the protein kinase Akt (8, 9), as
well as the activation of Ras-related proteins of the Rho/Rac/Cdc42
family involved in controlling the polymerization state of the actin
cytoskeleton, cell adhesion, and gene transcription (10, 11). Ras is
also able to activate the related GTPase Ral through RalGEFs that
constitute direct effectors of Ras (12-15); three such proteins,
namely RalGDS (16), RGL (17), and Rlf (18) have been extensively
characterized, and the isolation of another member of this family has
been recently reported (19). Elegant genetic experiments have shown
that in most cellular systems, the complementary action of at least two
of these three pathways (Raf, PI3K, and RalGEFs) is necessary for Ras
to transform murine fibroblasts in culture (11, 13, 20-22). Other Ras
effectors, such as the
isoform of protein kinase C (23), Rin (24), and AF-6/Canoe (25) have been identified; their involvement in Ras
action has not yet been documented.
The Rap group of Ras-related proteins is composed of the two closely
related Rap1A and Rap1B (which are 95% identical) and the two 90%
identical Rap2A and Rap2B proteins (26-28); overall, Rap1 and Rap2
share 60% identity. They constitute the closest Ras relatives because
they share more than 50% overall identity with Ras proteins and
exhibit very similar three-dimensional structures (29, 30). Moreover,
Rap1 contains the same effector domain as Ras, which has prompted
speculations that Rap proteins may behave as Ras antagonists. Indeed,
the Krev-1 gene encoding Rap1A had been isolated on the
basis that its overexpression was able to revert the phenotype of
Ras-transformed NIH 3T3 fibroblasts (31); since then, several
laboratories have provided evidence that overexpression of Rap1 can
indeed interfere with Ras function. Molecular basis for such findings
resides in the fact that Rap1 is capable of binding with affinities
similar to or in some cases even higher than Ras with Ras effectors,
such as Raf-1 kinase, PI3K, and RalGEFs (7, 18, 32). Yet the
physiological role of Rap1 appears to vary according to the biological
model studied. Rap1 seems to promote cAMP-dependent as well
as NGF-stimulated B-Raf and mitogen-activated protein kinase activation
in PC12 cells (33, 34). Conversely, it has also been shown to
participate in the maintenance of T cell anergy by acting as a negative
regulator of T cell receptor (TCR)-mediated interleukin-2 gene
transcription (35) and to inhibit Raf kinase by forming a catalytically
inactive complex in quiescent Chinese hamster ovary cells that
is reversed upon insulin stimulation (71). It is also
possible that the function of Rap1 is independent of regulating Ras
signaling, because activation of endogenous Rap1 by extracellular
signals fails to interfere with Ras effector signaling in fibroblasts
(72).
In contrast with Rap1, no function has yet been attributed to Rap2.
Although it also contains the same effector domain as Ras, except for a
single substitution of a serine to phenylalanine at position 39 (a
similar substitution in Ras only moderately affects its transforming
potential), its overexpression does not antagonize Ras signaling (36).
In an effort to uncover the function of Rap2, we searched for potential
effectors by using the yeast two-hybrid system. This enabled us to
identify a novel protein, RPIP8, that specifically interacts with Rap2
and is principally expressed in brain (37). As described in this paper,
we also isolated partial cDNAs encoding the C-terminal region of
the RalGEFs RalGDS, RGL, and Rlf. These three related proteins, which
constitute effectors of Ras, are capable of inducing activation of the
Ras-related Ral GTPase, i.e. nucleotide exchange leading to
the formation of active Ral-GTP complexes (12, 38, 39). Although Ras
and Rap1 can both interact with RalGDS and Rlf in cells, only Ras is
capable of inducing activation of the GTPase Ral in vivo
(12, 14). By themselves, RalGEFs exhibit little biological activity, only slightly stimulating transcription from the c-fos
promoter; however, upon co-expression with activated Raf, RalGDS
greatly synergizes to activate c-fos promoter activity, as
well as cell proliferation and morphological transformation (13, 40).
Moreover, targeting Rlf to the plasma membrane constitutively activates the protein, which is then able to stimulate gene induction and cell
growth (38). RalGEFs exhibit considerable homology among each other in
their 130 most C-terminal residues, which constitute the Ras and Rap1
interaction domain (RID) (18). They contain a conserved central domain
homologous to the RasGEF CDC25 that is responsible for their exchange
factor activity toward Ral as well as their stimulating effects on cell
growth and gene induction (16, 38).
In this study, we show that Rap2 binds to full-length RalGEFs in
vitro as well as in vivo. This interaction only occurs
with active Rap2; Rap2-RalGEF complexes are found in the particulate fraction of transfected cells, and active Rap2 is capable of recruiting RalGDS and Rlf to its resident compartment, the endoplasmic reticulum, suggesting that RalGEFs may indeed constitute effectors of Rap2 function. However, ectopic expression of activated Rap2 does not lead
to the activation of the GTPase Ral, nor does it interfere with the
ability of Ras to activate Ral. These results suggest that RalGEFs
could also serve a function other than activating Ral in cells and that
this novel function could be regulated by their interaction with the
GTPase Rap2.
