(Received for publication, November 7, 1996, and in revised form, December 16, 1996)
From the ¶ Departments of Pharmacology and Medicine,
University of California, San Diego, La Jolla, California 92093, ** Whitehead Institute for Biomedical Research, Cambridge, Massachusetts
02142, Thyroid-stimulating hormone stimulates
proliferation through both the cAMP-dependent protein
kinase and Ras but not through Raf-1 and mitogen-activated and
extracellular signal-related kinase kinase. We now report that
thyroid-stimulating hormone represses mitogen-activated protein kinase
activity and that microinjection of an effector domain mutant Ha-Ras
protein, Ras(12V,37G), defective in Raf-1 binding and mitogen-activated
protein kinase activation, stimulates DNA synthesis in quiescent and
thyroid-stimulating hormone-treated thyrocytes. A yeast two-hybrid
screen identified RalGDS as a Ras(12V,37G) binding protein and
therefore a potential effector of Ras in these cells. Associations
between Ras and RalGDS were observed in extracts prepared from thyroid
cells. Microinjection of a mutant RalA(28N) protein thought to
sequester RalGDS family members reduced DNA synthesis stimulated by Ras
as well as cAMP-mediated DNA synthesis in two cell lines which respond
to cAMP with mitogenesis. These results support the idea that RalGDS
may be an effector of Ras in cAMP-mediated growth stimulation.
The elucidation of the critical role played by Ras in growth
control initiated an intensive effort to identify Ras binding effector
molecules. The first Ras binding protein identified was p120GAP (1),
which functions both as an effector and down-regulator of Ras. The best
characterized Ras effector is the cytoplasmic serine/threonine protein
kinase Raf-1 (2, 3). Direct interaction between the N-terminal domain
of Raf-1 and the effector loop of Ras was first demonstrated using the
yeast two-hybrid system (4, 5), while later studies reported the
coprecipitation of Ras and Raf (6, 7). A number of other proteins bind
directly to GTP-bound Ras, including the p110 catalytic subunit of
phosphatidylinositol 3-kinase (8), AF6 (9), Rin-1 (10), NF1 (11), MEKK1
(12), protein kinase C TSH1-stimulated DNA synthesis requires both
the cAMP-dependent protein kinase and Ras (20). Although
Ras-dependent, mitogenic signaling induced by TSH does not
require Raf-1 or MEK (21), suggesting that Ras utilizes alternate
effectors in the presence of cAMP. To identify such effectors, an
extensive two-hybrid screen of a thyroid cell cDNA library was
performed with an effector domain mutant of Ras which does not bind
Raf-1 (22) but stimulates DNA synthesis in thyrocytes. This screen
repeatedly identified RalGDS as a potential Ras effector. Consistent
with such a role, interaction between Ras and RalGDS was detected in
extracts prepared from thyroid cells. Further, microinjection of a
mutant RalA(28N) protein thought to sequester RalGDS (19, 23)
specifically reduced cAMP-mediated DNA synthesis in both rat thyrocytes
and Schwann cells as well as DNA synthesis stimulated by Ras. These results are consistent with others (19, 23-26) which suggest that
RalGDS functions as an effector of Ras in some cellular signaling pathways. Our results suggest that RalGDS may be an important component
of cAMP-stimulated mitogenesis.
The
conditions under which Wistar rat thyrocytes (WRT) were propagated have
been described previously (27). Thyrocytes were rendered quiescent by
incubation in TSH- and serum-free medium containing 0.3% bovine serum
albumin and 0.5 µg/ml insulin for 48 h. Secondary rat Schwann
cells were propagated in Dulbecco's modified Eagle's medium
supplemented with glial-derived growth factor and forskolin as
described for human cells (28). One week prior to injection, Schwann
cells were plated on coverslips in Dulbecco's modified Eagle's medium
containing 10% fetal bovine serum. Proteins were coinjected with
rabbit IgG into the cytoplasm. Injected cells were identified by
staining with an fluorescein isothiocyanate-labeled anti-rabbit
antibody, and DNA synthesis was assessed by bromodeoxyuridine (BrdUrd)
incorporation as described previously (20).
cDNA
corresponding to the C-terminal 98 amino acids of RalGDSb was kindly
provided by Dr. F. Hofer. (University of California, Berkeley). The
C-terminal domain of RalGDSb isolated in the two-hybrid screen was
cloned into pGEX2T (Pharmacia Biotech Inc.). pGEX-Ha-Ras(12V), Ras(12G), and Ras(12V,37G) were provided by Dr. M. White (University of
Texas Southwestern Medical Center, Dallas). Vectors encoding RalA,
RalA(72L), and RalA(28N) fused to GST were kindly provided by Dr.
