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(Received for publication, April 29, 1996, and in revised form, May 21, 1996)
From the Department of Cell Biology and Neuroscience, University of
Texas Southwestern Medical Center, Dallas, Texas 75235-9039, § INSERM U-248, Section de Recherche, Institut Curie, Paris
75231, France, ¶ Signal Transduction, Promega Corporation,
Madison, Wisconsin 53711, and ABSTRACT Oncogenic Ras transforms cells through the
activation of multiple downstream pathways mediated by separate
effector molecules, one of which is Raf. Here we report the
identification of a second ras-binding protein that can induce cellular
transformation in parallel with activation of the Raf/mitogen-activated
protein kinase cascade. The Ral guanine nucleotide dissociation
stimulator (RalGDS) was isolated from a screen for Ras-binding proteins
that specifically interact with a Ras effector-loop mutant,
ras(12V,37G), that uncouples Ras from activation of Raf1. RalGDS, like
ras(12V,37G), cooperates synergistically with mutationally activated
Raf to induce foci of growth and morphologically transformed NIH 3T3
cells. RalGDS does not significantly enhance MAP kinase activation by
activated Raf, suggesting that the cooperativity in focus formation is
due to a distinct pathway acting downstream of Ras and parallel to
Raf.
INTRODUCTION The Ras GTPases function as important nodes in signal transduction
networks regulating proliferation and differentiation, integrating
extracellular signals to a variety of downstream cellular responses.
The importance of this role is highlighted by oncogenic ras, which can
induce growth and morphological transformation of many cell lines (1).
It is becoming increasingly apparent that Ras activity is mediated
through multiple effector pathways (2, 3, 4). The best characterized
pathway is the Raf/MAP1 kinase
(mitogen-activated protein kinase) cascade where, upon Ras binding, Raf
is activated and in turn activates MAP kinase through the activation of
MEK (mitogen-activated or extracellular signal-regulated kinase kinase)
(5, 6, 7, 8). MAP kinase activation is a critical step in cellular
transformation induced by oncogenic ras (9, 10).
Through the use of effector-specific ras mutants, which separate the
ability of Ras to interact with different downstream targets, we have
previously shown that multiple Ras functions can contribute to cellular
transformation. Only one of these involves Raf activation (3).
ras(12V,37G) was defective in Raf1 binding and did not transform cells.
However, ras(12V,37G) retained activity that complemented the
transformation defect of a different ras mutant, ras(12V,35S).
ras(12V,35S) could bind Raf1 and activate MAP kinase, but was
presumably defective in other target interactions preserved by
ras(12V,37G). The cooperativity between ras(12V,37G) and ras(12V,35S)
suggested the presence of a novel pathway mediating Ras-induced
cellular transformation in parallel to Raf activation (3).
Here we report the identification of a mammalian ras-binding protein,
RalGDS (11, 12, 13), that interacts with ras(12V,37G). Using focus
formation assays in NIH 3T3 cells, we show that both ras(12V,37G) and
RalGDS cooperate synergistically with mutationally activated Raf to
transform cells. This cooperativity is not due to additive effects on
MAP kinase activation, suggesting that RalGDS, like ras(12V,37G),
contributes to cellular transformation through an activity distinct
from activation of the Raf/MAP kinase cascade.
EXPERIMENTAL PROCEDURES All ras variants used were in the
vector pDCR, for mammalian expression, or pBTM116, for expression in
yeast, as described previously (3). pBTM116-Lamin was provided by A. Vojtek (14). pSR The yeast reporter
strain L40 (14) was used for all two-hybrid analysis. A random-primed,
size-selected, mouse embryo cDNA library expressed as fusions to
the VP16 activation domain (14) and an oligo(dT)-primed PC12 cDNA
library expressed as fusions to the GAL4 activation domain in pGADGH
(provided by S. Tsui and S. Halegua) were screened for fusions that
interacted with ras(12V,37G) fused to the LexA DNA-binding domain.
Approximately 10 million transformants were screened from each library.
Positives were tested for specificity of interaction by standard
techniques and sequenced (16).
Stable transfections of NIH
3T3 cells were performed by the calcium phosphate precipitation method
as described (17). 24 h post-transfection, the cells were split
into Dulbecco's modified Eagle's medium (ICN Biomedicals, Inc.) plus
5% calf serum (Life Technologies, Inc.) for focus formation assays,
and Dulbecco's modified Eagle's medium plus 10% calf serum
supplemented with 0.5 mg/ml G418 sulfate (Life Technologies, Inc.).
Foci of growth and morphologically transformed cells were scored under
magnification after 14 days of incubation in 5% serum. G418-resistant
colonies were counted after 10 days of growth in 10% serum plus G418.
Transient transfections were performed using LipofectAMINE (Life
Technologies, Inc.) according to the manufacturer's protocols.
