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(Received for publication, May 21, 1996)
From the ABSTRACT The pleckstrin homology (PH) domain is an ~100
amino acid structural motif found in many cellular signaling molecules,
including the Dbl oncoprotein and related, putative guanine nucleotide
exchange factors (GEFs). Here we have examined the role of the Dbl PH
(dPH) domain in the activities of oncogenic Dbl. We report that the dPH
domain is not involved in the interaction of Dbl with small GTP-binding
proteins and is incapable of transforming NIH 3T3 fibroblasts. On the
other hand, co-expression of the dPH domain with oncogenic Dbl inhibits
Dbl-induced transformation. A deletion mutant of Dbl that lacks a
significant portion of the PH domain retains full GEF activity, but is
completely inactive in transformation assays. Replacement of the PH
domain by the membrane-targeting sequence of Ras is not sufficient for
the recovery of transforming activity. However, subcellular
fractionations of Dbl and Dbl mutants revealed that the PH domain is
necessary and sufficient for the association of Dbl with the Triton
X-100-insoluble cytoskeletal components. Thus, our results suggest that
the dPH domain mediates cellular transformation by targeting the Dbl
protein to specific cytoskeletal locations to activate Rho-type small
GTP-binding proteins.
INTRODUCTION The cytoskeletal-associated Dbl oncoprotein transforms NIH 3T3
cells (1) by activation of signaling pathways involving Rho-type
GTP-binding proteins (2). Proto-Dbl is a 115-kDa
cytoskeletal-associated protein that is found in brain, adrenal glands,
and gonads (1). Oncogenic activation occurs as an outcome of an
amino-terminal truncation of proto-Dbl, where a recombination event
fuses about 10 kDa of an unidentified human gene product (from
chromosome 3) on to the carboxyl-terminal half of Dbl (residues
498-925). The oncogenic Dbl protein contains at least two putative
signaling motifs. The first is a region of 176 amino acids
(residues 498-674) that was originally found to share significant
homology with the Saccharomyces cerevisiae
cell-division-cycle protein, Cdc24, and the breakpoint cluster region
protein, Bcr1 (3). This region, referred to
as the Dbl homology (DH) domain, has been shown to be essential both
for the transformation activity of oncogenic Dbl and for its ability to
act as a GEF by stimulating the guanine nucleotide exchange activity of
Cdc42Hs (4, 5). The second putative signaling motif is the pleckstrin
homology (PH) domain (6, 7) and includes residues 703-812. Although PH
domains appear to be relatively poorly conserved, both NMR and x-ray
crystallographic studies of the PH domains of pleckstrin, dynamin, and
spectrin indicate that they adopt a common three-dimensional structural
motif (8, 9, 10, 11).
Over the past few years, a growing family of oncogene products and
other growth regulatory proteins have been shown to contain a DH domain
in tandem with a PH domain. In addition to Cdc24 and Bcr, these include
the Vav, Ost, Ect-2, Lbc, Lfc, and Dbs oncoproteins (12, 13, 14, 15, 16, 17) and the
activators of the Ras proteins, Sos (18), and Ras-GRF (19). All
indications from previous studies are that the DH domain will form a
binding site and in many cases contain GEF activity for Rho-like
GTP-binding proteins (8, 9, 13, 14, 20, 21, 22). However, less is known
about the roles of the PH domains. In the present study, we have used
the Dbl oncoprotein as a model to examine the role of the PH domain in
cellular transformation and GEF activity.
EXPERIMENTAL PROCEDURES Transfection assays were done
on duplicate cultures by adding 0.001, 0.01, 0.1, and 1 µg of DNA to
the recipient NIH 3T3 cells using the Ca2+-phosphate
precipitation method (3). Foci (focus forming units) were scored 14 days after transfection, and the results were calculated as number of
foci/pmol of DNA. The results listed in Fig. 1 and shown in Fig.
2C are the mean values of three transfection assays. Growth
in soft agar was examined as described by Ron et al.
(3).
Control NIH 3T3 and
different NIH 3T3 transfectants were lysed and fractionated into
cytosolic (S), Triton X-100-solubilized membrane fractions (T), and
Triton X-100-insoluble fractions (I) as described by Graziani et
al. (1). Cells were labeled with [35S]methionine and
[35S]cysteine for 3 h at 37 °C. Specific Dbl
products were detected by immunoprecipitation using anti-Dbl-2
antibodies (3), electrophoresed through a 12% polyacrylamide gel and
autoradiographed. For the detection of the PH domains (e.g.
