From the Department of Immunology, The Scripps Research Institute, La Jolla, California 92037
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ABSTRACT |
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Human cells contain four homologous Ras proteins,
but it is unknown whether each of these Ras proteins participates in
distinct signal transduction cascades or has different biological
functions. To directly address these issues, we assessed the relative
ability of constitutively active (G12V) versions of each of the four
Ras homologs to activate the effector protein Raf-1 in
vivo. In addition, we compared their relative abilities to induce
transformed foci, enable anchorage-independent growth, and stimulate
cell migration. We found a distinct hierarchy between the four Ras
homologs in each of the parameters studied. The hierarchies were as
follows: for Raf-1 activation, Ki-Ras 4B > Ki-Ras 4A >>>
N-Ras > Ha-Ras; for focus formation, Ha-Ras Ras proteins function as molecular relay switches in signal
transduction cascades that regulate cell proliferation,
differentiation, and apoptosis. Extracellular ligands activate cell
surface receptors and thereby induce the activation and/or membrane
recruitment of guanine nucleotide exchange factors
(GEFs),1 which convert
inactive GDP-bound Ras into active GTP-bound Ras. To date, the known
GEFs for Ras proteins include Sos 1, Sos 2, Ras-GRF/CDC25Mm, Ras-GRF 2, and Ras GRP (reviewed in Ref.
1). Following activation, GTP-bound Ras binds to/activates one or more
effector proteins that subsequently initiate downstream signaling
pathways that ultimately induce a programmed cellular response.
Although members of the Raf serine/threonine kinase family (consisting
of Raf-1, A-Raf, and B-Raf) are the best characterized effectors of
Ras, numerous other putative effectors have been identified, including phosphatidylinositol 3-kinase, GEFs for the small GTPase Ral (Ral GDS,
RGL, RLF/RGL2), AF6, RIN1, MEK kinase 1, protein kinase C Human cells contain four Ras proteins, Ha-Ras, N-Ras, Ki-Ras 4A and
Ki-Ras 4B. (The Ki-ras gene encodes two proteins, 4A and 4B,
via alternative splicing of two fourth exons). These proteins are 85%
homologous (5, 6) and, with the exception of Ki-Ras 4A, appear to be
ubiquitously expressed (7-9). Although no studies have directly
compared the biological functions of each of the four Ras homologs,
several observations suggest that these proteins likely have different
biological roles. For instance, recent studies have shown that
Ki-ras, but not N-ras or Ha-ras, is
essential for mouse embryogenesis (reviewed in Ref. 1). Mouse embryos lacking a functional Ki-ras gene develop cardiac, liver,
neurologic, and hematologic defects and die before term, whereas mice
lacking a functional Ha-ras or N-ras gene are
born and grow normally. In addition, although mutations at codons 12, 13, or 61 constitutively activate each of the ras genes
in vitro, there is clear selectivity with regard to which
ras homolog is mutationally activated in a given type of
human cancer. Whereas ~90% of pancreatic, 50% of colon, and 30% of
lung adenocarcinomas harbor mutant Ki-ras genes, mutant
Ha-ras or N-ras genes are rarely detected in
these cancers (10). Likewise, whereas ~25% of acute myelogenous
leukemias and myelodysplastic syndromes contain mutant N-ras
genes, mutant Ha-ras and Ki-ras genes are not
frequently observed in these malignancies (11). Moreover, mutant
Ha-ras genes are rarely detected in any type of human cancer
(10).
Given the likelihood that the four Ras homologs have different
biological roles in vivo, we hypothesized that each of the four Ras homologs participates in distinct signal transduction pathways. Recent studies from our laboratory have shown that the GEF
Ras-GRF activates Ha-Ras but does not activate N-Ras or Ki-Ras 4B
protein in vivo (12). In addition, previous studies have shown that extracellular ligands can selectively trigger a specific GEF
to activate Ras proteins. For instance, ligands that activate tyrosine
kinase receptors induce Sos, but not Ras-GRF, to activate Ras, whereas
ligands that activate G protein-coupled muscarinic receptors induce
Ras-GRF, but not Sos, to activate Ras proteins (13-15). These combined
studies suggest that each of the Ras homologs could potentially be
selectively activated by a distinct upstream extracellular
ligand/receptor/GEF(s) in vivo. If, therefore, it could be
demonstrated that each of the four Ras homologs selectively activates a
distinct downstream effector protein(s) in vivo, these studies would support the premise that the four Ras homologs
participate in distinct signaling cascades. Accordingly, in this
report, we assessed whether the four Ras homologs could differentially
activate the best characterized effector protein, Raf-1, in
vivo. In addition, to directly test the notion that the four Ras
homologs have different biological roles in vivo, we
compared the ability of each of the Ras homologs to induce transformed
foci, enable anchorage-independent growth, and stimulate cell migration.