L. A. Feig (Tufts University). The GST fusion proteins were expressed
and purified as described by the manufacturer. For injection studies,
glutathione-Sepharose beads containing the fusion proteins were
incubated with 1.5 units of thrombin for 16 h at 4 °C.
Following centrifugation, the supernatant was incubated with 20 µl of
p-aminobenzamidine-agarose beads for 30 min at 4 °C.
Following centrifugation, the purified proteins were exchanged into
injection buffer (20 mM Tris, pH 7.5, 20 mM
NaCl, 2 mM MgCl2, 0.1 mM EDTA),
concentrated, and aliquots frozen at Immune complex kinase assays were performed
as described previously (29). WRT cells were lysed in 50 mM
HEPES, pH 7.4, 10 mM MgCl2, 10 µg/ml
aprotinin, 10 µg/ml leupeptin, 2.5 µg/ml pepstatin, 150 mM NaCl, 500 µM sodium orthovanadate, 100 nM staurosporine, 50 mM Total cellular RNA was prepared from WRT cells using
guanidinium hydrochloride and mRNA isolated using a Pharmacia
oligo(dT) purification kit (Pharmacia Biotech Inc.). cDNA was
synthesized using a Great Lengths cDNA synthesis kit (Clontech) and
cloned into EcoRI-digested yeast two-hybrid vector pGADGH
(Clontech) using EcoRI adapters. Mutant Ha-Ras(12V,37G) in
the yeast two-hybrid vectors pGBT9 and pBTM116 was kindly provided by
Dr. M. H. Wigler, Cold Spring Harbor Laboratory. The genotype of the
Saccharomyces cerevisiae reporter strain HF7c is MATa
ura3 his3 ade2 lys2 trp1 gal80
LYS2::GAL1-GAL1-HIS3-URA3::GAL4-CYC1-lacZ
(30). The genotype of the L40 reporter strain is MATa trp1 leu2
his3 LYS2::lexAHIS3-URA3::lexA-lacZ (5).
Approximately 2 × 106 clones were screened with both
Ras-LexA and Ras-Gal4 constructs. Positive clones were identified on
the basis of their ability to grow in the absence of histidine and to
stimulate Mice were
immunized following a standard regimen with a bacterially produced
protein encoding a portion of RalGDSa as described previously (34).
Spleens were fused to P3-X63-Ag8.653 cells to generate hybridomas
following standard protocols (44).
GST fusion proteins encoding Ras(12V) or
Rho(14V) were purified on glutathione-Sepharose. The proteins were
eluted with 2 bed volumes of 5 mM glutathione in 50 mM Tris, pH 7.5, 50 mM NaCl, 5 mM
MgCl2, 1 mM dithiothreitol, 1 mM
Pefabloc, 1 µM GTP or GDP for 10 min at 4 °C. The
eluate was dialyzed against 50 mM Tris, pH 7.5, 20 mM KCl, 2 mM MgCl2, 7.5 mM EDTA, 1 mM dithiothreitol, 1 mM
pefabloc, 1 µM GTP or GDP, concentrated, and protein
determinations made. Nucleotide loading was performed in the same
buffer supplemented with either 1 mM GTP or GDP for 30 min
at 30 °C. The reaction was terminated by addition of 20 mM MgCl2 and cooling to 4 °C. Log phase
thyrocytes were lysed by addition of 100 µl of ice-cold lysis buffer
(10 mM HEPES, 2 mM MgCl2, 30 mM NaCl, 0.5 mM NaVO3, 0.1 M NaF, 10 mM
Na2P2O7, 1% Nonidet P-40, 2 µg/ml leupeptin, 2 µg/ml aprotinin, and 0.5 µg/ml pepstatin) per
100-mm dish. Approximately 30 µg of GST fusion protein was incubated
with 1.2 mg of total cell protein for 1 h at 4 °C.