Stably transfected NIH 3T3 cells were
lysed in a modified RIPA buffer supplemented with protease and
phosphatase inhibitors (18). ERK1 and ERK2 were immunoprecipitated from
200 µg of total protein with the anti-ERK1 C-16 polyclonal antibody
(Santa Cruz Biotechnology, Inc.). The immunocomplex was incubated with
12 µg of myelin basic protein (MBP) in a kinase assay for 30 min at
room temperature (18). Reactions were separated by SDS-polyacrylamide
gel electrophoresis and visualized on a PhosphorImager (Molecular
Dynamics).
Protein expression from
exogenously introduced cDNAs was determined by Western blot
analyses of cell lysates. raf-BXB expression was detected, from 20 µg
of total protein, using the anti-Raf1 C-20 antibody (Santa Cruz
Biotechnology, Inc.). Endogenous, activated forms of ERK1 and ERK2 were
detected, from 20 µg of total protein, by using the 759B (Promega
Corp.) antibody that selectively recognizes activated MAP kinase. Total
ERK1/ERK2 levels were detected using the anti-ERK1 C-16 antibody. RalA
was detected, from 20 µg of total protein, using a mouse anti-RalA
monoclonal antibody from Transduction Laboratories.
RESULTS To identify the effector
molecule(s) that may mediate signaling from ras(12V,37G), we used the
yeast two-hybrid system to screen cDNA libraries for clones that
interact with ras(12V,37G) but not ras(12V,35S). In this way, the
previously characterized ras-binding protein RalGDS was isolated from
libraries derived from mouse embryo and PC12 cells. RalGDS was
originally identified as a guanine nucleotide dissociation stimulator
(GDS) for the Ral GTPase (19). Human and rodent RalGDS interact
directly with Ras in a GTP-dependent manner (11, 12, 13), and
RalGDS has been shown to associate with Ras in COS cells upon
stimulation with epidermal growth factor (20). The two-hybrid
interactions of full-length RalGDS with ras(12V,37G) versus
other ras effector-domain mutants are shown in Fig. 1.
Although ras(12V,35S) does not interact with full-length RalGDS, a
truncated version of RalGDS, expressing amino acids 703-814, was
isolated from the mouse embryo library which interacts with both
ras(12V,37G) and ras(12V,35S) (data not shown). This suggests that the
specificity of the RalGDS-Ras interaction is affected by residues
outside of the minimal ras interaction domain (12). A different ras
effector mutant rasV12C40 did not interact with any form of RalGDS
isolated (Fig. 1 and data not shown).
ras(12V,37G) is defective in Raf1 binding, but retains a
function that cooperates with Raf1 to transform cells (3). The binding
of RalGDS with ras(12V,37G) suggests the possibility that RalGDS may
partially or fully mediate this function. To test this, we compared the
abilities of RalGDS and ras(12V,37G) to enhance focus formation induced
by activated raf. raf-BXB is a Raf1 variant with a deletion of the
amino-terminal regulatory domain containing the Ras-binding site (14,
21). This results in a mutationally activated protein that signals
constitutively to downstream components (21). NIH 3T3 cells were
transfected with rasV12, raf-BXB, ras(12V,37G), and wild-type RalGDS
alone and in the indicated combinations (Fig. 2).
raf-BXB alone induced a low level of focus formation. Both RalGDS and
ras(12V,37G) had no focus forming activity when expressed alone or
together. However, ras(12V,37G) acted synergistically with raf-BXB to
transform cells, resulting in an approximately 8-fold induction of
focus formation above the level observed with raf-BXB alone. Similarly,
RalGDS acted synergistically with raf-BXB to transform cells, resulting
in an approximately 12-fold induction of focus formation above the
level observed with raf-BXB alone. raf-BXB expression levels, as
determined by Western blotting of pooled G418-resistant colonies, were
equivalent in cells transfected with raf-BXB alone and in combination
with ras(12V,37G) or RalGDS (data not shown).
The activation of MAP kinase is an
important step in cellular transformation induced by Ras and Raf (9,
10). It is possible that the cooperativity in focus formation between
ectopically expressed RalGDS and raf-BXB converges at the level of MAP
kinase activation. To test this, the MAP kinases ERK1 and ERK2 were
immunoprecipitated from pooled G418-resistant colonies derived from
cells transfected with pDCR-ras(12V) or pSR
Transient transfection assays yielded similar results. To detect
activation of endogenous MAP kinase in transiently transfected cells,
it was necessary to use the 759B antibody which selectively recognizes
the activated phosphorylated forms of ERK1 and ERK2. Cells transiently
expressing RalGDS or ras(12V,37G) showed no increase in the levels of
activated MAP kinase as compared to cells transfected with empty
vectors. Importantly, RalGDS and ras(12V,37G) did not significantly
enhance the level of activated MAP kinase induced by raf-BXB. Total
levels of cellular MAP kinase were equivalent among transfections (Fig.