Molecular Constructs--
To distinguish exogenous from
endogenous Ras proteins, a Glu-Glu (EE) epitope tag (EEEEYMPME) (16)
was added to the N termini of constitutively active (G12V) human
Ha-ras, N-ras, Ki-ras 4A, and
Ki-ras 4B cDNAs. EE-tagged G12V ras cDNAs
and wild type raf-1 cDNA were cloned into the mammalian
expression vectors pZIP-Neo-SV(x)1 (17), pRK5 Myc, which contains an
SV40 origin of replication and adds a 10 amino acid N-terminal
Myc tag to expressed proteins (18), and/or pEGFP-C1, which fuses green
fluorescent protein (GFP) to the N termini of expressed proteins
(CLONTECH).
Raf-1 Kinase Assays--
Raf-1 kinase activity was assessed by
coupled MEK/ERK 2 kinase assays, according to previously described
methods (19, 20) with minor modifications. Briefly, COS-1 cells (8 × 105 cells/100-mm dish) were co-transfected with 2 µg
of wild type raf-1/pRK5 Myc and 0.5 µg of G12V
Ha-ras, N-ras, Ki-ras 4A, or Ki-ras 4B/pRK5 Myc (or empty pRK5 Myc) plasmid DNA using 60 µl of SuperFect (Qiagen). 24 h following transfection, cells
were serum-starved for 16 h in serum-free medium containing 4 mM mevalonolactone (COS-1 cells do not contain sufficient
amounts of endogenous isoprenoid precursors to farnesylate
overexpressed Ras proteins, so medium was supplemented with
mevalonolactone to augment available precursors). Cells were
subsequently lysed in 100 µl of lysis buffer (20 mM Tris-HCl, pH 7.5; 1% Triton X-100; 1 mM EDTA;
1.5 M KCl; 0.1 mM phenylmethylsulfonyl
fluoride; 5 µg/ml leupeptin; 1 mM benzamidine; 5%
glycerol; 0.3% Transformation Assays--
Cell transformation was assessed by
focus-forming assays and soft agar colony formation assays. For
focus-forming assays, NIH 3T3 mouse fibroblasts (5 × 105 cells/60-mm dish), Rat-1 fibroblasts (1 × l05 cells/60-mm dish), and RIE-1 rat intestinal epithelial
(5 × 105 cells/60-mm dish) cells were transfected by
calcium phosphate precipitation (NIH 3T3 and Rat-1) or LipofectAMINE
(RIE-1) with 50 ng, 2 µg, and 2.5 µg, respectively, of G12V
Ha-ras, N-ras, Ki-ras 4A, or
Ki-ras 4B/pZIP-Neo-SV(x)1 plasmid DNA; after 14 (NIH 3T3) or
21 (Rat-1, RIE-1) days, dishes were stained with crystal violet, and
the number of transformed foci was counted (21, 22).
For soft agar colony formation assays, Rat-1 and RIE-1 cells,
transfected as described above, were selected in G418, suspended in
reduced-serum (2%) medium containing 0.3% agar, and overlaid onto a
0.6% agar base at a density of 2 × 104 cells/60-mm
dish. Colony formation was monitored for up to 1 month (22, 23).
Cell Migration Assays--
Cell migration was assessed by
quantitating the number of cells that directionally migrate through
membranes to a collagen undercoating (24). Briefly, COS-7 cells (5 × 105/100-mm dish) were transfected with 0.1 µg of
Ha-ras or 0.2 µg of N-ras, Ki-ras
4A, or Ki-ras 4B/pEGFP-C1 plasmid DNA using 20 µl of
LipofectAMINE. (Amounts of transfected DNA represent amounts required
to achieve comparable expression levels). 24 h following transfection, cells were incubated in serum-free medium containing 4 mM mevalonolactone for 16 h and loaded (1 × l05 cells) into modified Boyden chambers containing
collagen type I-undercoated membranes. Cells were allowed to migrate
through membranes for 3 h at 37 °C, and the number of
GFP-Ras-expressing cells that migrated was quantitated on a
fluorescence microscope. At least five fields (magnification, × 10)
were counted for each membrane.
Immunodetection of Raf-1 and/or Ras Homologs--
Expression
levels of Raf-1 and/or Ras homologs in COS-1 and COS-7 cells were
assessed by Western blot, and expression levels of Ras homologs in
RIE-1 cells were assessed by immunoprecipitation. Briefly, for Western
blots, lysates of COS-1 and COS-7 cells (5 µg) were resolved on 15%
SDS-polyacrylamide gels and transferred to nitrocellulose. Raf-1 and
Ras proteins contained in COS-1 cell lysates were probed with Raf-1 C12
(Santa Cruz) and Myc 9E10.2 antibodies, respectively. GFP-Ras fusion
proteins contained in COS-7 cell lysates were probed with monoclonal
GFP antibody (CLONTECH). Immunoblots were developed
using enhanced chemiluminescence (Pierce). For immunoprecipitations,
G418-selected RIE-1 cells were labeled overnight with 100 µCi/ml
[35S]methionine/cysteine (ICN) and lysed in high
SDS/Tris/radio-immunoprecipitation assay buffer. Ras proteins were
immuno- precipitated from cleared cell lysates with EE antibodies,
resolved on 15% SDS-polyacrylamide gels, and visualized by
fluorography (21, 23).
