From the
The lbc oncogene is tumorigenic in nude mice,
transforms NIH 3T3 fibroblasts, and encodes a Dbl homology domain found
in several transforming gene products including the dbl oncogene product. While both lbc- and
dbl-transformed NIH 3T3 foci exhibited a comparable gross
appearance, lbc-transformed cell morphology was clearly
distinct from that of dbl-transformed cells. Given these
differences, we investigated the biochemical activity and target
specificity of the Lbc oncoprotein both in vivo and in
vitro. Here we show that Lbc associates specifically with the
GTP-binding protein Rho in vivo, but not with the Ras, Rac, or
Cdc42Hs GTP-binding proteins, and that recombinant, affinity-purified
Lbc specifically catalyzes the guanine-nucleotide exchange activity of
Rho in vitro. Consistent with an in vivo role for Lbc
in Rho regulation, we further demonstrate that micro-injected
onco- lbc potently induces actin stress fiber formation in
quiescent Swiss 3T3 fibroblasts indistinguishable from that induced by
Rho. Finally, lbc-induced NIH 3T3 focus formation is inhibited
by co-transfection with a rho dominant-negative mutant. These
results strongly indicate that the lbc oncogene encodes a
specific guanine nucleotide exchange factor for Rho and causes cellular
transformation through activation of the Rho signaling pathway.
The Rho-related GTP-binding proteins function as molecular
switches in a diversity of cellular signaling pathways, many of which
influence the cellular cytoskeleton, as well as cell polarity and
motility
(1, 2, 3) . Members of this family of
GTP-binding proteins include the Rho proteins (RhoA, RhoB, RhoC, and
RhoG) and the Rac1, Rac2, Cdc42, and TC10 proteins. As is the case for
all members of the Ras superfamily of GTP-binding proteins, the
GTP-binding/GTPase cycles of the Rho-related proteins are tightly
controlled, with guanine nucleotide exchange factors (GEFs)
Recently, the product of the dbl oncogene
(5, 6) was shown to serve as a GEF for the human Cdc42 (Cdc42Hs)
and Rho proteins
(7) . There now appear to be a number of
proteins, suspected to be involved in cell growth regulation, that
share significant sequence similarity with oncogenic Dbl within an
In this study, we now demonstrate that
the newly identified Dbl family member, the lbc oncogene
product
(14) , acts as a highly specific GEF for the Rho
proteins. In addition, we show that Lbc specifically associates with
Rho in vivo and potently induces actin stress fiber formation
in fibroblasts, similar to activated forms of Rho. Finally, we
demonstrate that Lbc-induced cellular transformation can be blocked by
a dominant-negative Rho mutant that would be predicted to bind with
high affinity to GEFs.
The lbc cDNA predicts a 424-amino acid protein and
encodes a DH (Dbl homology) domain that is present in oncogenic Dbl and
a number of other potential growth regulatory proteins
(14) .
Since the Lbc DH domain shares the highest degree of sequence
similarity (
Members of the Dbl family share the common structural feature of the
DH domain, a region of
The observation that Lbc potently induces actin re-organization also
provides the first direct demonstration of a DH domain-encoding protein
which regulates cytoskeletal changes in vivo, and provides a
physiological role for Lbc that is corroborated by the biochemical data
presented here. Members of the Rho subgroup of Ras-related GTP-binding
proteins are known to be essential in yeast
(26, 27, 28) and are involved in the organization of the mammalian
cytoskeleton
(1, 18, 29) . Various lines of
evidence also have suggested that the loss of regulation of Rho
GTP-binding proteins can result in some degree of cellular
transformation
(30, 31) . The current studies showing
that the potent oncogene lbc encodes a specific GEF for Rho
suggest a mechanism by which a growth regulatory protein can directly
activate Rho and contribute to the full complement of effects
associated with cellular transformation, namely cytoskeletal changes
that result in the loss of contact-growth inhibition.
We thank Dr. Alessandra Eva for
dbl-transfected cells, Drs. Roya Khosravi-Far and Channing J.
Der for pZipneo/ rho and pZipneo/N19 rho plasmids,
Peggy Atkinson for guidance in preparing insect cell-expressed
recombinant GST-Lbc, and Cindy Westmiller for expert technical
assistance.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
catalyzing their conversion to the GTP-bound active-state
and GTPase-activating proteins ensuring their return to an inactive,
basal state through the stimulation of GTP hydrolysis
(4) .