Figs. 2B and 4D, below), cells were
immunoprecipitated with anti-Flag M5 antibodies and electrophoresed
through a 15% polyacrylamide gel followed by immunoblotting with
anti-Flag M5 antibodies.
The
[3H]GDP dissociation assays were carried out as described
previously (4, 5). In Fig. 3A, the amounts of GST-Dbl and
GST-DH (see Fig. 1) purified from Sf9 insect cells were estimated by
Coomassie Blue staining after SDS-polyacrylamide gel electrophoresis.
~200 nM of GST-Dbl or GST-DH were incubated with 1 µg
of RhoA protein preloaded with [3H]GDP in 100 µl of
reaction buffer at room temperature, and 16-µl aliquots were diluted
into 5 ml of ice-cold termination buffer (20 mM Tris-HCl,
pH 7.4, 10 mM MgCl2, and 100 mM
NaCl) at various time points. In Fig. 3B, 1 µg of
[3H]GDP-bound RhoA was incubated with 2 µM
GST, 2 µM GST-PH (a fusion protein containing GST and the
pleckstrin homology domain from the Dbl protein), 300 µM
GST-Dbl, or 300 µM GST-Dbl and 2 µM GST-PH
in a 100-µl reaction mixture.
RESULTS AND DISCUSSION To investigate the role of the PH domain in cellular
transformation mediated by the oncogenic Dbl protein, we analyzed
several Dbl mutants for transforming activity in NIH 3T3 cells (Fig.
1). We found that while the transforming capability of a
deletion mutant containing primarily the DH and PH domains (designated
as pMA4 in Fig. 1) was similar to that of the Dbl oncogene
product, neither the DH domain nor the PH domain (pdDH and
pdPH, respectively) alone showed any detectable effects on
the growth of 3T3 fibroblasts. However, when Dbl and pdPH were
co-expressed in NIH 3T3 cells (dbl+pFlag/PHdbl, Fig.
2C), we observed a significant reduction of
the transforming activity by Dbl. Co-expression of Dbl with a
Flag-tagged PH domain of the Dbl-related Vav oncoprotein (12), on the
other hand, showed effects comparable with those obtained with the
pFlag/neo vector control (Fig. 2C), even though it appeared
to be more highly expressed (Fig. 2B, lane 2) than the PH
domain of Dbl (Fig. 2B, lane 1). The level of expression of
the Dbl oncoprotein was essentially equivalent in all cases (Fig.
2A).
To further confirm the selective inhibition of Dbl-induced
transformation by the dPH domain, we used a second mammalian expression
vector, pKH3 (23), to express the PH domain from either Dbl, Vav, or
from the related yeast Cdc24 protein (20), together with the Dbl
oncogene product. As shown in Fig. 2C, the expression of the
dPH domain inhibited the focus-forming activity of oncogenic Dbl by
~40%, whereas co-expression of Dbl with the PH domain of Vav
(pKH3/PHvav) had little effect. Mass populations of these transfected
cells also were examined for their ability to display
anchorage-independent growth. We observed that cells co-expressing Dbl
and the dPH domain lost the ability to grow in soft agar (data not
shown). In some cases, we found that expression of a Flag-tagged PH
domain of Vav caused some inhibition of Dbl-induced growth in soft
agar, suggesting that the Vav PH domain (perhaps when expressed at
sufficient levels) was capable of competing with the PH domain of Dbl
for a cellular target. However, it is likely that the Vav PH domain is
a weak competitor, since we often observed no detectable effects with
either the Flag-tagged protein or when expressing the PH domain of Vav
from the pKH3 vector. We also have found no detectable effects on Dbl
transformation when expressing the PH domain from Cdc24 (data not
shown). Mass cultures of Dbl transfectants expressing the dPH domain
also displayed a less transformed phenotype compared with Dbl
transfectants alone or compared with cells co-expressing Dbl and the PH
domain of Vav (data not shown). Taken together, these results
suggest that the PH domain of Dbl behaves as a selective antagonist of
Dbl-induced transformation, possibly by binding to a saturable
and specific ligand in cells.