Differential Activation of Raf-1 by Ras Homologs--
To determine
whether the four Ras homologs might differentially activate Raf-1
in vivo, we transiently co-transfected COS-1 cells with wild
type raf-1 and constitutively active (G12V)
Ha-ras, N-ras, Ki-ras 4A, or
Ki-ras 4B expression plasmids and assessed the activity of
Raf-1 immunoprecipitated from these cells by quantitating MBP substrate
phosphorylation in coupled MEK/ERK2 in vitro kinase assays.
As shown in Fig. 1, A and
B, Ki-Ras 4B activated Raf-1 8.4-, 4.4-, and 2.3-fold more
efficiently than Ha-Ras, N-Ras, or Ki-Ras 4A protein, respectively
(p < 0.005). Importantly, the differential abilities
of the Ras homologs to activate Raf-1 were not due to variations in Ras
homolog or Raf-1 expression levels (Fig. 1C). (Levels of
expression of exogenous Ras and Raf-1 proteins were approximately
5-6-fold and 6-8-fold higher, respectively, than endogenous Ras and
Raf-1 proteins). For instance, although Ki-Ras 4B had an enhanced
ability and Ha-Ras had a reduced ability to activate Raf-1, Raf-1
expression levels were comparable and exogenous Ha-Ras expression was
significantly higher than exogenous Ki-Ras 4B protein expression. (Note
that the small, slower-migrating Ha-Ras band in Fig. 1C
represents non-posttranslationally modified Ha-Ras protein). Likewise,
although Raf-1 levels were comparable and exogenous N-Ras was expressed
at significantly higher levels than exogenous Ki-Ras 4A protein, Ki-Ras
4A activated Raf-1 significantly better than N-Ras protein.
Furthermore, although Ki-Ras 4A expression was slightly lower than
Ki-Ras 4B expression, the relative ability of Ki-Ras 4A
versus Ki-Ras 4B protein to activate Raf-1 did not significantly change when the amount of transfected Ki-ras
4A plasmid DNA was titrated to achieve comparable Ki-Ras 4A and Ki-Ras 4B expression levels (data not shown). Our results indicate, therefore, that the four Ras homologs significantly differ in their abilities to
activate Raf-1 in COS-1 cells. Ki-Ras 4B efficiently activates Raf-1,
and Ki-Ras 4A, N-Ras, and Ha-Ras proteins show progressively decreasing
abilities to activate Raf-1.
Differential Biological Properties of Ras Homologs--
To
determine whether the four Ras homologs might have different biological
functions in vivo, we assessed their relative abilities to
induce transformed foci, enable anchorage-independent growth, or
stimulate cell migration.
For focus-forming assays, NIH 3T3 mouse fibroblast, Rat-1 fibroblast
and RIE-1 rat intestinal epithelial cells were transfected with
constitutively active (G12V) Ha-ras, N-ras,
Ki-ras 4A, or Ki-ras 4B expression plasmids, and
the number of transformed foci that formed after a period of 14-21
days was counted. As indicated in Fig. 2,
the focus-forming abilities of Ha-Ras and Ki-Ras 4A were ~2-2.5-fold
higher than Ki-Ras 4B or N-Ras in NIH 3T3 and Rat-1 cells, and ~8.3-
and 6.3-fold, respectively, higher than Ki-Ras 4B or N-Ras in RIE-1
cells (p < 0.005). (Results are normalized and
indicate focus-forming activity relative to Ha-Ras. Ha-Ras induced an
average of 3365 ± 150, 178 ± 3, and 13 ± 2 foci/µg of transfected DNA in NIH 3T3, Rat-1, and RIE-1 cells,
respectively).
To assess anchorage-independent growth, RIE-1 or Rat-1 cells
transfected with each of the G12V ras homologs were selected in G418 and suspended in reduced-serum soft agar medium. Colony formation was monitored for up to 1 month. As shown in Figs. 2 and
3A, the ability of Ki-Ras 4A
or Ki-Ras 4B to enable RIE-1 cells to grow in soft agar paralleled
their ability to induce transformed foci in these cells. Ki-Ras 4A
efficiently induced foci and enabled soft agar growth, whereas Ki-Ras
4B neither efficiently induced foci nor enabled soft agar growth. In
contrast, although Ha-Ras efficiently induced foci in RIE-1 cells,
Ha-Ras-expressing RIE-1 cells were completely unable to grow in soft
agar. Moreover, although N-Ras demonstrated little ability to induce
foci in RIE-1 cells, N-Ras efficiently enabled RIE-1 cells to grow in
soft agar. As shown in Fig. 3B, in RIE-1 cells, expression
levels of exogenous Ha-Ras, Ki-Ras 4B, and N-Ras proteins were
comparable and slightly higher than exogenous Ki-Ras 4A protein. It is
unlikely, therefore, that differences in protein expression accounted
for the ability of Ki-Ras 4A and N-Ras or inability of Ha-Ras and
Ki-Ras 4B proteins to enable soft agar growth. However, we are unable
to definitively conclude whether there may be differences in the
relative ability of Ki-Ras 4A or N-Ras protein to enable RIE-1 cells to
grow in soft agar, due to the slight differences in expression between these homologs. Interestingly, although only Ki-Ras 4A and N-Ras enabled RIE-1 epithelial cells to grow in soft agar, each of the four
Ras homologs enabled Rat-1 fibroblast cells to grow in soft agar (data
not shown).