Glutathione-Sepharose (250 µl) was added to each mixture and
incubated for a further 60 min. The beads were washed with 3 × 1 ml ice-cold Tris-buffered saline (25 mM Tris, pH 7.5, 140 mM NaCl, 2 mM KCl), boiled in sample buffer,
separated on an 8% SDS-polyacrylamide gel, and immunoblotted for Ras
(SC29 Santa Cruz), GST (SC138 Santa Cruz), or RalGDS.
Quiescent
WRT cells stably transfected with a cAMP response element-regulated
lacZ gene were injected with the RalGDS Ras binding domain
together with rabbit IgG and subsequently stimulated with TSH (10 mU/ml) or 8-Br-cAMP (1 mM) for 6 h. Following
fixation, the cells were stained with an fluorescein
isothiocyanate-anti-rabbit antibody (to identify injected cells) and
5-bromo-4-chloro-3-indoyl Since Raf-1 is
apparently not required for the mitogenic effects of TSH, we assessed
whether microinjection of Ras(12V,37G), an effector domain mutant
defective in binding Raf-1, was sufficient to stimulate DNA synthesis.
Data shown in Table I demonstrate that injection of
Ras(12V,37G) stimulated DNA synthesis in quiescent WRT cells. Although
Ras(12V,37G) encoded an activating mutation, it appeared to be less
active than Ras(12V) in these assays but similar in activity to
cellular Ras(12G). Ras(12G,37G) also stimulated DNA synthesis, although
this single mutant was less active than both its activated counterpart
and cellular Ras (data not shown). Unlike the Ras proteins, activated
forms of Rho and Rac as well as GST alone (data not shown) failed to
stimulate DNA synthesis, although they were purified in parallel with
the Ras proteins. Like cellular Ras (21), Ras(12V,37G) retained its
mitogenic activity in the presence of cAMP stimulated by TSH,
consistent with its effects being independent of Raf-1.
Effects of Ras and Rho proteins on DNA synthesis in thyroid cells
Department of Cell Biology and
Neuroscience, University of Texas Southwestern Medical Center, Dallas,
Texas 75235, ¶¶ Department of Pediatrics,
Pharmacology,
University of Pennsylvania School of Medicine, Philadelphia,
Pennsylvania 19104-6084
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES
(13), a herpesvirus-encoded protein (14), and a guanine nucleotide dissociation stimulator for the Ras-related protein Ral (RalGDS) and a closely related protein (RGL) (15-18). While interaction of Ras with Raf-1, p110 (8), and RalGDS (19) results
in increased enzyme activity, the biological outcomes of these other
Ras-protein interactions are less understood.
Cell Culture, DNA Synthesis, and Microinjection
80 °C.
-glycerophosphate,
1% Nonidet P-40, and 500 µM Pefabloc. MAPK was
immunoprecipitated using an anti-MAPK antibody (SC93 Santa Cruz).
Immunoprecipitates were washed twice in 0.1 × phosphate-buffered saline and resuspended in 50 µl of kinase buffer (30 mM
HEPES, pH 7.4, 10 mM MgCl2, 1 mM
dithiothreitol, 50 µM ATP, and 500 µg/ml myelin basic
protein). 15 µCi of [32P]ATP was added and the samples
incubated at 30 °C for 10 min. The reactions were stopped by the
addition of 5 × Laemmli sample buffer, boiled, and analyzed on
12% SDS-polyacrylamide gels. The gels were dried and subjected to
autoradiography.
-galactosidase expression.
-D-galactoside (to assess
-galactosidase expression) and scored as described previously
(20).
Ras(12V,37G) Stimulates DNA Synthesis
Proteina
Treatment
N (inj)b
%BrdUrd + (inj)c
%BrdUrd + (C)d
Ras(12V, 37G), 0.75 mg/mle
Starved
555
65
15
Ras(12V), 0.075 mg/mle
Starved
875
78
24
Ras(12G), 0.5 mg/mle
Starved
1099
57
8
Rac(12V), 5.0 mg/ml
Starved
275
14
5
Rho(14V), 5.0 mg/ml
Starved
341
9
6
Ras(12V, 37G), 4.0 mg/ml
Starved
249
73
6
Ras(12V, 37G), 4.0 mg/ml
TSH
(0.05 mU/ml)
694
74
55
a
Purified proteins were coinjected with rabbit IgG and
DNA synthesis examined as described (see Ref. 66).
b
N(inj), number of injected cells examined.
c
%BrdUrd + (inj), proportion of injected
(FITC+) cells which synthesized DNA (Texas Red+).
d
C refers to control cells or uninjected cells adjacent to
injected cells on the same coverslip. In all cases, the number of
control cells examined was greater than or equal to the number of
injected cells examined.
e
For these proteins, the minimal effective concentration is
shown.