3B).
RalGDS stimulates guanine nucleotide exchange on the
Ras-related GTPase Ral (19). Therefore, the promotion of cellular
transformation by RalGDS may be a consequence of Ral activation. To
test this, we examined the contribution of Ral to focus formation by
Ras and Raf.
ralA(23V) has a mutation resulting in a defective intrinsic GTPase
activity homologous to the ras(12V) activating mutation (22).
Expression of pCEP4-ralA(23V) resulted in a 3-fold elevation of total
RalA protein but did not cooperate with raf-BXB to transform cells
(Fig. 4). This lack of cooperativity suggests that
RalGDS may contribute to raf-BXB-induced focus formation through a
RalA-independent mechanism. Alternatively, the 23V mutation may not
result in the same degree of functional activation of RalA as ectopic
expression of RalGDS.
ralA(26A) contains a mutation with structure and sequence homology to
the dominant negative mutant ras(15A) (23). ras(15A) has been shown to
form unproductive complexes with Ras guanyl nucleotide exchange factors
and is predominantly in the GDP-bound state (24, 23). We hoped that
expression of ralA(26A) would inhibit cellular Ral function by
sequestering Ral guanyl nucleotide exchange factors in a manner
analogous to the block of cellular Ras function that occurs upon
expression of ras(15A). Expression of pCEP4-ral(26A) resulted in a
2.5-fold elevation in total detectable RalA protein. ralA(26A) had no
significant effect on focus formation induced by raf-BXB. ralA(26A)
expression did result in a small but reproducible reduction in foci
induced by ras(12V) and by coexpression of raf-BXB and ras(12V,37G)
(Fig. 4). This reduction may be a result of sequestration of RalGDS
through unproductive binding to ralA(26A). Consistent with this,
ralA(26A) interacts with RalGDS in the yeast two-hybrid system whereas
wild-type RalA and ralA(23V) give no detectable interaction (data not
shown).
DISCUSSION ras(12V,37G) is defective in Raf1 interaction and cellular
transformation. However, ras(12V,37G) does retain an activity that
complements the transformation defect of a different Ras mutant,
ras(12V,35S). ras(12V,35S) binds Raf1, but is presumably defective in
other target interactions that mediate cellular transformation. The
transformation defect of ras(12V, 37G) is rescued by coexpression of a
Raf1 mutant that rescues binding to ras(12V,37G). We have interpreted
this as evidence that Ras transforms cells through the activation of
Raf1 as well as other, as yet unidentified, molecules (3). Here, we
show that the ras-binding protein RalGDS interacts with ras(12V,37G)
but not ras(12V,35S), suggesting that it may mediate the complementary
transformation activity of ras(12V,37G). Consistent with this, RalGDS,
like ras(12V,37G), cooperates with mutationally activated raf to
transform cells.
While the results described here establish RalGDS as a positive
regulator of transformation, it remains to be demonstrated that this
activity is mediated by Ras interaction. In addition, RalGDS is not the
only candidate Ras effector other than Raf which may mediate
Ras-induced cellular transformation. Additional Ras-binding proteins
have been identified, including phosphatidylinositol-3-OH kinase (2),
AF6 (25), Rin-1 (26), NF1 (27), Raf1 activates MAP kinase through direct binding and activation of MEK,
a MAP kinase kinase (8). MAP kinase activation is an important step in
the induction of cellular transformation by oncogenic ras (9, 10). The
complementary transforming activity of RalGDS does not appear to
converge at the level of MAP kinase activation, as it does not activate
MAP kinase and does not enhance activation of MAP kinase by activated
Raf. This suggests that RalGDS contributes to cellular transformation
by activating a pathway that is distinct from the Raf-MAP kinase
cascade.
Obvious candidates for targets that mediate RalGDS transforming
activity are the Ral GTPases (30). The use of ``activated'' and
``dominant negative'' mutants of RalA was unsuccessful in revealing a
role for Ral GTPases in Ras-induced cellular transformation. Similar
experiments with RalB mutants were also negative (data not shown).
However, while this manuscript was in preparation, Urano et
al. (31) published observations that ralA(72L) cooperated with
ras(12V) to transform cells, and ralA(28N) interfered with cellular
transformation by both ras(12V) and v-raf. Parallel studies with these
same reagents have not been done by us, and we cannot rule out a role
for RalA in mediating the activities we observe with RalGDS. Further
studies are underway, using mutants of RalGDS that effect Ral binding
and guanyl nucleotide exchange, as well as RalGDS mutants that restore
interaction with ras mutants, that should clarify the roles of RalGDS
and Ral in mediating Ras-induced cellular transformation.