For cell migration haptotaxis assays, COS-7 cells were transiently
transfected with G12V GFP-ras fusion constructs,
serum-starved, and loaded into Boyden chambers. Numbers of
GFP-Ras-expressing cells that migrated through polycarbonate membranes
to a collagen-undercoating during a 3 h incubation were counted on
a fluorescence microscope. As demonstrated in Fig.
4A, in comparison to GFP
alone, Ki-Ras 4B efficiently and Ha-Ras minimally induced cell
migration (p < 0.005), but Ki-Ras 4A and N-Ras did not
significantly induce cell migration (p > 0.3). As
shown in Fig. 4B, the relative inability of Ha-Ras, Ki-Ras
4A, or N-Ras to stimulate cell migration was not due to differences in
protein expression. (Levels of expression of exogenous GFP-Ras proteins
were approximately 1-2-fold higher than endogenous Ras proteins).
Furthermore, even when increasing amounts of GFP-ras
constructs were transfected into COS-7 cells, Ki-Ras 4A and N-Ras
continued to show little or no ability to induce cell migration in
comparison to Ki-Ras 4B protein (data not shown). Importantly, similar
migration results were observed with native Ras proteins that were not
fused to GFP (data not shown).
These combined studies clearly indicate that the four Ras homologs have
different biological properties.
Our results indicate that the four human Ras homologs
significantly differ in their abilities to activate the effector
protein, Raf-1, in vivo. Ki-Ras 4B efficiently activates
Raf-1, Ki-Ras 4A is intermediate, and N-Ras and Ha-Ras have
progressively decreasing and inefficient abilities to activate Raf-1.
Our studies also demonstrate that the four Ras homologs can induce
significantly different biological responses in vivo. Ha-Ras
and Ki-Ras 4A induce 6-8 times more transformed foci than Ki-Ras 4B or
N-Ras in RIE-1 cells, but only Ki-Ras 4A and N-Ras enable RIE-1 cells
to undergo anchorage-independent growth in soft agar. Moreover, Ki-Ras
4B efficiently stimulates and Ha-Ras inefficiently stimulates COS-7 cell migration, but N-Ras and Ki-Ras 4A proteins do not.
Although differences in the abilities of the four Ras homologs to
activate Raf-1 were quantitative rather than qualitative, it is likely
that the quantitative differences observed under our "ideal"
experimental conditions significantly underestimate actual differences
between the Ras homologs. For instance, our studies utilized mutant
(G12V) Ras homologs that are constitutively active, and this
constitutive activity might have enabled Ha-Ras and N-Ras proteins to
promiscuously, albeit inefficiently, activate Raf-1. Likewise, because
the exogenous Ras homologs were expressed at significantly higher
levels than their endogenous counterparts, this overexpression might
also have enabled Ha-Ras and N-Ras to promiscuously activate Raf-1.
Furthermore, because each of the exogenous Ras homologs was expressed
in cells individually, the exogenous Ras homologs did not have to
"compete" with each other for available effector proteins. This
lack of competition could also have artifactually enabled some Ras
homologs to activate Raf-1. It is possible, therefore, that under
normal cellular conditions, wild type endogenous Ha-Ras or N-Ras
proteins may have little or no ability to activate Raf-1. We would like
to emphasize, however, that even if each of the four wild type
endogenous Ras proteins can activate Raf-1 in vivo,
differences in their relative abilities to activate Raf-1 could still
enable each of the Ras homologs to induce different biological
responses. For instance, recent studies have shown that low levels of
Raf kinase activity induce proliferation, whereas high levels of Raf
kinase activity induce cell cycle arrest and/or differentiation
(reviewed in Ref. 1). Furthermore, although our studies did not address
the question of whether the homologs might activate Raf-1 with
different time courses, previous studies have shown that
differences in the duration of Raf kinase activation
can also influence whether a cell undergoes proliferation or
differentiation (25, 26).