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EXPERIMENTAL PROCEDURES |
Two-hybrid Screens--
Screening of a mouse brain cDNA
library with the first 168 residues of Rap2A containing a Gly-12 to Val
substitution fused to the C terminus of the bacterial LexA protein has
already been described (37). The coding sequence for the full-length
Rap2B protein was obtained by reverse transcription-polymerase chain reaction from total RNA of the human pro-erythrocyte line HEL (a
generous gift from Dr. Dominique Dumesnil) using oligonucleotides 5'-ACT GGG ATC CAC CAT GAG AGA GTA CAA AGT GGT GGTG-3' and 5'-CCC TCG
TCG ACG GAC TAC GCC GCG TAG TTC ATC TGC CGC AC-3' as forward and
reverse amplimers respectively and Pfu polymerase
(Stratagene); the resulting product was digested with BamHI
and SalI and cloned into pGBT10 (3) restricted with the same
enzymes. The absence of mutations was verified by DNA sequencing. This
construct was transformed into Saccharomyces cerevisiae
strain HF7c (MATa, ura3-52, his3-200, lys2-801, ade2-101,
trp1-901, leu2-3, 112, gal4-452, gal80-538,
LYS2::GAL1-HIS3,
URA3::(GAL17-mers)3-CYCI-lacZ) and
used to screen a cDNA library from human Jurkat cells as described (41).
Interactions between identified partners were performed by mating
with Ras family proteins expressed as fusions with the DNA binding domain of GAL4 in HF7c yeast, and putative effectors fused to
the activation domain of GAL4 in yeast of the strain Y187
(MAT
, ura3-52, his3, ade2-101, trp1-901, leu2-3, 112, met-, gal4
, gal80
,
URA3::GAL1-lacZ). Interaction was assessed by the
capacity of diploid conjugants to grow in the absence of histidine and
to express
-galactosidase activity, with similar results. pGAD-RalGDS was a generous gift from Dr. Michael White (13), pGAD-Raf
was from L. van Aelst (42), pGAD-B-Raf was from A. Eychène (43),
and pPC86-Rlf was described previously (18).
In Vitro Interactions--
Ras family proteins were expressed as
GST fusions, purified on glutathione-Sepharose beads, and loaded with
GDP or Gpp(NH)p prior to binding reactions as described previously
(37). Potential partners were transcribed and translated in
vitro in the presence of [35S]methionine in a
coupled reticulocyte lysate using the bacteriophage T7 RNA polymerase
(TNT, Promega) from templates obtained as follows. The RIDs of RalGEFs
(obtained from the two-hybrid screens described in this study) were
amplified with polymerase chain reaction with Pfu polymerase
and subcloned at the 5' EcoRI site of the pGEMMyc4 vector (a
generous gift from Harald Stenmark and Marino Zerial) in frame with a
Myc epitope and downstream of the bacteriophage T7 promoter. The 5' and
3' oligonucleotide primers used to amplify these sequences were as
follows: for RalGDS, 5'-TGTG GAA TTC GCC TCC ACC ACG CCC GTG GCT GCC-3'
and 5'-CCTTG CTC GAG TCA GAA GAT GCC CTT GGC AAA TCTT-3'; for RGL,
5'-TGTG GAA TTC AAC AAT CCT AAA ATC CAC AAG CGC-3' and 5'-CCTTG CTC GAG
TCA GAG GGT GAT CTT GCT GTG CCT-3'; and for Rlf, 5'-TGTC GAA TTC TCC
CCT AGG CCT TCT CGG GGT-3' and 5'-GTCT GTC GACTCAG AAC AGT GCC
CGTGC-3'. The RBD of c-Raf, obtained as a GST fusion from A. Wittinghofer (44), was excised with BamHI and
EcoRI, subcloned into the pMalC2 vector (New England
Biolabs), amplified with oligonucleotides homologous to vector
sequences providing a 3' XhoI site, and cloned as above in
pGEMMyc4. The absence of mutations in all clones was assessed by DNA
sequencing. RPIP8 was transcribed and translated from pBS-RPIP8 as
described (37). Full-length RalGDS and RGL coding sequences were
excised from pCEP4RalGDS and pCEP4RGL, respectively (generous gifts
from M. White (13)) with BamHI and inserted into the
BamHI site of pGEMMyc4; full-length Rlf was excised from
pPC86-Rlf with SalI and NotI, blunted with Klenow
at its 3' extremity, and inserted into the 5' SalI and 3'
PstI (blunted with T4 DNA polymerase) sites of pGEMMyc4.
For binding experiments, 10 µl of glutathione-Sepharose 4B beads
bound to 250 ng of GST, GST-Rap2A, GST-Ha-Ras, GST-Rap1A, and GST-RalA
proteins were washed three times in ice-cold exchange buffer (25 mM Tris-HCl, pH 7.5, 2 mM EDTA, and 5 mM dithiothreitol) and incubated for 30 min at 37 °C in
20 µl of exchange buffer containing 150 mM of Gpp(NH)p or
GDP. The beads were then diluted in 180 µl of interaction buffer (25 mM Tris-HCl, pH 7.5, 1 mM EDTA, 5 mM MgCl2, and 5 mM dithiothreitol)
containing 200 µM of the appropriate nucleotide and
incubated for 3 h at 4 °C with 1 µl of in vitro
translated [35S]methionine-labeled potential effector.
Beads were washed four times with 1 ml of interaction buffer and then
boiled in SDS gel sample buffer to recover bound proteins. Samples were
analyzed by SDS-polyacrylamide gel electrophoresis on 10%
polyacrylamide gels; after staining with Coomassie Blue to detect the
GST and GST fusion proteins, gels were treated with Amplify (Amersham Pharmacia Biotech), dried, and exposed to film.
In Vivo Interactions--
The cDNAs encoding Ras and Rap2
GTPases as well as RalGEFs were subcloned into mammalian expression
vectors under the control of the cytomegalovirus promoter as follows.