250-amino acid region termed the Dbl homology (DH) domain. These
include the breakpoint-cluster region (Bcr) protein
(8) , the
Saccharomyces cerevisiae cell-division-cycle protein Cdc24
(9) , the T-lymphoma invasion gene product Tiam-1
(10) ,
and the vav (11) , ect2 (12) , ost (13) , and lbc (14) oncogene products. The
DH domain has been shown to be essential both for the transforming
activity and GEF activity of oncogenic Dbl
(7) , which has led
to the suspicion that each of the Dbl-related proteins will have GEF
activity. However, thus far, this has only been demonstrated for the
Dbl
(7) and Ost
(13) oncoproteins, and for Cdc24, which
is a specific GEF for Cdc42
(9) . Moreover, it has been
difficult to obtain in vivo data that supports a role for the
Dbl family members as GEFs.
Cell Culture and Western Blot Analysis
Stable
lbc-transformed NIH 3T3 transfectants were derived as
previously detailed
(14) and grown on tissue culture dishes in
Dulbecco's modified Eagle's medium and 10% calf serum.
dbl-transfected NIH 3T3 cells were obtained from A. Eva
(5) . Flag: lbc-transfected NIH 3T3 cells (confluent)
were washed once with ice-cold buffer A containing 20 m
M
Tris-HCl, pH 7.4, 100 m
M NaCl, 1 m
M EDTA, 1
m
M phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin and
aprotinin before lysing with 0.4% Triton X-100 in buffer A for 30 min
at 4 °C. The clarified supernatant was either directly subjected to
SDS-polyacrylamide gel electrophoresis (lysate) or incubated with 3
µg of M5 anti-Flag monoclonal antibody (IBI) for 1 h, followed by 2
h of additional incubation, after addition of 3 µg of rabbit
anti-mouse antibody precoupled to anti-rabbit IgG-agarose beads
(Sigma). Duplicate samples in which the M5 antibody was omitted were
used as controls. The immunocomplexes were collected by centrifugation
and washed three times with buffer A. The immunoprecipitates were
blotted either with M5 antibody or with anti-Ras, -RhoA, -Rac1, and
-Cdc42Hs antibodies (Santa Cruz Biotech), respectively. The primary
antibodies were detected with anti-mouse, anti-rabbit, or anti-rat
immunoglobulins coupled to horseradish peroxidase. The bands were
visualized by the ECL system (Amersham Corp.).
Expression of Glutathione S-Transferase Fusion
Proteins
The GST-RhoA, GST-Cdc42Hs, GST-Rac1, GST-TC10, and
GST-RhoG were expressed in E. coli as described previously
(7) . The GST-Ha-Ras construct and RhoB were kind gifts from
Drs. H. Maruta (University of Melbourne) and J. Settleman (Harvard
Medical School). RhoC was expressed as a GST fusion protein by
inserting the cDNA encoding RhoC (using the polymerase chain reaction)
into the pGEX-2T vector (Pharmacia Biotech Inc.). The GST fusion
proteins were purified as described
(15) and used without
removal of the GST for the in vitro GDP/GTP exchange assays. A
1.3-kilobase BamHI- EcoRI fragment of the cDNA
containing the entire coding sequence for lbc was ligated with
a 600-base pair XbaI- BamHI fragment of the cDNA
encoding the GST protein and inserted into the baculovirus transfer
vector pVL1392. Recombinant virus was generated according to Ref. 9.
Sf9 cells were harvested 70 h after infection and the cell pellets were
resuspended in ice-cold buffer A supplemented with 0.4% Triton X-100,
after a quick freeze-thaw. The resuspended cells were homogenized with
a Dounce homogenizer and then centrifuged for 15 min at 4 °C in a
microcentrifuge. The supernatants were further incubated with
glutathione-agarose beads (Sigma) for 2 h, and the beads were washed
four times before the GST fusion proteins were eluted with 10
m
M glutathione in buffer A. Excess glutathione was removed
from the GST-Lbc and GST-Dbl proteins by dialysis.