FOOTNOTES Acknowledgments We thank A. Vojtek, A. Minden, M. Karin, S. Tsui, and S. Halegua for some of the reagents used in this study.
REFERENCES
Volume 271, Number 28,
Issue of July 12, 1996
pp. 16439-16442
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
COMMUNICATION:
,
''
Cold Spring Harbor Laboratories,
Cold Spring Harbor, New York 10021
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES
-raf-BXB was provided by A. Minden and M. Karin.
pCEP4-RalGDS contains the entire coding sequence of mouse
ralGDS inserted as a BamHI fragment into the
BamHI site of pCEP4 (Invitrogen Corp.). pCEP4-ralA(26A) and
pCEP4-ralA(23V) were derived from pBTM116-ralA(26A) and
pBTM116-ralA(23V), respectively (15). Ral coding sequences were removed
as blunt-ended XbaI-KpnI fragments and ligated
into pCEP4 at blunt-ended BamHI and KpnI
sites.
Fig. 1.
Two-hybrid interactions between ras mutants
and RalGDS. Ras variants and lamin, as a control, were expressed
as fusions to the LexA DNA-binding domain. RalGDS was expressed as a
fusion to the GAL4 activating domain. Transformants, from the yeast
reporter strain L40, selected to express GAL4 activating domain-RalGDS
and the indicated LexA DNA-binding domain fusions, were tested for the
ability to grow on media lacking histidine. Growth on the selective
plate indicates a positive two-hybrid interaction. Transformants that
were His+ also gave a positive indication of
-galactosidase activity using filter assays (data not shown). At
least four independent transformants were tested for each pair.
Fig. 2.
ras(12V,37G) and RalGDS cooperate with
raf-BXB to transform cells. NIH 3T3 cells were transfected with
the constructs expressing the indicated proteins. Each transfection
included the appropriate empty vector(s) as needed to achieve
equivalent DNA concentrations and to allow G418 selection. Focus
formation frequency for each transfection was determined as number of
foci per number of G418-resistant colonies, normalized to the focus
formation frequency induced by ras(12V), which was set at 100. Ras
variants were introduced (100 ng/transfection) in pDCR which contains
the G418 resistance gene. RalGDS was introduced (500 ng/transfection)
in pCEP4. raf-BXB was introduced (100 ng/transfection) in pSR.
Transfections with only empty vectors yielded no foci. Results shown
represent one experiment with each transfection performed in duplicate.
Error bars indicate the variation observed between the
duplicate transfections. Three additional experiments yielded similar
results.
-raf-BXB alone and in
combination with pCEP4-RalGDS. The number of colonies derived from
individual transfections were equivalent (~250/plate). Activity of
the immunoprecipitated ERK1 and ERK2 was assayed by in vitro
phosphorylation of MBP. The level of MAP kinase activation in cells
expressing raf-BXB was similar to that in cells expressing ras(12V).
Coexpression of RalGDS with raf-BXB resulted in no detectable induction
of MAP kinase above the level observed with raf-BXB alone (Fig.
3A).
Fig. 3.
RalGDS does not contribute to MAP kinase
activation by activated raf. A, MAP kinase activity in
stable transfected cell lines, expressing the indicated proteins, was
measured by MBP phosphorylation using anti-ERK1/ERK2 immune complexes.
Numbers indicate fold elevation relative to vector-transfected cells.
raf-BXB expression levels were equivalent in cells transfected with
raf-BXB alone or in combination with RalGDS (data not shown). Two
additional experiments yielded similar results. B, NIH 3T3
cells were transiently transfected with the indicated constructs (1 µg/transfection) and empty vector as required (2 µg/transfection of
total DNA). The level of activated cellular MAP kinase was measured by
Western blotting using the polyclonal antibody 759B which selectively
recognizes the activated phosphorylated forms of ERK1 and ERK2. Blots
were stripped and reprobed with the C-16 antibody which recognizes both
active and inactive forms of ERK1 and ERK2. A repeated experiment
yielded similar results.
Fig. 4.
Contribution of ral(26A) and ral(23V) mutants
to focus formation induced by activated ras and raf. NIH 3T3 cells
were transfected with plasmids expressing the indicated proteins, and
focus formation frequencies were determined as described in Fig. 2. Ras
variants, RalGDS, and raf-BXB were introduced as described in Fig. 2.
Ral mutants were introduced (500 ng/transfection) in pCEP4, resulting
in a 2- to 3-fold elevation of total Ral protein as determined by
Western blot (data not shown). Results represent one experiment with
each transfection performed in duplicate. Error bars
indicate the variation observed between the duplicate transfections.
Two additional experiments yielded similar results.
PKC (28), and MEK kinase 1 (29),
some or all of which may have roles in mediating Ras activity,
including induction of cellular transformation.