The coding sequences for Rap2 proteins carrying a Gly to Val
substitution at position 12 (Rap2Val-12) and a Thr to Ala substitution
at position 35 (Rap2Ala-35) (45) were amplified by polymerase chain
reaction with Pfu using primers 5'-GTGT GGA TCC ACC ATG CGC
GAG TAC AAA GTG GTG GTG-3' and 5'-TCTT CTC GAGC CTA TTG TAT GTT ACA TGC
AGA ACA-3' and inserted into the BamHI and XhoI
sites of pcDNA3 (Invitrogen). Full-length RalGDS and RGL sequences
excised as indicated above were inserted into the BamHI site
of a pRK5 vector engineered to encode an N-terminal Myc epitope fused
in frame to the N terminus of the protein of interest (a generous gift
from Dr. Alan Hall).
HeLa cells were co-transfected with 4 µg of Rap2 and 8 µg of RalGDS
or Rlf expression constructs (or the relevant empty vector) per 8.5-cm
dish with calcium phosphate; 40 h after transfection, cells were
washed twice with phosphate-buffered saline and processed as follows.
For experiments performed with total cell extracts, cells were lysed on
ice in 25 mM Hepes buffer, pH 7.5, containing 0.1 M NaCl, 1% Nonidet P-40, and protease inhibitors, and
debris were removed by a 15-min centrifugation at 14,000 × g. In order to prepare membranes, cells were swollen on ice
in hypotonic buffer (25 mM Hepes, pH 7.5, containing
protease inhibitors) and lysed by 100 strokes of the tight-fitted
pestle of a Dounce homogeneizer. The postnuclear supernatant obtained
after centrifugation at 1000 × g for 3 min was
submitted to further centrifugation at 45,000 rpm for 30 min in a
Beckman TLA 45 rotor; membranes were resuspended in hypotonic buffer
containing 0.1 M NaCl and recentrifuged as described above.
They were solubilized in the same buffer containing 1% Nonidet P-40
(30 min on ice), and insoluble material was eliminated by a final
centrifugation as described above.
Solubilized extracts were precleared with protein A-Sepharose and
immunoprecipitated with 5 µg of anti-Myc 9E10 antibody (Boehringer Mannheim) followed by protein A-Sepharose as described previously (46);
the presence of Rap2 in immune complexes was revealed by Western
blotting with affinity-purified polyclonal anti-Rap2 antibodies (47)
and visualized by ECL (Amersham Pharmacia Biotech).
Intracellular Localization of Co-expressed GTPases and
RalGEFs--
5 × 106 exponentially growing HeLa
cells were electroporated in a final volume of 200 µl of culture
medium supplemented with 15 mM Hepes buffer, pH 7.5, at 960 microfarads and 240 V in the 0.4-cm electrode gap cuvettes of a Bio-Rad
Gene Pulser with 6 µg of each expression construct for Myc-tagged
RalGDS or Rlf, and Ras or Rap2 GTPase as indicated. After
electroporation, cells were washed once and plated in four 35-mm dishes
containing glass coverslips. 24 h later, they were washed twice
with phosphate-buffered saline, fixed for 6 min at
20 °C in
methanol, and simultaneously incubated with affinity-purified anti-Rap2
or anti-Ha-Ras (Santa-Cruz, catalog no. sc-520) and 9E10 anti-Myc (1 µg/ml, Boehringer Mannheim) antibodies as described previously (47).
Complexes were stained with fluorescein isothiocyanate-coupled
anti-mouse and tetramethylrhodamine isothiocyanate-coupled anti-rabbit
antibodies (Jackson ImmunoResearch Laboratories, Inc.) and visualized
with a Leica scanning laser confocal microscope.
Activation of Ral by Rap2 in Vivo--
In order to assess
whether Rap2 could activate Ral via RalGDS or Rlf, COS-7 cells grown in
5-cm dishes were transfected with 1.5 µg of pMT2-HA-Ral together with
2 µg of expression vector for RasVal-12 or Rap2Val-12 and/or 1 µg
of pcDNA3-Rlf or pcDNA-Myc-RalGDS (a generous gift from Thomas
Linnemann and Alfred Wittinghoger) as indicated. After metabolic
labeling of cells with [32P]orthophosphate, HA-Ral bound
nucleotides were analyzed and quantitated as described (38).
Ras-dependent Ral activation in the response to
insulin, was measured in A14 cells, which are NIH 3T3 fibroblasts
expressing the human insulin receptor (48), grown in 5-cm dishes, and
transfected with 1 µg of pMT2HA-Ral together with 1 µg of
pcDNA3 or 1 µg of pcDNA3-Rap2-Val-12. After serum
starvation for 16 h, the cells were stimulated for 5 min with 1 µM of insulin and lysed. Ral-GTP levels were determined
by trapping the active complex on beads covered with GST-RalBD fusion
proteins (GST fused to the Ral binding domain of the Ral effector
RLIP76 (49)) as described (15). Rap2 and Ral proteins were detected on
Western blots using monoclonal antibodies (Transduction Laboratories).
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RESULTS |
Rap2-GTP Interacts with the C-terminal Ras Interaction Domains of
RalGEFs--
In order to search for potential effectors of Rap2,
through which it may exert its biological effects, we performed two
independent screens using the two-hybrid method in the yeast S. cerevisiae. We screened a mouse brain cDNA library with
residues 1-168 of Rap2A carrying a Gly to Val substitution at position
12 (Rap2AVal-12) fused to the C terminus of the DNA binding domain of
the bacterial transcription activator LexA as a bait, and a human
Jurkat T lymphoma cDNA library with the full-length Rap2B protein
fused to the C terminus of the DNA binding domain of the yeast
transcription activator GAL4 as a bait. Among potential effectors, we
isolated the C-terminal regions of the RalGEFs RalGDS (residues
702-852), RGL (residues 618-768), and Rlf (residues 607-778); Rlf
was isolated in both screens, whereas RGL and RalGDS were obtained from
the mouse brain cDNA library and the Jurkat library, respectively. Because these molecules are known effectors of Ras and have been shown
to bind Rap1, we proceeded to investigate the biochemical and
biological significance of their interaction with Rap2.