GDP/GTP Exchange Assays
The GDP dissociation and
GTP binding assays were carried out at 24 °C as described
(7) . The quantities of GTP-binding proteins and GST, GST-Lbc,
GST-Dbl, or GST-Ras-GRF used for each individual experiment are
indicated in the figure legends.
Microinjections of Swiss 3T3 Fibroblasts
pSR
lbc 9a cDNA
(14) was microinjected at 100 ng/µl
into the nuclei of serum-starved quiescent Swiss 3T3 cells. After 40 h,
cells were fixed in 4% paraformaldehyde, permeabilized in 0.2% Triton
X-100, treated with 1 mg/ml sodium borohydride, and blocked with 10%
fetal calf serum, 0.1% bovine serum albumin. Actin filaments were
detected with TRITC-labeled phalloidin as described previously
(1) . Lbc expression was detected by incubating with a
1:250 dilution of rabbit anti-Lbc antibody followed by a 1:300 dilution
of fluorescein isothiocyanate-labeled goat anti-rabbit IgG antibody. C3
transferase at 0.1 mg/ml and rat IgG at 0.5 mg/ml were co-microinjected
into cells that had been microinjected 40 h previously with lbc cDNA. Cells were then incubated for an additional 10 min. C3- and
rat IgG-injected cells were identified using a 1:100 dilution of
Cascade Blue-labeled goat anti-rat IgG antibody. Cells were
photographed using a Zeiss Axiophot microscope with Kodak TMY-400 film.
Focus Formation Assay
NIH 3T3 cells were
transfected using calcium phosphate precipitation according to standard
procedures detailed previously
(14) with 15 ng of pSR
lbc 9a cDNA either alone or together with 300 ng of the
rhoA mutant encoding asparagine at position 19 (instead of
threonine) or with the wild type rhoA cDNA in pZipneo in the
presence of 20 µg of genomic NIH 3T3 carrier DNA/dish. After day
14, the plates were stained with crystal violet and foci were counted.
24% identity,
42% homology) with the Dbl
oncoprotein, we compared the morphology of lbc-transfected
cells with that of dbl-transfected cells. lbc transfectants exhibited an obvious transformed phenotype of an
intersecting bundle- and stretching spindle-form (Fig. 1, compare A and B), and subconfluent cells had a higher cytoplasm to
nucleus ratio than dbl-transfected cells and displayed
striking ``feet-like'' projections (see arrows in
Fig. 1C) similar to that described for cells
overexpressing the Rho GTP-binding protein
(16) .
dbl-overexpressing cells showed a clearly distinctive
morphology, having a more rounded shape and growing to a higher density
in a more detached manner; the characteristic giant multinuclear cells
(5) were observed (Fig. 1 D). In contrast, while
multinuclear lbc-transformed cells were also noted, giant
syncytia were not as readily apparent. These subtle yet distinct
morphological differences suggested that the mechanism of lbc transformation may be different from that caused by dbl.
Figure 1:
Distinct morphologies of lbc-
versus dbl-transformed NIH 3T3 fibroblasts. Figure
shows parental NIH 3T3 fibroblasts ( A),
lbc-transfected cells ( B), subconfluent
lbc-transfected cells ( C), dbl-transfected
cells ( D). Magnification: 10 for panels A,
B, and D;
20 for panel C.
Given that Dbl and Cdc24 have been recently shown to serve as
guanine nucleotide exchange factors (GEFs)
(7, 9) , we
set out to determine whether the biological activity of the Lbc
oncoprotein is manifested by its ability to interact with low molecular
weight GTP-binding proteins. To test whether Lbc can directly bind to
Ras or Rho family GTP-binding proteins in vivo, we utilized a
stable NIH 3T3 transfectant cell line transformed by carboxyl-terminal
Flag-tagged lbc cDNA in pSRneo plasmid. Western blotting of
transfectant cell lysates detected the presence of a 49-kDa Lbc-Flag
band and various small GTP-binding proteins such as Ha-Ras, Rho, Rac,
and Cdc42Hs (Fig. 2; first lane in all panels). When
the anti-Flag antibody was used toimmunoprecipitate Lbc from
the lysates, and the precipitates were Western blotted with anti-Ras,
-Rho, -Rac, and -Cdc42Hs antibodies, only Rho was observed to
co-precipitate with Lbc (Fig. 2, second lane in
all panels). Typically, 10% of the total Rho and
60% of the
Lbc-Flag protein detected in the whole cell lysates were present in the
immunoprecipitates. Thus, despite the high degree of sequence
similarity (50-70%) among the different members of the Rho
subfamily that were examined, Lbc appears to exclusively associate with
the Rho GTP-binding protein in vivo.