We do not currently know the mechanism(s) underlying the differential
abilities of the four Ras homologs to activate Raf-1. Raf-1 contains a
C-terminal catalytic domain and an N-terminal regulatory domain that
inhibits its catalytic activity (1, 4, 27). Ras binds to two distinct
regions within the N-terminal regulatory domain. Specifically, the
switch 1 region of Ras (amino acids 30-37) binds to the minimal Ras
binding domain of Raf-1 (amino acids 55-131), and the switch 2 region
of Ras (amino acids 59-76) binds to the cysteine-rich domain (CRD) of
Raf-1 (amino acids 139-184) (1, 4, 27). In current models, Ras is
thought to recruit Raf-1 (and the acidic protein, 14-3-3, which is
bound to the CRD and other sites of Raf-1) to plasma membranes by
binding to the Ras binding domain of Raf-1. Following membrane
recruitment, negatively charged membrane phospholipids, in particular
phosphatidylserine, are believed to competitively displace 14-3-3 from
the CRD of Raf-1. This enables Ras to bind the CRD and ultimately
permits full Raf-1 activation by other factors (such as
membrane-associated tyrosine kinases, serine/threonine kinases, hsp90
and p50 molecular chaperones, and/or the kinase suppressor of Ras,
etc.) (1, 4, 27). Because the four Ras homologs are identical in their switch 1 and switch 2 regions, it is unlikely that differential binding
to the Ras binding domain or CRD of Raf-1 accounts for the discrepant
abilities of the Ras homologs to activate Raf-1. The hypervariable
domain, consisting of the 25 C-terminal amino acids of Ras proteins, is
the only region where the highly homologous Ras proteins substantially
differ from each other. It seems likely, therefore, that residues
within this domain might account for differential Raf-1 activation.
This domain contains a CAAX motif (where C indicates
cysteine, A indicates an aliphatic amino acid, and
X indicates serine or methionine) that signals the Ras
proteins to undergo a series of posttranslational modifications. Each
of the Ras proteins becomes farnesylated, truncated, and
carboxylmethylated at its CAAX residues, and these
modifications are required for Ras proteins to become associated with
plasma membranes (6). In addition, Ha-Ras, N-Ras, and Ki-Ras 4A
proteins become palmitoylated on one or two non-CAAX
cysteine residues within their hypervariable domains (see Fig.
5) (6). Ki-Ras 4B lacks these cysteine
residues and does not become palmitoylated. Rather, Ki-Ras 4B contains a polybasic domain, consisting of six contiguous lysines, within its
hypervariable domain (see Fig. 5). Previous studies have shown that
palmitoylation (Ha-Ras, N-Ras, and Ki-Ras 4A) or a polybasic domain
(Ki-Ras 4B) is required for Ras proteins to become fully membrane
associated (21, 28). Palmitoyl moieties likely facilitate Ha-Ras,
N-Ras, or Ki-Ras 4A membrane association through hydrophobic or van der
Waals interactions, whereas the polybasic domain likely facilitates
Ki-Ras 4B membrane association through ionic interactions with
negatively charged membrane phospholipid head groups. These hypervariable domain differences (and perhaps other hypervariable domain differences) could, therefore, enable each of the four Ras
homologs to bind to distinct regions of the plasma membrane and/or to
distinct membrane target proteins. It is possible, therefore, that
Ki-Ras 4B activates Raf-1 more efficiently than the other Ras homologs
because Ki-Ras 4B recruits Raf-1 to a membrane site where other
components required for Raf-1 activation (such as negatively charged
membrane phospholipids) are co-localized. Similarly, the two contiguous
basic lysines upstream of the CAAX of Ki-Ras 4A (see Fig. 5)
might partially enable Ki-Ras 4A to bind to membrane phospholipids and
account for its intermediate ability to activate Raf-1. Alternatively,
it is possible that hypervariable domain differences enable Ki-Ras 4B
(and to a lesser extent Ki-Ras 4A) to recruit Raf-1 to the membrane
and/or activate Raf-1 more efficiently than the other Ras homologs.
Recent studies have shown that Raf-1 must be complexed with 14-3-3 for
efficient membrane recruitment by Ras (29), and it has been
demonstrated that two basic contiguous residues within the CRD domain
of Raf-1 (Arg-143 and Lys-144) are required for 14-3-3 binding (30). It
is possible, therefore, that Ki-Ras 4B and Ki-Ras 4A activate Raf-1
more efficiently than Ha-Ras or N-Ras because their polybasic residues
bind to the acidic 14-3-3 protein and thereby facilitate Raf-1 membrane
recruitment, and/or their polybasic residues competitively displace
14-3-3 from the CRD of Raf-1 and thus allow their switch 2 regions to bind the CRD and facilitate Raf-1 activation. The recent demonstration that Ki-Ras 4B membrane recruits and activates Raf-1 more efficiently than Ha-Ras is consistent with this possibility (31). Moreover, our
finding that a Ki-Ras 4B mutant that contains six neutral glutamines
substituted for the six contiguous lysines of the polybasic domain of