The interaction spectrum of RalGEFs with Ras family proteins was
assessed by mating derivatives of yeast strain HF7c (mat a) expressing
GTPases fused to the C terminus of the DNA binding domain of GAL4, with
derivatives of yeast strain Y187 (mat
) expressing the various
effector proteins fused to the C terminus of the activation domain of
GAL4 and testing the resultant diploid strains for growth in the
absence of histidine and
-galactosidase activity. As shown in Table
I, the C-terminal domains of all three
RalGEFs (RalGDS, RGL, and Rlf) interacted as strongly with Rap2A and
Rap2B as they did with Ras and Rap1A; among other Ras-related proteins,
they interacted less well with R-Ras and not at all with Ral. In
contrast, Raf-1 kinase interacted strongly with Ras and poorly with
Rap1 and Rap2, whereas RPIP8 (a potential Rap2 effector expressed in
brain (37)) bound specifically to Rap2 (A and B). The same pattern of
interaction was observed irrespective of whether Rap1A and Rap2B
proteins were complete or truncated of their 18 C-terminal residues. No
interaction occurred when Rap1A and Rap2 (A and B) proteins carried a
Ser to Asn mutation at position 17, and they had therefore lost their
high affinity for GTP (data not shown); such a pattern of interaction
is characteristic of potential effectors of Ras-related proteins.
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Table I
Interaction of RIDs and full-length effectors with Ras and Rap proteins
in the yeast two-hybrid system
HF7c yeast expressing Ras family proteins fused to the C terminus of
the DNA binding domain of GAL4 were mated with Y187 yeast expressing
potential effectors fused to the C terminus of the activation domain of
GAL4. The capacity of diploids to grow in the absence of histidine was
scored; similar results were obtained by measuring -galactosidase
activity. ND, not done.
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An in vitro binding assay was devised in order to
investigate these interactions by a more direct and independent method. It consisted in assessing the ability of potential effectors, transcribed and translated in vitro in the presence of
[35S]methionine, to bind to the various Ras-related
proteins fused to glutathione S-transferase, immobilized on
glutathione-Sepharose beads, and loaded either with GDP or with a
nonhydrolyzable analogue of GTP, Gpp(NH)p. Fig.
1 shows that this assay displayed results similar to those obtained by the yeast two-hybrid method: RPIP8 and the
RBD of Raf-1 interacted specifically with Rap2 and Ras, respectively,
whereas the C-terminal domains of all three RalGEFs bound to Ras, Rap1,
Rap2A, and Rap2B indiscriminately. In all cases, the interaction only
occurred with the GTP-bound form of the GTPases. Hence, RalGEFs,
through the direct binding of their C-terminal domain to Rap2-GTP
complexes, constitute potential effectors of Rap2A and Rap2B
GTPases.

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Fig. 1.
Interaction of the RIDs of RalGEFs with Ras,
Rap1, and Rap2 GTPases. The RIDs of RalGDS, RGL, and Rlf, along
with the RBD of c-Raf and the Rap2 effector RPIP8 were transcribed and
translated in vitro in the presence of
[35S]methionine. They were incubated with control GST or
with Ras, Rap1, Rap2A, and Rap2B proteins fused to GST and bound to
glutathione-Sepharose beads that had previously been loaded with GDP
(lanes a) or Gpp(NH)p (lanes b). After extensive
washing, the beads were boiled in SDS-sample buffer, and the extracts
submitted to SDS-polyacrylamide gel electrophoresis and fluorography.
Lane T, one-half of the input labeled proteins.
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Interactions with Full-length RalGEFs--
Because the RIDs of
RalGEFs only represent the C-terminal one-third of these molecules, we
investigated whether the full-length proteins might contain regulatory
regions that would introduce some differential specificity in their
interactions with Ras, Rap1A, Rap2A, and Rap2B. As shown in Table I,
using the yeast two-hybrid system, full-length RalGDS and Rlf (we were
unable to express a functional RGL protein in yeast) interacted with all four GTPases, albeit slightly less efficiently with Rap1A than with
Ras and Rap2 (A and B). As with their RIDs, the interaction of
full-length RalGEFs with Rap1 and Rap2 was unaffected by the presence
or absence of the 18 C-terminal residues of these GTPases (not shown).
It is noticeable that, in contrast with the results obtained with their
RIDs, full-length RalGDS and Rlf could no longer interact with R-Ras,
suggesting that RalGEFs do not constitute physiological effectors of
the R-Ras GTPase.
Using the same in vitro binding assay as above, we extended
the results obtained with the yeast two-hybrid system: Fig.
2 shows that full-length RalGDS, RGL, and
Rlf interacted with Ras, Rap1, Rap2A, and Rap2B in their active
GTP-bound form. In this assay, Rlf interacted better with Ras than with
Rap1 and Rap2, in agreement with the reported high affinity of the
Ras-GTP-Rlf interaction (18). Although some interaction of RalGDS and
RGL occurred in certain experiments with the GDP-forms of Rap GTPases, it was always much weaker than that observed with their GTP-bound form.
In summary, the data obtained with the yeast two-hybrid system as well
as with an in vitro binding assay suggest that RalGEFs could
indeed constitute effectors of Rap2A and Rap2B GTPases.