In some cells, cAMP inhibits
Raf activation without similar effects on MAPK activity (31, 32). Since
TSH-stimulated DNA synthesis appears both Raf-1- and MEK-independent,
the effects of TSH on MAPK activity were examined. Treatment with TSH
for 5-120 min (Fig. 1A, lanes 2-6) failed
to stimulate MAPK activity, although
12-O-tetradecanoylphorbol-13-acetate stimulated a dramatic increase in activity (lane 7). Similar to the effects of
TSH, which elevates cAMP, forskolin and 8-Br-cAMP also failed to
increase MAPK activity in these cells (data not shown). These results
are in good agreement with those published earlier in canine thyrocytes (33). Not only did TSH fail to increase MAPK activity, pretreatment with TSH repressed serum-stimulated (Fig. 1B) and
IGF-1-stimulated activity (data not shown). This data further supports
our hypothesis that TSH acts through a novel pathway which is
Ras-dependent but independent of Raf, MEK, and MAPK.
Identification of RalGDS as a Ras(37G) Binding Protein
To
identify potential Ras effectors expressed in thyroid cells, a yeast
two-hybrid screen was performed to identify Ras binding proteins.
Ras(12V,37G) was used in this screen in order to identify proteins
other than Raf-1 which bind to Ras. Approximately 2 × 106 clones were screened and a total of 4 positive clones
were obtained that permitted growth under selective conditions and
which expressed -galactosidase (Fig. 2). The cDNA
inserts from all 4 clones encoded a single sequence identical to the
C-terminal Ras binding domain (RBD) of RalGDSb (15-18, 34).
In some (25, 26) but not all (35) instances, expression of the RalGDS
RBD or that from a RalGDS-related protein (RGL) interfered with
Ras-mediated signaling. To test its effects on Ras-mediated signaling
in thyroid cells, the RalGDS RBD was coinjected with Ras proteins and
DNA synthesis examined. Injection of this peptide markedly reduced DNA
synthesis stimulated by both Ras(12V) and Ras(12V,37G), suggesting that
this domain binds Ras in living cells as it does in vitro
(17) (Fig. 3A). Injection of the RalGDS RBD
also abolished TSH-, cholera toxin-, and 8-Br-cAMP-stimulated DNA
synthesis, confirming the importance of Ras in these signaling pathways
(Fig. 3B). In contrast, this injected peptide had no effect
on TSH- or 8-Br-cAMP-stimulated cAMP response element-regulated gene
expression (Fig. 3C), results which are identical to those obtained following injection of a dominant interfering mutant Ras(17N) protein (20).
To assess whether RalGDS binds to Ras in thyroid cells, in
vitro binding experiments were performed. Lysates from
exponentially growing thyroid cells were incubated with immobilized GST
fusion proteins encoding activated forms of Ras and Rho, washed, and associated proteins were analyzed by Western blotting with a RalGDS monoclonal antibody (Fig. 4A) or mouse IgG
(Fig. 4B). RalGDS was detected only in lysates incubated
with GTP-bound Ras and probed with the RalGDS antibody. Neither
GDP-bound Ras nor GTP-bound Rho interacted detectably with RalGDS.
Probing with anti-GST (Fig. 4C) and anti-Ras (Fig.
4D) antibodies revealed that the abundance of the
immobilized GST fusion proteins was similar.
Ral Activity Is Required for Ras- and cAMP-stimulated DNA Synthesis
To assess whether RalGDS was required for Ras-stimulated DNA synthesis, a dominant interfering mutant RalA(28N) protein (19, 23) was utilized. Similar to dominant interfering Ras(17N), this mutant Ral protein exhibits a preferential affinity for GDP (36) and should therefore act to sequester molecules which bind to the GDP-bound form of RalA, including RalGDS and related molecules such as RGL (16). Injection of RalA(28N) protein reduced DNA synthesis stimulated by cellular, activated, and the effector domain Ras proteins in thyroid cells (Table II). Ras(12G) and Ras(12V,37G) appeared more sensitive to repression by RalA(28N) than was Ras(12V). The reason for this difference remains to be determined.