Previously we have shown that the DH domain alone is sufficient for the
GEF activity for Cdc42Hs (5). Since oncogenic Dbl also stimulates the
guanine nucleotide exchange activity of Rho, we examined whether the
Dbl domain is sufficient for stimulating the activation of Rho. To do
this, we compared Rho-GEF activities of approximately equal amounts
(~200 nM) of insect cell-expressed, purified GST-Dbl and
GST-DH domain. No significant differences were observed for the
abilities of the GST-Dbl and GST-DH to stimulate [3H]GDP
dissociation from RhoA (Fig. 3A). These
results suggest that the PH domain does not contribute to the GEF
function of Dbl. This is further reinforced by the results in Fig.
3B, which show that the addition of excess Escherichia
coli recombinant PH domain to GEF assay mixtures containing
[3H]GDP-bound RhoA and recombinant GST-Dbl has no
detectable effect on the time-course of GST-Dbl-stimulated
[3H]GDP dissociation from RhoA. The GST-PH domain, alone,
also shows no ability to stimulate [3H]GDP dissociation
from RhoA (compared with GST alone). Similar results were also obtained
with [3H]GDP-bound Cdc42Hs (data not shown). Thus, the
dPH domain is not involved in the interactions of Dbl with RhoA and
Cdc42Hs or in the direct regulation of the GEF catalytic activity of
the DH domain.
The membrane association of
We have reported previously that significant portions of both proto-
and oncogenic Dbl are localized to the Triton X-100-insoluble fractions
of transfected NIH 3T3 cells, suggesting an association with the
cytoskeletal matrix (1). To address the possible role of the dPH domain
in mediating this pattern of localization for the Dbl protein, stable
transfectants of Dbl and Dbl deletion mutants (Fig. 1) were subjected
to subcellular fractionation. The crude membrane fractions (P100) of
the cells were solubilized either by 1% Triton X-100 or by treatment
with 0.1% SDS and 0.25% sodium deoxycholate. Anti-Dbl
immunoprecipitates revealed that a percentage of both the intact
oncogenic Dbl protein and a truncation mutant pMA4 associated with the
Triton X-100-insoluble fractions of cells (designated by I
in Fig. 4, B and C). The amounts of oncogenic Dbl
and pMA4 that were present in the Triton X-100-insoluble fraction
typically varied between 50 and 70% of the total detectable protein,
although in some cases (Fig. 4B) the percentage of oncogenic
Dbl in this fraction was less than 50%. However, the DH domain of Dbl,
which lacks transforming ability, was localized exclusively to the
cytosolic fraction (designated S in Fig. 4C).
When cells expressing the Flag-tagged PH domains were subjected to
similar fractionation, the PH domains were found associated with the
Triton X-100-insoluble fractions (Fig. 4D). These results
suggest that the dPH domain is directly responsible for the association
of oncogenic Dbl with the Triton X-100-insoluble cytoskeletal fraction
and thus may serve to target the catalytic DH domain to the
cytoskeleton.
We have reported previously that the DH domain is responsible for Dbl
GEF function and is required for Dbl transforming activity (3, 5).
Here, we demonstrate that while the dPH domain does not seem to be
involved in the interactions of Rho-type small GTP-binding proteins
with Dbl, it is essential for Dbl transforming activity. Thus, our
present findings establish that both the DH and PH domains are required
for the cellular function of Dbl. Indeed, the minimum structural unit
(pMA4) of oncogenic Dbl conferring complete transforming activity just
encompasses the DH domain and the PH domain. The finding that plasma
membrane-targeting of Dbl is not sufficient to confer transforming
activity, coupled with the requirement of the dPH domain as the
necessary and sufficient element for association of the Dbl protein
with the Triton X-100-insoluble component, suggests that the function
of the PH domain resides in its ability to target the catalytic DH
domain to the cytoskeletal matrix. Whether this targeting function
holds for other members of Dbl-related GEF family proteins remains to
be seen. However, based on the observation that the PH domains of
Dbl-related molecules Vav and Cdc24 do not act effectively as
inhibitors of Dbl-induced transformation, it is an attractive
possibility that different members of the family of Dbl-related
proteins may be targeted by their PH domains to distinct cellular
locations to activate various Rho-type GTP-binding proteins, in
response to different extracellular stimuli such as epidermal growth
factor, platelet-derived growth factor, lysophosphatidic acid, and
bradykinin. This may also explain the finding that substitution of a
membrane-targeting (i.e. Ras-farnesylation) sequence for the
PH domain of Lfc restored its transformation capability (32), whereas
this substitution did not restore transforming activity to a Dbl
protein that just contains the DH domain. It may be that Lfc needs to
be targeted to the plasma membrane to optimally couple to other protein
components in its signaling pathway while Dbl needs to be targeted to a
cytoskeletal location.