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Fig. 2.
Interaction of full-length RalGEFs with Ras,
Rap1, and Rap2 GTPases. Full-length RalGEFs were transcribed and
translated in vitro, and their binding to the GDP or
Gpp(NH)p form of Ras, Rap1, and Rap2 was assessed as described in Fig.
1.
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Interaction of Rap2 with RalGEFs in Mammalian Cells--
In order
to assess whether such interactions between RalGEFs and Rap2 could also
occur in mammalian cells, we co-transfected HeLa (Figs.
3 and 4) or
HEK 293 cells (not shown) with expression vectors for the
constitutively active (carrying a Gly to Val substitution at position
12) and inactive (carrying a Thr to Ala substitution at position 35 (45)) mutants of Rap2A together with constructs encoding full-length
RalGDS or Rlf tagged at their N terminus with an exogenous Myc epitope.
Under these conditions, Rap2Val-12 was present in complexes
immunoprecipitated from whole cell lysates with anti-Myc antibodies
(Fig. 3); such was not the case when the inactive Rap2Ala-35 mutant was
expressed, showing that RalGDS and Rlf only associated in mammalian
cells with the active form of Rap2. These results are similar to those
obtained in control experiments with Ras (not shown).

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Fig. 3.
Co-immunoprecipitation of the active form of
Rap2 with RalGDS and Rlf from whole cell lysates. HeLa cells were
co-transfected with expression vectors encoding inactive (Ala-35) or
constitutively active (Val-12) mutant Rap2 proteins along with vectors
encoding RalGDS or Rlf carrying an exogenous Myc epitope at their N
terminus. 40 h after transfection, cells were lysed with 1%
Nonidet P-40. Aliquots were removed and tested for the expression of
transfected constructs by Western blotting with anti-Myc or anti-Rap2
antibodies. The bulk of the extract was submitted to
immunoprecipitation with anti-Myc antibodies, and the presence of Rap2
in these complexes was revealed by Western blotting. Similar results
were obtained by transfecting HEK 293 cells.
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Fig. 4.
A complex between Rap2 and RalGEFs is formed
in the particulate fraction of cells. HeLa cells were
co-transfected with expression vectors for Rap2 and Myc-tagged RalGEFs
as in Fig. 3. 40 h after transfection, cells were mechanically
lysed in hypotonic buffer, and the particulate fraction of cells was
isolated by ultracentrifugation, solubilized with 1% Nonidet P-40, and
submitted to immunoprecipitation and Western blotting as in Fig. 3. In
the middle panels, the particulate fraction was prepared
from cells transfected with Rap2 or Myc-Rlf vectors only; these
fractions were then mixed, solubilized, and immunoprecipitated as
above. Similar results were observed after transfection of HEK 293 cells.
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Although they are synthesized as soluble precursors, mature Ras family
GTPases are bound to cellular membranes, Ras to the plasma membrane and
Rap2 to the endoplasmic reticulum, following a series of
posttranslational modifications that involve prenylation and
palmitoylation in the case of Ras and Rap2 (47, 50). In order to
establish that the complexes between RalGEFs and Rap2 did not involve
unprocessed cytosolic precursors of the GTPase but bona fide
processed and membrane-associated protein, the particulate fraction of
transfected cells was isolated by ultracentrifugation prior to the
immunoprecipitation of RalGEFs with anti-Myc antibodies as above (Fig.
4). As previously reported (38), a significant proportion of
transfected RalGDS and Rlf (10-20%) was associated with the
particulate fraction of cells; this proportion did not vary with the
co-expression of Ras (not shown) or Rap2 (Fig. 4) proteins and
represented nonspecific association with cellular membranes (see below
and Fig. 6). As with whole cell extracts, the active Rap2 protein (but
not the inactive Ala-35 mutant, not shown) was co-immunoprecipitated
with RalGDS and Rlf from solubilized membranes of co-transfected cells.
In contrast, when membranes prepared from cells only transfected with
Rap2 or RalGEFs were mixed, solubilized, and submitted to
immunoprecipitation, only a very minor amount of Rap2 was recovered in
the immunoprecipitates (Fig. 4); similar results were obtained after
transfection in HEK 293 cells, as well as in control experiments
performed with Ras (not shown). Hence, these complexes between Rap2 and
RalGEFs were formed on membrane structures prior to cell lysis,
suggesting that the interaction between active Rap2 and RalGEFs may
indeed occur in mammalian cells.
Rap2 Does Not Lead to Activation of the Ral GTPase--
In order
to assess whether the observed interaction between Rap2 and members of
the RalGEF family resulted in activation of the Ral GTPase, we measured
the levels of Ral-GTP in transfected COS-7 cells after labeling of the
nucleotide pools with [32P]orthophosphate. Ectopic
expression of either RalGDS or Rlf enhanced the level of Ral-GTP from
4-6% (relative to the total amount of Ral-GDP + Ral-GTP) to 15-20%
(Fig. 5, A and B);
co-expression of activated Ras further stimulated Ral-GTP formation up
to 30-50% (Fig. 5A and Refs. 38 and 39). However, in
contrast to activated Ras, overexpression of activated Rap2 did not
further enhance the level of Ral-GTP induced by RalGDS (Fig.
5A) or Rlf (Fig. 5B). These results demonstrate
that despite the ability of active Rap2 to form complexes with RalGEFs
in vivo, active Rap2 does not stimulate the ability of
RalGDS or Rlf to activate Ral under these circumstances. Therefore, it
is unlikely that the Ral GTPase is involved in signal transduction
downstream of Rap2.

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Fig. 5.