|
Because TSH-stimulated DNA synthesis is Raf-1-, MEK-, and
MAPK-independent, we assessed whether RalA(28N) would reduce it. Microinjection of RalA(28N) markedly reduced TSH-stimulated (Fig. 5,
a and b), cholera
toxin-stimulated, and 8-Br-cAMP-stimulated DNA synthesis, all mitogens
which act through elevations in cAMP, to near background levels (Table
III). In contrast, DNA synthesis stimulated by IGF-1
(Fig. 5, c and d) or fetal calf serum (FCS) was
not reduced by injection of RalA(28N). Similarly, injection of
RalA(28N) did not reduce FCS-stimulated DNA synthesis in NIH3T3 or
REF52 fibroblasts, pathways which are partially
Ras-dependent but independent of cAMP (data not shown).
|
These results suggested that Ral activity is critical for cAMP-mediated DNA synthesis. To examine this further, we extended our analysis to proliferating rat Schwann cells, which respond to cAMP with mitogenesis (28, 37, 38). Injection of RalA(28N) profoundly reduced DNA synthesis stimulated by cholera toxin and 8-Br-cAMP in these cells (Table III). Because RalA(28N) is thought to act by sequestering Ral exchange factors, including RalGDS (39), the repressive effects of RalA(28N) on cAMP- and Ras-mediated DNA synthesis suggest that a member or members of the RalGDS family play an essential role in Ras-dependent, cAMP-mediated mitogenic signaling pathways.
TSH stimulates proliferation through a cAMP-dependent pathway which requires Ras activity (20). Although Ras-dependent, TSH does not utilize Raf-1 or MEK in mitogenic signaling (21). We now demonstrate that TSH fails to stimulate MAPK activity and represses serum-stimulated activity. Consistent with these results, microinjection of a Ras effector domain mutant Ras(12V,37G), defective in Raf-1 interaction, stimulated DNA synthesis in quiescent and TSH-treated thyrocytes. Based on these results, this mutant Ras protein was used in a yeast two-hybrid screen of a thyroid cell library. This screen identified RalGDS as a Ras(12V,37G) binding protein and therefore a potential effector of Ras. Similar results were recently reported by White et al., who found that Ras(12V,37G) binds RalGDS expressed in a fibroblast library (18). RalGDS was first isolated from a mouse cell library using sequences derived from yeast Ras guanine nucleotide dissociation stimulator proteins as probes (34). Although its repeated identification as a Ras binding protein (15-17) suggests that RalGDS acts in Ras-dependent signaling pathways, its biological roles remain unknown. Kikuchi et al. reported that protein kinase A enhanced Ras association with RalGDS by decreasing Ras-Raf-1 interaction (24). Because TSH-stimulated DNA synthesis is both protein kinase A- and Ras-dependent, we examined whether RalGDS played a role in mitogenic signaling initiated by TSH.
In vitro binding data revealed that Ras binds to RalGDS in thyroid cell extracts. Consistently, microinjection of the RalGDS RBD reduced Ras-stimulated DNA synthesis, suggesting that this domain also binds to Ras in living cells. As expected, injection of the RalGDS RBD also reduced TSH- and cAMP-stimulated DNA synthesis in these cells, confirming the importance of Ras in this signaling pathway. A mutant RalA(28N) protein exhibiting a preferential affinity for GDP (36) analogous to the dominant interfering Ras(17N) mutant was used to examine whether RalGDS or a related activity was required for TSH-stimulated mitogenesis. RalA(28N) has been used previously to document a requirement for Ral activity in Src-stimulated phospholipase D activation (23) and in Ras-stimulated transformation (19). Microinjection of RalA(28N) dramatically reduced DNA synthesis stimulated by TSH, cholera toxin, and 8-Br-cAMP, all mitogens which act through elevations in cAMP. The level of DNA synthesis in quiescent thyrocytes varies between 0 and 30% depending upon how long the cells have been in culture. In most experiments, injection of RalA(28N) very nearly abolished DNA synthesis stimulated by agents which act through cAMP. In contrast, IGF-1- and FCS-stimulated DNA synthesis was little affected by injection of RalA(28N). These results suggest that a greater proportion of cAMP-mediated mitogenic signaling proceeds through a RalGDS-sensitive pathway in these cells than does signaling stimulated by IGF-1 or FCS. This would be expected given that IGF-1 and FCS do not elevate cAMP and therefore can transduce Ras-dependent signals through Raf-1 and the MAPK cascade. Unlike TSH, which fails to stimulate MAPK activity in either rat (Fig. 2) or canine thyrocytes (33), both IGF-1 and FCS increased MAPK activity in thyroid cells. Pretreatment with TSH abolished this activation, demonstrating that TSH interferes with signaling through the Raf-MAPK cascade and suggesting that TSH channels its Ras-mediated signals to a novel pathway involving RalGDS or a related molecule.