To whom correspondence should be addressed: Dept. of Cell Biology
and Neuroscience, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-9039. Tel.: 214-648-2861; Fax:
214-648-8694; E-mail: white08{at}utsw.swmed.edu.
''
American Cancer Society Research Professor.
1
The abbreviations used are: MAP,
mitogen-activated protein; MEK, mitogen-activated or extracellular
signal-regulated kinase kinase; MBP, myelin basic protein; RalGDS, Ral
guanine nucleotide dissociation stimulator.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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G. R. Post, C. Swiderski, B. A. Waldrop, L. Salty, C. C. Glembotski, R. M. F. Wolthuis, and N. Mochizuki Guanine Nucleotide Exchange Factor-like Factor (Rlf) Induces Gene Expression and Potentiates alpha 1-Adrenergic Receptor-induced Transcriptional Responses in Neonatal Rat Ventricular Myocytes J. Biol. Chem., May 3, 2002; 277(18): 15286 - 15292. [Abstract] [Full Text] [PDF]
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J. J. Fiordalisi, S. P. Holly, R. L. Johnson II, L. V. Parise, and A. D. Cox A Distinct Class of Dominant Negative Ras Mutants. CYTOSOLIC GTP-BOUND Ras EFFECTOR DOMAIN MUTANTS THAT INHIBIT Ras SIGNALING AND TRANSFORMATION AND ENHANCE CELL ADHESION J. Biol. Chem., March 22, 2002; 277(13): 10813 - 10823. [Abstract] [Full Text] [PDF]
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G. A. Murphy, S. M. Graham, S. Morita, S. E. Reks, K. Rogers-Graham, A. Vojtek, G. G. Kelley, and C. J. Der Involvement of Phosphatidylinositol 3-Kinase, but Not RalGDS, in TC21/R-Ras2-mediated Transformation J. Biol. Chem., March 15, 2002; 277(12): 9966 - 9975. [Abstract] [Full Text] [PDF]
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J. C. Wolfman, T. Palmby, C. J. Der, and A. Wolfman Cellular N-Ras Promotes Cell Survival by Downregulation of Jun N-Terminal Protein Kinase and p38 Mol. Cell. Biol., March 1, 2002; 22(5): 1589 - 1606. [Abstract] [Full Text] [PDF]
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Y. Wang, R. T. Waldron, A. Dhaka, A. Patel, M. M. Riley, E. Rozengurt, and J. Colicelli The RAS Effector RIN1 Directly Competes with RAF and Is Regulated by 14-3-3 Proteins Mol. Cell. Biol., February 1, 2002; 22(3): 916 - 926. [Abstract] [Full Text] [PDF]
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N. D. De Ruiter, B. M. T. Burgering, and J. L. Bos Regulation of the Forkhead Transcription Factor AFX by Ral-Dependent Phosphorylation of Threonines 447 and 451 Mol. Cell. Biol., December 1, 2001; 21(23): 8225 - 8235. [Abstract] [Full Text] [PDF]
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M. Karasarides, B. Anand-Apte, and A. Wolfman A Direct Interaction between Oncogenic Ha-Ras and Phosphatidylinositol 3-Kinase Is Not Required for Ha-Ras-dependent Transformation of Epithelial Cells J. Biol. Chem., October 19, 2001; 276(43): 39755 - 39764. [Abstract] [Full Text] [PDF]
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Y. Ward, W. Wang, E. Woodhouse, I. Linnoila, L. Liotta, and K. Kelly Signal Pathways Which Promote Invasion and Metastasis: Critical and Distinct Contributions of Extracellular Signal-Regulated Kinase and Ral-Specific Guanine Exchange Factor Pathways Mol. Cell. Biol., September 1, 2001; 21(17): 5958 - 5969. [Abstract] [Full Text] [PDF]
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M. Rosário, H. F. Paterson, and C. J. Marshall Activation of the Ral and Phosphatidylinositol 3' Kinase Signaling Pathways by the Ras-Related Protein TC21 Mol. Cell. Biol., June 1, 2001; 21(11): 3750 - 3762. [Abstract] [Full Text]
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 A. S. Gladfelter, J. J. Moskow, T. R. Zyla, and D. J. Lew Isolation and Characterization of Effector-Loop Mutants of CDC42 in Yeast Mol. Biol. Cell, May 1, 2001; 12(5): 1239 - 1255. [Abstract] [Full Text]
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M. Spoerner, C. Herrmann, I. R. Vetter, H. R. Kalbitzer, and A. Wittinghofer Dynamic properties of the Ras switch I region and its importance for binding to effectors PNAS, April 24, 2001; 98(9): 4944 - 4949. [Abstract] [Full Text] [PDF]