Rap2 does not activate Ral in
vivo. A, COS 7 cells were transfected with pMT2-HA-Ral
together with expression constructs for RasVal-12, Rap2Val-12, and
Myc-RalGDS as indicated. Prior to lysis, cells were labeled with
[32P]orthophosphate for 5 h. HA-Ral protein was
immunoprecipitated from the precleared lysates using 12CA5 protein
A-Sepharose, the beads were extensively washed, and bound nucleotides
were eluted and separated using TLC. The left panel shows
the amounts of GDP and GTP bound to Ral; the arrows indicate
the positions of the nucleotides after separation. The
percentages of GTP were quantified using a PhosphorImager. Similar
results were obtained in at least two different experiments. The
right panel shows the expression levels of Myc-RalGDS, Ras,
and Rap2 in the lysates as assessed by Western blotting.
B, the same experiments were performed as in A
except that Rlf was transfected instead of Myc-RalGDS together with
Rap2Val-12 as indicated.
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Rap2 Recruits RalGEFs to Its Resident Compartment, Which Is
Different from That of Ras--
As a possible hint to the actual
inability of Rap2 to activate Ral despite its interaction in
vivo with RalGEFs, we examined by confocal microscopy whether the
subcellular localization of RalGDS and Rlf was similar in cells
expressing activated forms of Ras and Rap2. Immunofluorescence
experiments were performed on HeLa cells co-transfected with expression
constructs for Myc-tagged RalGEFs and constitutively inactive (Fig.
6A) or activated (Fig. 6B) GTPases, which were fixed with cold methanol, conditions
that eliminate cytosolic proteins and enable to visualize only
molecules present on structures such as the cytoskeleton and cellular
membranes. In the absence of co-expressed activated Ras or Rap2, a
faint and diffuse localization of ectopically expressed RalGDS and Rlf to intracellular membranes was observed in transfected cells (Fig. 6A, a and b). Upon co-expression with activated
Ras, the membrane-associated fraction of RalGDS (Fig. 6B, a
and b), as well as Rlf (Fig. 6B, c and
d), was localized with Ras at the plasma membrane. In
contrast, when they were co-expressed with the active form of Rap2, the membrane-associated fraction RalGDS, as well as Rlf, co-localized with
this GTPase at the endoplasmic reticulum (Fig. 6B, e-h). In
control experiments, co-expression of Ral GEFs with the inactive RasN17
(Fig. 6A, c-f) and Rap2Ala-35 (Fig. 6A, g-j)
mutants did not cause them to co-localize with the GTPases but rather
to maintain the same faint and nonspecific membrane labeling as when
they were expressed alone. It is noteworthy that RasN17 was localized at the plasma membrane similarly to RasVal-12, whereas inactive Rap2Ala-35 (as well as Rap2AAsn-17, not shown) did not specifically label the endoplasmic reticulum as transfected Rap2Val-12 (Fig. 6B, f and h) or endogenous protein (47), but it
did label various cellular membranes, including the plasma membrane.
Hence, the co-localization of ectopically expressed RalGEFs with the
active GTPase co-expressed in transfected cells represents recruitment of RalGDS and Rlf by active Ras and Rap2 at their resident compartment, i.e. the plasma membrane for Ras and the endoplasmic
reticulum for Rap2. Such a recruitment further hints that RalGEFs may
indeed act as effectors of Rap2 function and also provides an
explanation for why active Rap2 does not lead to the activation of
Ral.

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Fig. 6.
Active Ras and Rap2 recruit RalGDS and Rlf to
their respective resident compartments. A, HeLa cells
were transfected by electroporation with expression constructs for
Myc-tagged RalGDS (a) or Myc-tagged Rlf (b)
alone, or co-transfected with the following constructs encoding
Myc-tagged RalGEFs and inactive GTPase mutants: RalGDS and RasN17
(c and d), Rlf and RasN17 (e and
f), RalGDS and Rap2Ala-35 (g and h),
and Rlf and Rap2Ala-35 (i and j). 24 h
later, cells were processed for double immunofluorescence as described
under "Experimental Procedures" in order to simultaneously
visualize RalGEFs, stained in green, with monoclonal
anti-Myc followed by fluorescein isothiocyanate-coupled anti-mouse
antibodies (c, e, g, and i), and GTPases, stained
in red, with rabbit polyclonal anti-Ras or anti-Rap2
antibodies followed by tetramethylrhodamine isothiocyanate-coupled
anti-rabbit antibodies (d, f, h, and j) in
co-transfected cells. B, HeLa cells were co-electroporated
as in A with the following expression constructs for
Myc-tagged RalGEFs and constitutively activated GTPase mutants: RalGDS
and RasVal-12 (a and b), Rlf and RasVal-12
(c and d), RalGDS and Rap2Val-12 (e
and f), Rlf and Rap2Val-12 (g and h).
They were processed for double immunofluorescence as in A in order to simultaneously
stain RalGEFs (a, c, e, and g) with fluorescein
isothiocyanate and GTPases (b, d, f, and h) with
tetramethylrhodamine isothiocyanate-coupled secondary antibodies in
co-transfected cells. Images were obtained by confocal
immunofluorescence microscopy; similar results were observed in several
experiments.