To determine whether the apparent requirement for Ral activity extended to other cells in which cAMP stimulates mitogenesis, RalA(28N) was injected into secondary rat Schwann cells. In the presence of serum, these cells cycle only very slowly. Addition of cholera toxin, 8-Br-cAMP, or forskolin stimulates a dramatic increase in DNA synthesis (28, 37, 38). Similar to the effects observed in rat thyrocytes, injection of RalA(28N) profoundly reduced both cholera toxin- and 8-Br-cAMP-stimulated DNA synthesis in secondary Schwann cells. These results support the hypothesis that RalGDS plays an important role in cAMP-stimulated mitogenesis.
Although in vitro RalGDS catalyzes nucleotide exchange on Ral, its effectors in vivo are unknown. By analogy with Ras, it is possible that recruitment of RalGDS to the plasma membrane through association with Ras activates Ral. However, microinjection of RalA or a GTPase-defective mutant RalA(72L) protein failed to stimulate DNA synthesis in quiescent thyrocytes. This may indicate that other factors, i.e. cAMP or Ras, are required for Ral to exert its mitogenic effects. This would be consistent with recent results demonstrating that RalA(72L) failed to stimulate phospholipase D activity, although it potentiated this activity in src-transformed cells (23), and that it failed to stimulate focus formation although it enhanced Ras-mediated transformation (19). If Ral is the effector of RalGDS, then other small G proteins are likely to function in this signaling pathway. Three laboratories have identified Ral binding proteins which exhibit characteristics expected for a Ral effector and cdc42GAP activity (40-42). It is also conceivable that there are targets of RalGDS other than RalA and RalB. In fibroblasts, RalGDS but not Ral collaborated with Raf in focus-forming assays (18). In further support of this idea, microinjection of RalA protein repressed TSH-stimulated DNA synthesis, perhaps indicating that RalA interferes with RalGDS-mediated signaling to another effector. Attempts to isolate full length RalGDS protein for microinjection studies have not yet been successful. Since thyrocytes arrest in G1 following intranuclear DNA injections and exhibit very low transfection efficiencies in transient assays, it will be necessary to generate stable lines overexpressing RalGDS and Ral in order to determine whether they exert similar effects on thyrocyte growth control.
In addition to Ha- and K-Ras, RalGDS also binds Rap1A (19), R-Ras (43), and TC21 (35), although binding to these molecules does not stimulate RalGDS activity in vitro (19). Several lines of evidence support the idea that TSH transduces its mitogenic signals through Ras, rather than one of these other proteins, to RalGDS or a related protein. First, purified Ras protein binds to RalGDS in thyroid cell extracts. Second, microinjection of the RalGDS RBD reduced DNA synthesis stimulated by Ras and TSH, suggesting that this domain binds to Ras in living cells. Third, similar to the effects of TSH, overexpression of Ras stimulates DNA synthesis in thyroid cells. It remains to be determined whether any of the other small G proteins which bind RalGDS exhibit similar effects. Additional support for signaling from Ras to RalGDS has been reported. In Xenopus oocytes, expression of the Ras-interacting domain of RGL interfered with Ras-mediated maturation (25). In NIH3T3 cells, this domain reduced Ras-mediated transformation (26), although in another study, expression of the RBD of RalGDS reportedly had no inhibitory effect on Ras- or TC21-stimulated transformation (35). Last, the observation that protein kinase A stimulates Ras-RalGDS association (24) further supports the hypothesis that Ras is capable of signaling through RalGDS and that this signaling pathway might predominate in the presence of cAMP.
We thank L. VanAelst and M. Wigler for help with the yeast two-hybrid screen, F. Hofer and G. Steven Martin for GST-RalGDS constructs, L. A. Feig for Ral constructs and helpful discussions regarding the manuscript, and S. Cannon for technical assistance.