Ki-Ras 4B is unable to activate Raf-1 also supports this
notion.2
Ki-Ras 4A
>>> N-Ras = Ki-Ras 4B; for anchorage-independent growth, Ki-Ras
4A
N-Ras >>> Ki-Ras 4B = Ha-Ras = no growth; and
for cell migration, Ki-Ras 4B >>> Ha-Ras > N-Ras = Ki-Ras
4A = no migration. Our results indicate that the four Ras homologs
significantly differ in their abilities to activate Raf-1 and induce
distinctly different biological responses. These studies, in
conjunction with our previous report that demonstrated that the Ras
homologs can be differentially activated by upstream guanine nucleotide
exchange factors (Jones, M. K., and Jackson, J. H. (1998)
J. Biol. Chem. 273, 1782-1787), indicate that each of
the four Ras proteins may qualitatively or quantitatively participate in distinct signaling cascades and have significantly different biological roles in vivo. Importantly, these studies also
suggest for the first time that the distinct and likely cooperative
biological functions of the Ki-ras-encoded Ki-Ras 4A and
Ki-Ras 4B proteins may help explain why constitutively activating
mutations of Ki-ras, but not N-ras or
Ha-ras, are frequently detected in human carcinomas.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
, and Nore1
(reviewed in 1-4). In addition, the GTPase-activating proteins, p120
GTPase-activating protein and neurofibromin/NF1-GTPase-activating protein, which inactivate Ras proteins by catalyzing GTP hydrolysis, have also been implicated as Ras effectors (1-4).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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-mercaptoethanol; 5 mM NaF; 0.2 mM Na3VO4; 0.5 µM
okadaic acid) and diluted 1:10 in lysis buffer containing 10% glycerol
and no KCl. Cleared cell lysates (25 µg) were subsequently incubated
with 0.3 µg of Raf-1 C12 antibody (Santa Cruz) for 1 h on ice,
followed by 50 µl of Pansorbin Staph A cells (Calbiochem) for 1 h at 4 °C. Immunoprecipitates were washed successively one time each
in wash buffer 1 (30 mM Tris-HCl, pH 7.5; 0.1% Triton
X-100; 0.2 mM EDTA; 0.3%
-mercaptoethanol; 1 M KCl; 5 µg/ml leupeptin; 1 mM benzamidine;
0.1 mM phenylmethylsulfonyl fluoride; 5 mM NaF;
0.2 mM Na3VO4; 0.5 µM
okadaic acid), wash buffer 2 (same as wash buffer 1 except KCl is
reduced to 0.1 M), and wash buffer 3 (same as wash buffer 1 except KCl is excluded); resuspended in 20 µl of reactivation buffer
(30 mM Tris-HCl, pH 7.5; 0.1% Triton X-100; 0.03% Brij
35; 10 mM MgCl2, 0.3%
-mercaptoethanol; 5 mM NaF; 0.2 mM Na3VO4;
0.5 µM okadaic acid) containing 6.5 µg/ml MEK-1 (Santa
Cruz), 100 µg/ml ERK2 (Santa Cruz), and 0.8 mM ATP; and
incubated/vortexed for 15 min at 37 °C. Reactions were terminated by
the addition of 20 µl of reactivation buffer containing 20 mM EDTA and no MgCl2. Supernatants (10 µl) of
terminated reactions were added to 40 µl of ice-cold reaction buffer
(50 mM Tris, pH 7.5; 0.1 mM EGTA; 12.5 mM MgCl2) containing 16 µg of myelin basic protein (MBP) and 0.125 mM [
-32P]ATP
(106 cpm/nmol), and incubated/vortexed for 30 min at
37 °C. Reactions were terminated by the addition of 50 µl of 2×
SDS-polyacrylamide gel electrophoresis sample buffer, boiled for 3 min,
and resolved on 15% SDS-polyacrylamide gels. The radioactivity
incorporated into MBP was visualized by autoradiography and quantitated
on a PhosphorImager (Molecular Dynamics). PhosphorImager counts due to
Raf-1 (and the empty Ras vector) were subtracted from counts due to
Raf-1 and each of the four Ras homologs.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Fig. 1.
Ki-Ras 4B activates Raf-1 more efficiently
than Ha-Ras, Ki-Ras 4A, or N-Ras. Raf-1 was immunoprecipitated
from COS-1 cells transiently transfected with wild type
raf-1 and constitutively active (G12V) Ha-ras,
Ki-ras 4A, Ki-ras 4B, or N-ras
expression plasmids. The activity of immunoprecipitated Raf-1 was
assessed by quantitating MBP substrate phosphorylation in coupled
MEK/ERK2 in vitro kinase assays. Data shown are
representative of four independent experiments done in triplicate.
A, autoradiogram of MBP phosphorylation. B,
PhosphorImager quantitation of MBP phosphorylation. PhosphorImager
counts shown are arbitrary and represent the relative intensity of
photon emissions. Counts due to Raf-1 alone, in the absence of
exogenous Ras homologs (282 × 102), have been
subtracted. Data shown represent the mean ± S.E. C,
immunoblot of COS-1 cell lysates showing expression levels of Raf-1 and
exogenous Ras homolog proteins.
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Fig. 2.
Ha-Ras and Ki-Ras 4A induce transformed foci
more efficiently than Ki-Ras 4B or N-Ras. NIH 3T3, Rat-1, and
RIE-1 cells were transfected with constitutively active (G12V)
Ha-ras, Ki-ras 4A, Ki-ras 4B,
N-ras, or empty (vector alone) expression plasmids, and the
number of foci that formed after a period of 14 (NIH 3T3) or 21 (Rat-1
and RIE-1) days was counted. Data shown are normalized and represent
the mean ± S.E. of five independent transfections done in
quadruplicate.