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Rap2 Does Not Interfere with Ras-mediated Ral
Activation--
Given that the overexpression of activated Rap2 can
recruit RalGEFs yet does not lead to Ral activation, we wished to test the hypothesis that in such a manner, activation of Rap2 could regulate
Ras signaling to Ral, possibly by sequestering RalGEFs. To this end, we
used a cell line derived from NIH 3T3 overexpressing the insulin
receptor (A14), in which insulin stimulation leads to
Ras-dependent Ral activation (15), and investigated whether overexpression of activated Rap2 was able to interfere with this response (Fig. 7). In these experiments,
Ral activation was qualitatively measured by trapping the active
Ral-GTP complex on a GST-RalBD fusion protein as in (15). When A14
cells were transfected with HA-tagged Ral alone, insulin stimulated
both the activation of endogenous Ral, representing the whole cell
population, and HA-Ral ectopically expressed in transfected cells,
confirming that this method may indeed be used to assess Ras to Ral
signaling in transfected cells. Transfection of Rap2 expression vector
caused a vast overexpression of the protein but did not affect the
expression level of co-transfected HA-Ral as compared with control
cells transfected with empty vector. Under basal conditions,
overexpression of Rap2 was associated with a slight activation of
HA-Ral. Quite remarkably, the level of HA-Ral activation in response to
insulin stimulation was similar in cells overexpressing or not
overexpressing activated Rap2; further enhancement of Rap2 expression
level had no noticeable effect on insulin-stimulated Ral activation as
well (not shown). It should be noted that after insulin stimulation,
Ras becomes strongly activated, so GTP-bound Ras and Rap2Val-12 would
be expected to compete for RalGEFs binding. Our results indicate that,
under these conditions, and despite its vast overexpression, Rap2 is unable to sequester the entire endogenous pool of RalGEFs, and Ras-RalGEF complexes are formed as attested by the activation of Ral in
response to insulin. In conclusion, we have shown that although
activated Rap2 is able to interact with RalGEFs in cells, neither does
it lead to Ral activation, nor can it interfere with signaling from Ras
to Ral.

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Fig. 7.
Overexpression of Rap2-Val-12 fails to
interfere in insulin-induced Ral activation. Subconfluent
serum-starved A14 cells, transfected with 1 µg of pMT2HA-Ral together
with either 1 µg of pcDNA3 (left two lanes) or 1 µg
of pcDNA3-Rap2-Val-12 (right two lanes), were stimulated
with insulin (1 µM for 5 min), which activates Ral in a
Ras-dependent manner (15). Activated Ral was isolated from
the cell extracts by GST-RalBD precoupled to glutathione beads and
detected using a monoclonal RalA antibody (upper panel). As
a consequence, activation of both transfected HA-Ral (upper
band) and endogenous Ral (lower band) was detected. The
expression of transfected HA-Ral and Rap2-Val-12 in total lysates was
analyzed by Western blotting using anti-RalA and anti-Rap2 monoclonal
antibodies (lower panel).
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DISCUSSION |
In this study, we show that the Ras-related GTPase Rap2 is capable
of binding to proteins, the normal function of which is to activate
another Ras-related GTPase, Ral, yet this interaction, observed in
cells overexpressing Rap2 and RalGEFs, does not lead to formation of
the active Ral-GTP complex. This situation is reminiscent of what is
observed with another Ras-related GTPase, Rap1, which binds RalGDS
under similar experimental set-ups but does not either lead to Ral
activation (12, 14). This latter observation was somewhat surprising in
view of the fact that biochemical studies had revealed that the
C-terminal RID of RalGDS binds Rap1 with a 10-fold higher affinity than
Ras, which had led to speculate that RalGDS might well act as an
effector of Rap1 function (32). The two qualitative yeast two-hybrid
and in vitro assays described here that we performed with
the C-terminal RIDs and with the full-length RalGEFs suggest that there
is little differential specificity of the Ras, Rap1, and Rap2 GTPases
for the three RalGEFs, RalGDS, RGL and Rlf, respectively. This observed
promiscuity is not an artifact of the methods used, because Ras and
Rap2 interacted specifically with Raf-1 and RPIP8, respectively, in
both assays; moreover, RalGEFs did not behave as potential effectors of
another Ras-related GTPase, R-Ras (Table I). Besides the near identity of their effector regions, Ras, Rap1, and Rap2 present a high degree of
overall sequence conservation (45-55%); moreover, comparison of the
three-dimensional structures of Ras and Rap1 (29, 51) with the recently
established one of Rap2 (30) shows that all three proteins, in their
GTP-bound forms, are very similar, especially in their switch I, switch
II, and effector regions. Therefore, the apparent molecular promiscuity
demonstrated above reflects the biochemical possibility of all three
RalGEFs to interact with the GTP-bound form of the three GTPases, Ras,
Rap1, and Rap2.
One can, however, formulate several hypotheses as to why Rap proteins
are apparently able to bind RalGEFs yet cannot lead to the activation
of Ral. The first one is that Rap proteins and RalGEFs may never
actually interact in cells. This would be surprising because RalGEFs
are principally cytosolic (although we and others consistently find
that 10-20% transfected RalGDS and Rlf are associated with cellular
membranes; see Fig. 4 and 6) (38, 52) and should therefore be able to
access Rap proteins that, similarly to Ras, are bound to membranes via
their C-terminal extremities and remain on the cytoplasmic face of
their respective compartments (47, 53, 54). In fact, in the experiment
that attempts to uncover Rap-stimulated Ral activation, Rap2 and Rlf
are overexpressed and can be co-immunoprecipitated attesting of their
effective interaction (not shown).
The logical second hypothesis is that Rap-RalGEF interactions are
unproductive, i.e. that contrarily to Ras, Rap proteins cannot induce activation of the GEF activity of RalGDS and Rlf on Ral.