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A. Self, E Caron, H. Paterson, and A Hall Analysis of R-Ras signalling pathways J. Cell Sci., January 4, 2001; 114(7): 1357 - 1366. [Abstract] [PDF]
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D. O. Henry, S. A. Moskalenko, K. J. Kaur, M. Fu, R. G. Pestell, J. H. Camonis, and M. A. White Ral GTPases Contribute to Regulation of Cyclin D1 through Activation of NF-kappa B Mol. Cell. Biol., November 1, 2000; 20(21): 8084 - 8092. [Abstract] [Full Text]
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C. Peyssonnaux, S. Provot, M. P. Felder-Schmittbuhl, G. Calothy, and A. Eychène Induction of Postmitotic Neuroretina Cell Proliferation by Distinct Ras Downstream Signaling Pathways Mol. Cell. Biol., October 1, 2000; 20(19): 7068 - 7079. [Abstract] [Full Text]
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S. A. S. Johnson, N. Mandavia, H.-D. Wang, and D. L. Johnson Transcriptional Regulation of the TATA-Binding Protein by Ras Cellular Signaling Mol. Cell. Biol., July 15, 2000; 20(14): 5000 - 5009. [Abstract] [Full Text]
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J. Suzuki, Y. Yamazaki, L. Guang, Y. Kaziro, and H. Koide Involvement of Ras and Ral in Chemotactic Migration of Skeletal Myoblasts Mol. Cell. Biol., July 1, 2000; 20(13): 4658 - 4665. [Abstract] [Full Text]
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C. Missero, M. T. Pirro, and R. Di Lauro Multiple Ras Downstream Pathways Mediate Functional Repression of the Homeobox Gene Product TTF-1 Mol. Cell. Biol., April 15, 2000; 20(8): 2783 - 2793. [Abstract] [Full Text]
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L. Xue, J. H. Murray, and A. M. Tolkovsky The Ras/Phosphatidylinositol 3-Kinase and Ras/ERK Pathways Function as Independent Survival Modules Each of Which Inhibits a Distinct Apoptotic Signaling Pathway in Sympathetic Neurons J. Biol. Chem., March 17, 2000; 275(12): 8817 - 8824. [Abstract] [Full Text] [PDF]
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V Jullien-Flores, Y Mahe, G Mirey, C Leprince, B Meunier-Bisceuil, A Sorkin, and J. Camonis RLIP76, an effector of the GTPase Ral, interacts with the AP2 complex: involvement of the Ral pathway in receptor endocytosis J. Cell Sci., January 8, 2000; 113(16): 2837 - 2844. [Abstract] [PDF]
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M. Vo{beta}, P. A. O. Weernink, S. Haupenthal, U. Moller, R. H. Cool, B. Bauer, J. H. Camonis, K. H. Jakobs, and M. Schmidt Phospholipase D Stimulation by Receptor Tyrosine Kinases Mediated by Protein Kinase C and a Ras/Ral Signaling Cascade J. Biol. Chem., December 3, 1999; 274(49): 34691 - 34698. [Abstract] [Full Text] [PDF]
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M. Therrien, A. M. Wong, E. Kwan, and G. M. Rubin Functional analysis of CNK in RAS signaling PNAS, November 9, 1999; 96(23): 13259 - 13263. [Abstract] [Full Text] [PDF]
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R. H. Cool, G. Schmidt, C. U. Lenzen, H. Prinz, D. Vogt, and A. Wittinghofer The Ras Mutant D119N Is Both Dominant Negative and Activated Mol. Cell. Biol., September 1, 1999; 19(9): 6297 - 6305. [Abstract] [Full Text] [PDF]
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M. Osada, T. Tolkacheva, W. Li, T. O. Chan, P. N. Tsichlis, R. Saez, A. C. Kimmelman, and A. M.-L. Chan Differential Roles of Akt, Rac, and Ral in R-Ras-Mediated Cellular Transformation, Adhesion, and Survival Mol. Cell. Biol., September 1, 1999; 19(9): 6333 - 6344. [Abstract] [Full Text] [PDF]
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L. A. Quilliam, A. F. Castro, K. S. Rogers-Graham, C. B. Martin, C. J. Der, and C. Bi M-Ras/R-Ras3, a Transforming Ras Protein Regulated by Sos1, GRF1, and p120 Ras GTPase-activating Protein, Interacts with the Putative Ras Effector AF6 J. Biol. Chem., August 20, 1999; 274(34): 23850 - 23857. [Abstract] [Full Text] [PDF]
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L. M'Rabet, P. J. Coffer, R. M. F. Wolthuis, F. Zwartkruis, L. Koenderman, and J. L. Bos Differential fMet-Leu-Phe- and Platelet-activating Factor-induced Signaling Toward Ral Activation in Primary Human Neutrophils J. Biol. Chem., July 30, 1999; 274(31): 21847 - 21852. [Abstract] [Full Text] [PDF]