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Fig. 3.
Ki-Ras 4A and N-Ras, but not Ha-Ras or Ki-Ras
4B, enable anchorage-independent growth in soft agar. RIE-1 cells
were transfected with constitutively active (G12V) Ha-ras,
Ki-ras 4A, Ki-ras 4B, N-ras, or empty
(vector alone) expression plasmids; selected in G418; and suspended in
reduced-serum soft agar medium. Colony formation was monitored for up
to 1 month. Data shown are representative of four independent
experiments done in duplicate. A, × 10 magnification of
soft agar colonies. B, immunoblot of RIE-1 cell lysates
showing expression levels of exogenous Ras homolog proteins.
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Fig. 4.
Ki-Ras 4B stimulates cell migration more
efficiently than Ha-Ras, Ki-Ras 4A, or N-Ras. COS-7 cells were
transiently transfected with constitutively active (G12V)
Ha-ras, Ki-ras 4A, Ki-ras 4B,
N-ras, or empty (vector alone) GFP fusion expression
plasmids; serum-starved; and loaded into Boyden chambers containing
collagen-undercoated membranes. Numbers of GFP-Ras-expressing cells
that migrated through the membrane to the collagen undercoating after
3 h were counted on a fluorescence microscope. Data shown
represent the mean of two independent experiments done in triplicate.
A, number of cells that migrated per × 10 magnification field. At least five fields were counted for each
membrane. B, immunoblot of COS-7 cell lysates showing
expression levels of exogenous GFP-Ras homolog fusion proteins.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 5.
25 C-terminal amino acids of Ha-Ras, N-Ras,
Ki-Ras 4A, and Ki-Ras 4B proteins. The palmitoylation sites of
Ha-Ras, N-Ras, and Ki-Ras 4A; the polybasic domain of Ki-Ras 4B; and
the CAAX motif of each of the Ras proteins are
indicated.
There was often little or no correlation between the abilities of each of the four Ras homologs to activate Raf-1 in vivo and their relative activities in the three biological assays studied. For instance, although Ki-Ras 4B had the greatest and Ha-Ras had the least ability to activate Raf-1 in COS-1 cells, Ki-Ras 4B had the least and Ha-Ras had the greatest ability to induce transformed foci in NIH 3T3, Rat-1, and RIE-1 cells. Our results with Ki-Ras 4B suggest that Raf-1 activation may not be sufficient for efficient focus formation, and our findings with Ha-Ras suggest that little or no Raf-1 activation may be required for efficient focus formation in the cell lines we employed. Our results are consistent with previous studies that demonstrated that Raf-1 activation is not sufficient or necessary for the induction of transformed foci in some cells (reviewed in Refs. 1-4 and 32). In addition, our studies support the notion that activation of multiple effectors is likely required for full Ras transformation (1-4, 32). Although Ki-Ras 4B activates Raf-1 efficiently, it may inefficiently activate other effectors required for focus formation. Likewise, although Ha-Ras activates Raf-1 inefficiently, it may efficiently activate other effectors capable of inducing focus formation. Similarly, although Ki-Ras 4A moderately activates Raf-1, Ki-Ras 4A likely also activates other effectors required for focus formation.
Interestingly, differences observed between the four Ras homologs
depended on the cell line utilized. For instance, although we
demonstrated that only two of the four Ras homologs, Ki-Ras 4A and
N-Ras, can enable RIE-1 epithelial cells to grow in soft agar, we found
that each of the four Ras homologs enables Rat-1 fibroblast cells to
grow in soft agar, and a previous report (33) showed that chimeric
Ha-Ras, N-Ras, and Ki-Ras 4B proteins enable Rat-2 and NIH 3T3
fibroblast cells to grow in soft agar. These results suggest,
therefore, that the effector(s) activated by a given Ras homolog may
vary in different cell types and/or the effector(s) required to induce
a given biologic response may vary in different cell types. We also
showed that differences between the focus-forming activities of each of
the four Ras homologs were much more marked in RIE-1 epithelial cells
than in NIH 3T3 or Rat-1 fibroblast cells. We found, however, that the
relative abilities of the four Ras homologs to induce transformed foci (Ha-Ras Ki-Ras 4A >>> N-Ras = Ki-Ras 4B) were
consistent among each of the cell lines we employed. Similarly,
although their nonstandard methodology made their results inconclusive,
a previous study (33), utilizing G418-selected Rat-2 cells infected
with ras retroviruses, suggested that chimeric Ha-Ras
proteins might have greater focus-forming activity than chimeric N-Ras
or Ki-Ras 4B proteins. Because the vast majority of human cancers are
of epithelial cell origin, our combined results underscore the crucial importance of using epithelial cells as model systems for
carcinogenesis studies.