Figure 2:
Association between Lbc and RhoA in
vivo. Western blot analysis of lbc-transfected NIH 3T3
cell lysates and anti-Flag immunoprecipitates probed with anti-Flag,
anti-Ras, anti-Rho, anti-Rac, and anti-Cdc42Hs antibodies as described
under ``Experimental Procedures.'' The results shown are
representative of three independent
experiments.
We next examined
whether Lbc acts as a GEF toward Rho and other members of the Rho
subfamily. To do this, the transforming Lbc protein was expressed as a
glutathione S-transferase (GST) fusion product in Sf9 insect
cells using a baculovirus expression system, and purified by
glutathione-agarose affinity chromatography (Fig. 3 A, lane 2). The ability of purified GST-Lbc to stimulate
guanine-nucleotide exchange on Rho was measured with the recombinant
RhoA protein. The rate of dissociation of [H]GDP
from RhoA was stimulated
10-fold by the GST-Lbc protein
(Fig. 3 B). Similarly, the rate of
[
S]GTP
S binding, which directly reflects
the GDP-GTP
S exchange activity of RhoA, was also stimulated
10-fold by GST-Lbc (Fig. 3 C). Therefore, Lbc does
act as a GEF. Lbc also showed comparable effects on the guanine
nucleotide exchange activities of the recombinant RhoB and RhoC
proteins (data not shown). Fig. 3 D compares the
abilities of purified GST-Lbc to stimulate GDP dissociation from
recombinant Ha-Ras, RhoA, and its close homologs Rac1, Cdc42Hs, TC10,
and RhoG. Unlike Dbl, which stimulates the rate of GDP dissociation
from both Cdc42Hs and RhoA
(7) without affecting Ras, Rac1, and
TC10, Lbc specifically accelerated the rate of GDP dissociation from
RhoA while showing no detectable effects on GDP dissociation from
Cdc42Hs or from Ras, Rac1, or TC10 (Fig. 3 D). The Ras-GRF,
which specifically catalyzes Ras GDP/GTP exchange
(17) , was
used as a positive control for stimulated GDP dissociation from Ras,
while oncogenic Dbl served as a positive control for a Cdc42Hs-GEF. We
also tested for possible regulatory effects of Lbc on the GTPase
activities of the above GTP-binding proteins, and found that Lbc had no
effect on their GTP hydrolytic activities (data not shown). Taken
together, these results indicate that oncogenic Lbc functions as a
specific GEF for Rho by stimulating the release of its tightly bound
GDP.
Figure 3:
Evidence that recombinant Lbc functions as
a guanine-nucleotide exchange factor for Rho. A, expression
and purification of Lbc and Dbl as GST fusion proteins.
SDS-polyacrylamide gel electrophoresis (9% polyacrylamide) of the
purified GST-Lbc and GST-Dbl prepared from Sf9 insect cell lysates
infected with recombinant viruses encoding the Lbc and Dbl proteins.
B, effects of purified GST-Lbc on the kinetics of GDP
dissociation from Rho. 2 µg of recombinant GST-RhoA were preloaded
with [H]GDP and incubated with 5 µg of GST
( open squares) or 1 µg of GST-Lbc ( filled
triangles) in reaction buffer for the indicated time before
termination of the reactions by the nitrocellulose filter binding
method. C, effects of purified GST-Lbc on the kinetics of GTP
binding. 5 µg of GST ( open squares) or 1 µg
GST-Lbc ( filled triangles) were added to the GDP-bound
GST-RhoA (2 µg) in a reaction mixture containing
[
S]GTP
S as described. D,
specificity of Lbc-stimulated GDP dissociation from the Ras and
Rho-type GTP-binding proteins. 2 µg of various recombinant
GST-GTP-binding proteins were incubated with 5 µg of GST ( solid
bars), 1 µg of GST-Lbc ( dark striped bars), or 1
µg of GST-Dbl ( light striped bars) before termination of
the reaction after 10 min. 1 µg of GST-Ras-GRF ( light hatched bar) was used as a control for assaying
(stimulated) GDP dissociation from Ras.