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 K. Sawamoto, P. Winge, S. Koyama, Y. Hirota, C. Yamada, S. Miyao, S. Yoshikawa, M.-h. Jin, A. Kikuchi, and H. Okano The Drosophila Ral GTPase Regulates Developmental Cell Shape Changes through the Jun NH2-terminal Kinase Pathway J. Cell Biol., July 26, 1999; 146(2): 361 - 372. [Abstract] [Full Text] [PDF]
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B. Bauer, G. Mirey, I. R. Vetter, J. A. Garcia-Ranea, A. Valencia, A. Wittinghofer, J. H. Camonis, and R. H. Cool Effector Recognition by the Small GTP-binding Proteins Ras and Ral J. Biol. Chem., June 18, 1999; 274(25): 17763 - 17770. [Abstract] [Full Text] [PDF]
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K. L. Wang and B. D. Roufogalis Ca2+/Calmodulin Stimulates GTP Binding to the Ras-related Protein Ral-A J. Biol. Chem., May 21, 1999; 274(21): 14525 - 14528. [Abstract] [Full Text] [PDF]
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N. van den Berghe, R. H. Cool, and A. Wittinghofer Discriminatory Residues in Ras and Rap for Guanine Nucleotide Exchange Factor Recognition J. Biol. Chem., April 16, 1999; 274(16): 11078 - 11085. [Abstract] [Full Text] [PDF]
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 C. A. Singer, X. A. Figueroa-Masot, R. H. Batchelor, and D. M. Dorsa The Mitogen-Activated Protein Kinase Pathway Mediates Estrogen Neuroprotection after Glutamate Toxicity in Primary Cortical Neurons J. Neurosci., April 1, 1999; 19(7): 2455 - 2463. [Abstract] [Full Text] [PDF]
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V. Nancy, R. M. F. Wolthuis, M.-F. de Tand, I. Janoueix-Lerosey, J. L. Bos, and J. de Gunzburg Identification and Characterization of Potential Effector Molecules of the Ras-related GTPase Rap2 J. Biol. Chem., March 26, 1999; 274(13): 8737 - 8745. [Abstract] [Full Text] [PDF]
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T. Goi, G. Rusanescu, T. Urano, and L. A. Feig Ral-Specific Guanine Nucleotide Exchange Factor Activity Opposes Other Ras Effectors in PC12 Cells by Inhibiting Neurite Outgrowth Mol. Cell. Biol., March 1, 1999; 19(3): 1731 - 1741. [Abstract] [Full Text] [PDF]
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Y. Tang, J. Yu, and J. Field Signals from the Ras, Rac, and Rho GTPases Converge on the Pak Protein Kinase in Rat-1 Fibroblasts Mol. Cell. Biol., March 1, 1999; 19(3): 1881 - 1891. [Abstract] [Full Text] [PDF]
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I. Treinies, H. F. Paterson, S. Hooper, R. Wilson, and C. J. Marshall Activated MEK Stimulates Expression of AP-1 Components Independently of Phosphatidylinositol 3-Kinase (PI3-Kinase) but Requires a PI3-Kinase Signal To Stimulate DNA Synthesis Mol. Cell. Biol., January 1, 1999; 19(1): 321 - 329. [Abstract] [Full Text] [PDF]
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M. B. Ramocki, M. A. White, S. F. Konieczny, and E. J. Taparowsky A Role for RalGDS and a Novel Ras Effector in the Ras-mediated Inhibition of Skeletal Myogenesis J. Biol. Chem., July 10, 1998; 273(28): 17696 - 17701. [Abstract] [Full Text] [PDF]
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M. J. Miller, L. Rioux, G. V. Prendergast, S. Cannon, M. A. White, and J. L. Meinkoth Differential Effects of Protein Kinase A on Ras Effector Pathways Mol. Cell. Biol., July 1, 1998; 18(7): 3718 - 3726. [Abstract] [Full Text]
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J.-J. Yang, J.-S. Kang, and R. S. Krauss Ras Signals to the Cell Cycle Machinery via Multiple Pathways To Induce Anchorage-Independent Growth Mol. Cell. Biol., May 1, 1998; 18(5): 2586 - 2595. [Abstract] [Full Text]
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Y.-T. Liu and H. L. Yin Identification of the Binding Partners for Flightless I, A Novel Protein Bridging the Leucine-rich Repeat and the Gelsolin Superfamilies J. Biol. Chem., April 3, 1998; 273(14): 7920 - 7927. [Abstract] [Full Text] [PDF]
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M. Shibatohge, K.-i. Kariya, Y. Liao, C.-D. Hu, Y. Watari, M. Goshima, F. Shima, and T. Kataoka Identification of PLC210, a Caenorhabditis elegans Phospholipase C, as a Putative Effector of Ras J. Biol. Chem., March 13, 1998; 273(11): 6218 - 6222. [Abstract] [Full Text] [PDF]