Notably, there was often no correlation between the abilities of the Ras homologs to induce focus formation and their abilities to enable anchorage-independent growth. For instance, whereas Ha-Ras efficiently and N-Ras inefficiently induced focus formation in RIE-1 cells, N-Ras, but not Ha-Ras, enabled RIE-1 cells to grow in soft agar. Our results suggest, therefore, that in epithelial cells, the effector(s) required for efficient focus formation may differ from the effector(s) required for anchorage-independent growth. Consistent with this premise, previous studies have shown that Ras effector domain mutants that have lost their ability to bind to some, but not all, of their effectors fail to induce transformed foci, but still enable growth in soft agar (34).
Multiple signaling pathways are believed to play a role in directed
cell migration. For instance, mitogen-activated protein kinase has been
shown to promote cell migration by phosphorylating myosin light chain
kinase (35); Rac, Rho, and Cdc42 facilitate cell motility through their
abilities to reorganize the actin cytoskeleton (36); and
phosphatidylinositol 3-kinase, Ral, and phospholipase C have also
been implicated as potential mediators of cell migration (37, 38).
Because Ki-Ras 4B activates Raf-1 more efficiently than Ha-Ras, N-Ras,
or Ki-Ras 4A, it is possible that Raf-1-induced mitogen-activated
protein kinase activation accounts for the enhanced ability of Ki-Ras
4B to induce cell migration. It should be noted, however, that although
Ki-Ras 4A activates Raf-1 more potently than Ha-Ras, Ha-Ras induces
cell migration significantly better than Ki-Ras 4A. Our results support the notion, therefore, that multiple signaling pathways contribute to
cell migration. Future studies will be required to delineate which
signaling component(s) accounts for the enhanced ability of Ki-Ras 4B
to induce cell migration.
Interestingly, although the Ki-Ras 4A and Ki-Ras 4B proteins are alternative splice variants of the same gene, their biological activities are significantly different. Ki-Ras 4A, but not Ki-Ras 4B, efficiently induces transformed foci and enables anchorage-independent growth, whereas Ki-Ras 4B, but not Ki-Ras 4A, induces cell migration. These different biological properties suggest that in addition to Raf-1, Ki-Ras 4A and Ki-Ras 4B likely differ in their abilities to qualitatively or quantitatively activate other effector proteins. As noted previously, Ki-ras gene mutations are detected in human malignancies much more frequently than N-ras or Ha-ras gene mutations. Because Ki-ras gene mutations occur at codons 12, 13, or 61, Ki-ras gene-encoded Ki-Ras 4A and Ki-Ras 4B proteins are both constitutively active. If, therefore, Ki-Ras 4A and Ki-Ras 4B proteins activate different, but cooperative, effector pathways, the combination of these constitutively active signaling pathways could account, at least in part, for the increased frequency of Ki-ras gene mutations in human cancers. For instance, Ki-Ras 4A could activate effector pathways that transform cells and enable anchorage-independent growth, whereas Ki-Ras 4B could activate effector pathways that enable cell motility and thereby facilitate angiogenesis, invasion, and ultimately metastasis. Consistent with this premise, a recent study, utilizing Ras effector domain mutants, has shown that the effector(s) required for metastasis is distinct from the effector(s) required for tumorigenicity (39). Thus, whereas the constitutive activity of only one of the two Ki-Ras proteins might not be sufficient to efficiently enable a malignant tumor to develop, cooperativity of the two Ki-Ras proteins (in conjunction with other oncogene/tumor suppressor gene abnormalities) could efficiently enable the development of a clinically detectable malignancy. Importantly, although Ki-ras 4B was previously reported to be transcribed 10-20-fold more than Ki-ras 4A (40, 41), a recent study (9) demonstrated that in some tissues, such as the colon, transcription of Ki-ras 4A is almost comparable to Ki-ras 4B.
In summary, our results demonstrate that the four human Ras proteins
significantly differ in their abilities to activate Raf-1 and also vary
in their abilities to induce transformed foci, enable anchorage-independent growth, and stimulate cell motility. It will be
important, in future studies, to assess the relative abilities of each
of the four Ras homologs to activate other Raf kinase family members
(A-Raf and B-Raf), as well as other putative effector proteins. In
addition, it will be important to further define the biological
differences between the four Ras homologs and determine their
biological roles in vivo.
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ACKNOWLEDGEMENTS |
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We thank Dawn Warnock, Rachel Molander, and Val Hill for technical assistance; Jiing-Dwan Lee for helpful advice; Deborah Morrison for raf-1 cDNA; Paul Dent for MEK-1 protein; and Monica Cochrane for typing the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant CA 54298 (to J. H. J.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Immunology,
IMM-12, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La
Jolla, CA 92037. Tel.: 619-784-8748; Fax: 619-784-8150; E-mail:
jjackson{at}scripps.edu.
2 J. Jackson and S. Wong, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: GEF, guanine nucleotide exchange factor; GFP, green fluorescent protein; MBP, myelin basic protein; CRD, cysteine-rich domain; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase /ERK kinase.
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REFERENCES |
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