In mammalian cells, RhoA regulates actin stress fiber formation
and focal adhesion assembly following growth factor stimulation
(1, 18) . Consequently, the above results would suggest
that Lbc may induce the same cytoskeletal changes as Rho. In order to
test this, the nuclei of serum-starved quiescent Swiss 3T3 cells were
microinjected with onco- lbc cDNA in the same mammalian
expression vector used for focus formation. lbc expression
(Fig. 4 A) was found to be associated with the formation of
actin stress fibers (Fig. 4 B), a response typical of Rho
stimulation
(1, 18) . In addition, microinjection of the
Rho inhibitor C3 transferase
(19) eliminated stress fibers in
lbc-expressing cells (see Fig. 4 D). These
in vivo results demonstrate that Lbc activates a Rho-mediated
response.
Figure 4:
Effects of lbc and rho expression and C3 transferase on actin stress fiber formation and
focus formation. Panel A shows the expression of the
lbc oncogene product, as detected by immunofluorescence with
an anti-Lbc antibody, and panel B shows the formation
of actin stress fibers in the same cells as detected with
rhodamine-labeled phalloidin (see ``Experimental
Procedures''). Panels C and D represent
the same comparisons except that the cells have been micro-injected
with C3 transferase. E, the dominant-negative mutant RhoT19N
(designated as N19rho) inhibits lbc focus forming
activity. Overexpression of wild type RhoA (designated wt rho) shows no effect on lbc-induced focus
formation. The bars represent the results of three separate
experiments in which 4 dishes/group were
transfected.
Based on analogy with the Ras protein
(20) , we
further predicted that the dominant-negative RhoA mutant, RhoA-T19N,
where the threonine at position 19 is changed to an asparagine, would
bind to a Rho-GEF ( i.e. Lbc), and thereby prevent Lbc from
activating endogenous Rho proteins and inhibit Lbc-mediated
transformation of NIH 3T3 cells. The results presented in
Fig. 4E support this prediction. Co-expression of the
lbc oncogene with RhoA-T19N caused up to 80% reduction in the
number of foci induced by lbc, whereas no effect was observed
when lbc was co-expressed with the cDNA encoding wild type
RhoA (Fig. 4 E). Thus, these results are consistent with the
findings that the Lbc and Rho proteins interact functionally in
vivo and that Lbc-induced transformation proceeds through its
ability to stimulate the guanine nucleotide exchange activity of Rho.
250 amino acids, and there is considerable
evidence that the DH domain serves as a functional unit for GEF
activity for Rho subfamily GTP-binding proteins. For example, the
dbl oncogene product has in vitro GEF activity for
the Cdc42Hs and RhoA proteins
(7) , and Cdc24 has been shown to
be the GEF for the S. cerevisiae Cdc42 protein
(9) .
However, the specific roles of the DH domains in other members of this
family are not clear. The ect2 oncogene product binds to the
Rac and Rho proteins in vitro (12) , but thus far has
not been shown to serve as a GEF. The vav oncogene product
reportedly serves as a Ras-GEF
(21) ; however, this is
controversial because the best documented evidence for Ras-GEF activity
is displayed by the S. cerevisiae Cdc25 protein
(22) ,
and the Sos
(23, 24) and brain Ras-GRF proteins
(17) , and is associated with their encoded CDC25 domains, which
are clearly distinct from the DH domain. At present, the DH domains
within the Sos, Ras-GRF, and Bcr proteins have not been associated with
any specific activity. While RhoA appears to be the target for both Lbc
and Dbl GEF activity, their mutually exclusive tissue expression
( lbc is expressed in hematopoietic cells, muscle, heart and
lung (Ref. 14); dbl is expressed in adrenal gland, testes, and
fetal brain (Ref. 25)) preclude an overlapping function of these two
oncogenes in vivo. Thus, the ability of the Lbc oncoprotein to
bind to Rho and stimulate its guanine nucleotide exchange activity
provides the first demonstration of an interaction between a DH
domain-containing protein and a low molecular mass GTP-binding protein
that is specific, can occur in vivo, and has an impact on the
transformation capability of an oncogene, as illustrated by the results
of co-transfection with the dominant-negative rho mutant.
S, guanosine 5`-3- O-(thio)triphosphate.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.