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M. Ikeda, O. Ishida, T. Hinoi, S. Kishida, and A. Kikuchi Identification and Characterization of a Novel Protein Interacting with Ral-binding Protein 1, a Putative Effector Protein of Ral J. Biol. Chem., January 9, 1998; 273(2): 814 - 821. [Abstract] [Full Text] [PDF]
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A. Yamaguchi, T. Urano, T. Goi, and L. A. Feig An Eps Homology (EH) Domain Protein That Binds to the Ral-GTPase Target, RalBP1 J. Biol. Chem., December 12, 1997; 272(50): 31230 - 31234. [Abstract] [Full Text] [PDF]
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B. Anand-Apte, B. R. Zetter, A. Viswanathan, R.-G. Qiu, J. Chen, R. Ruggieri, and M. Symons Platelet-derived Growth Factor and Fibronectin-stimulated Migration Are Differentially Regulated by the Rac and Extracellular Signal-regulated Kinase Pathways J. Biol. Chem., December 5, 1997; 272(49): 30688 - 30692. [Abstract] [Full Text] [PDF]
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A. Khwaja and J. Downward Lack of Correlation between Activation of Jun-NH2-terminal Kinase and Induction of Apoptosis after Detachment of Epithelial Cells J. Cell Biol., November 17, 1997; 139(4): 1017 - 1023. [Abstract] [Full Text] [PDF]
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K. L. Wang, M. T. Khan, and B. D. Roufogalis Identification and Characterization of a Calmodulin-binding Domain in Ral-A, a Ras-related GTP-binding Protein Purified from Human Erythrocyte Membrane J. Biol. Chem., June 20, 1997; 272(25): 16002 - 16009. [Abstract] [Full Text] [PDF]
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L. Han, D. Wong, A. Dhaka, D. Afar, M. White, W. Xie, H. Herschman, O. Witte, and J. Colicelli Protein binding and signaling properties of RIN1 suggest a unique effector function PNAS, May 13, 1997; 94(10): 4954 - 4959. [Abstract] [Full Text] [PDF]
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C. Mineo, R. G.W. Anderson, and M. A. White Physical Association with Ras Enhances Activation of Membrane-bound Raf (RafCAAX) J. Biol. Chem., April 18, 1997; 272(16): 10345 - 10348. [Abstract] [Full Text] [PDF]
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H. Murai, M. Ikeda, S. Kishida, O. Ishida, M. Okazaki-Kishida, Y. Matsuura, and A. Kikuchi Characterization of Ral GDP Dissociation Stimulator-like (RGL) Activities to Regulate c-fos Promoter and the GDP/GTP Exchange of Ral J. Biol. Chem., April 18, 1997; 272(16): 10483 - 10490. [Abstract] [Full Text] [PDF]
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M. J. Miller, S. Prigent, E. Kupperman, L. Rioux, S.-H. Park, J. R. Feramisco, M. A. White, J. L. Rutkowski, and J. L. Meinkoth RalGDS Functions in Ras- and cAMP-mediated Growth Stimulation J. Biol. Chem., February 28, 1997; 272(9): 5600 - 5605. [Abstract] [Full Text] [PDF]
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K. M. T. de Bruyn, J. de Rooij, R. M. F. Wolthuis, H. Rehmann, J. Wesenbeek, R. H. Cool, A. H. Wittinghofer, and J. L. Bos RalGEF2, a Pleckstrin Homology Domain Containing Guanine Nucleotide Exchange Factor for Ral J. Biol. Chem., September 15, 2000; 275(38): 29761 - 29766. [Abstract] [Full Text] [PDF]
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H. Shao and D. A. Andres A Novel RalGEF-like Protein, RGL3, as a Candidate Effector for Rit and Ras J. Biol. Chem., August 25, 2000; 275(35): 26914 - 26924. [Abstract] [Full Text] [PDF]
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C. Song, C.-D. Hu, M. Masago, K.-i. Kariya, Y. Yamawaki-Kataoka, M. Shibatohge, D. Wu, T. Satoh, and T. Kataoka Regulation of a Novel Human Phospholipase C, PLCepsilon , through Membrane Targeting by Ras J. Biol. Chem., January 19, 2001; 276(4): 2752 - 2757. [Abstract] [Full Text] [PDF]
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A. J. Dupuy, K. Morgan, F. C. von Lintig, H. Shen, H. Acar, D. E. Hasz, N. A. Jenkins, N. G. Copeland, G. R. Boss, and D. A. Largaespada Activation of the Rap1 Guanine Nucleotide Exchange Gene, CalDAG-GEF I, in BXH-2 Murine Myeloid Leukemia J. Biol. Chem., April 6, 2001; 276(15): 11804 - 11811. [Abstract] [Full Text] [PDF]
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