Yet when posttranslationally modified forms of Rap1 and Ral were
incorporated into liposomes, Rap1 was able to stimulate through RalGDS
the dissociation of GDP from Ral (14). Moreover, ectopic overexpression
of Rap1 and Rlf in COS cells resulted in a 1.5-2-fold activation of
coexpressed Ral (72). However, in a similar situation, Rap2 did not
lead to Ral activation through RalGDS or Rlf (Fig. 5), although a vast
overexpression of activated Rap2 alone was associated with a very
modest increase in Ral-GTP (Fig. 7). Therefore, at the biochemical
level, Rap1 is able to activate the exchange factor activity of RalGDS
and Rlf toward Ral. Our data hint that Rap2 may not be able to do the
same, and biochemical experiments performed by reconstituting
posttranslationally modified Rap2 and Ral in micelles together with
recombinant RalGEFs would be required to formally address this
question. As in the case of the interaction of Ras with Raf, it is not
yet understood whether targeting effectors to the membrane is
sufficient to cause their activation as suggested by grafting membrane
targeting sequences to the C terminus of Raf-1 (55, 56), PI3K (57), and
Rlf (38) and the inability of non-prenylated Ras mutants to activate Rlf in cells (38), or whether conformational changes in the effector
induced by interaction with the GTP-bound GTPase also play a role in
the activation mechanism as suggested in transfection experiments with
Raf-1 (58, 59) and NMR spectroscopy experiments with the RID of Ral-GDS
(60) and Rlf (61).
There are structural differences in the effector and so-called
"extended effector" regions that distinguish Rap2 from Ras and Rap1
that may impair the ability of Rap2 to activate RalGEFs. Rap2 contains
a phenylalanine at position 39, in the effector region, instead of the
serine found in Ras and Rap1. Although this position does not appear
critical from the three-dimensional structure of Rap2 (30), it is
involved in the interaction of Rap1A with the Raf-1 RBD (29), and a
replacement of serine 39 by phenylalanine reduces the transforming
ability of Ras by 3-10-fold (36); one should examine whether this
effect is due to a reduced ability of Ras to activate one or several of
its effectors, such as Raf-1, RalGEFs, or PI3K. There are also
nonconservative substitutions at positions 25 and 43 that exhibit
glutamine to threonine and glutamine to glutamic acid substitutions,
respectively, in Rap2 as compared with Ras and Rap1, as well as the
conservative replacement of valine 44 in Ras and Rap1 by isoleucine in
Rap2. It is noteworthy that residues 25 and 43 do not appear to make
direct contact with the Raf RBD in its crystal structure complexed to
Rap1 (29); however, they could be involved in the interaction of Ras
and Rap1 with the cysteine rich domain of Raf-1 (62, 63), which is
necessary for optimal Ras binding and Raf-1 activation in cells (64,
65). The possibility that such residues are also necessary for Ras and
Rap1 to activate RalGEFs in cells could be investigated by making the
appropriate substitutions in Ras/Rap1 and Rap2 and assessing by
transfection their ability to activate Ral via RalGEFs.
Finally, and perhaps most importantly, Rap2 is able to recruit RalGEFs
to its resident compartment, the endoplasmic reticulum, as Ras recruits
them to the plasma membrane. Yet under these circumstances, Rap-RalGEF
complexes might not be able to meet Ral, the subcellular localization
of which is quite diverse, because it has been found in plasma membrane
fractions and cytoplasmic vesicles, including clathrin-coated and
secretory vesicles (66, 67). Therefore even if a Rap2-RalGEF
interaction were to occur under physiological conditions, the different
subcellular localizations of the Ras-related proteins, Ras at the
plasma membrane and Rap2 at the endoplasmic reticulum (47), would
ensure that Rap2 and Ral do not act in the same transduction pathways,
whereas activation of Ras leads to the activation of Ral.
Because overexpressed activated Rap2 is able to recruit overexpressed
RalGDS and Rlf to the endoplasmic reticulum, one could have expected
overexpression of activated Rap2 to sequester RalGEFs away from the
plasma membrane and therefore inhibit Ras-dependent Ral
activation. This could have represented a mechanism for Rap2, as
already suggested for Rap1, to control signaling downstream from Ras.
However, we have shown that in a cell line overexpressing the insulin
receptor, where Ral is activated in the response to insulin stimulation
via endogenous Ras and RalGEFs, the overexpression of Rap2 is unable to
interfere with Ras-dependent Ral activation. This is in
line with previous observations that overexpression of Rap2 has no
effect on the growth-promoting effects of Ras (36). Several plausible
explanations include the possibility that a pool of membrane-associated
RalGEFs remains in the vicinity of Ras and Ral, due to their
association with a membrane microdomain or molecular scaffold,
mechanisms that have been suggested to increase the efficiency of
signal transduction in mammalian cells (68-70). Our results,
showing that the biochemical interaction promiscuity of Ras, Rap1, and
Rap2 GTPases with RalGEFs does not lead to functional promiscuity
in cells, suggest that compartmentalization of signaling proteins is of
the greatest importance to ensure the functional specificity of
signaling pathways.
The possibility of a physiological Rap2-RalGEF interaction raises the
question of whether RalGEFs might have another physiological role in
addition to stimulating the activation of Ral. In fact, because
activated Ral mutants cannot substitute for RalGDS or Rlf to transform
cells or activate transcription from the c-fos promoter (13,
38, 40), RalGEFs probably also exert a Ral-independent function in the
Ras signaling pathway. In the case of Rap2 signaling, a yet to be
identified partner of RalGEFs, which might be specifically present at
the surface of the endoplasmic reticulum, could serve as a target of
active Rap2-RalGEF complexes. Whether RalGEFs play a role downstream of
Rap2 by activating cellular pathways other than those involving Ral is
currently under investigation.