The identity of the ligand(s) that binds to the PH domain of oncogenic
Dbl will represent an important focus of future studies. It seems
likely, that given the hypervariable nature of the putative
ligand-binding cleft in the PH domains that have thus far been
identified (33), a complex diversity of ligands may exist that are
responsible for mediating the actions of various PH domain-containing
signaling molecules, including Dbl and related regulatory molecules of
small GTP-binding proteins.
FOOTNOTES Acknowledgment We thank Cindy Westmiller for expert
secretarial assistance.
REFERENCES
Volume 271, Number 32,
Issue of August 9, 1996
pp. 19017-19020
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
COMMUNICATION:
§,
and
Department of Pharmacology, Cornell
University, Ithaca, New York 14853-6401 and the ¶ Laboratory of
Cellular and Molecular Biology, National Institute of Health,
Bethesda, Maryland 20892
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
Acknowledgment
REFERENCES
Fig. 1.
Schematic representations of oncogenic Dbl
and different mutants of Dbl used in this study. The abilities of
these constructs to serve as GEFs for Rho and Cdc42Hs and to transform
NIH 3T3 cells are summarized. pdDH represents the Dbl homology domain
and pdPH is the pleckstrin homology domain. The abbreviation ffu
represents focus forming units. 100% is 3 × 105
foci/pmol DNA. pDHF is a construct in which the PH domain sequences
downstream from residue 750 are replaced with the carboxyl-terminal 16 amino acids of Ha-Ras, which include both the palmitylation and
farnesylation sites (black rectangle). pdHF* is a construct
encoding the DH domain of Dbl and the carboxyl-terminal 16 amino acids
of Ha-Ras, except that the cysteine, which is normally farnesylated,
has been changed to serine (cross-hatched rectangle). The
GEF activity for these constructs has not been determined
(N/A). The pdDH, pdDHF, and pdDHF* were subcloned from
dbl by the polymerase chain reaction and inserted into the
mammalian expression vector pZipneo. The GEF activities were measured
as described (5) by the in vitro nitrocellulose filter
binding assay either using the anti-Dbl immunoprecipitates from the NIH
3T3 transfectants (proto-Dbl, Dbl, and pMA4) or using the insect cell
expressed peptides (DH and PH).
Fig. 2.
Expression of the PH domain inhibits
Dbl-induced transformation in NIH 3T3 cells. A, detection of
the Dbl oncoprotein (using an anti-Dbl antibody) in NIH 3T3
transfectants. Lane 1 represents cells expressing the Dbl
oncoprotein. Lane 2 represents cells co-expressing Dbl and
the Dbl PH domain (dPH). Lane 3 represents cells
co-expressing Dbl and the Vav PH domain. Lane 4 is a
control, i.e. cells transfected with the plasmid (pFlag)
used to express the PH domains. B, detection of the PH
domains of Dbl and Vav (using M5 anti-Flag antibody) in NIH 3T3 cells.
Lane 1 represents cells co-expressing Dbl and the Dbl PH
domain (dPH). Lane 2 represents cells co-expressing Dbl and
the Vav PH domain. Lane 3 is a control (cells transfected
with the pFlag plasmid). C, effects of the PH domains of Dbl
and Vav on Dbl-induced foci-formation. pFlag/PHdbl and pKH3/PHdbl
represent the mammalian expression vectors encoding the Dbl PH domain
and pFlag/PHvav and pKH3/PHvav are the expression vectors encoding the
Vav PH domain. The results shown represent the average of three
independent experiments.
Fig. 3.
The PH domain is not directly involved in the
regulation of the GEF activity of the Dbl oncoprotein. A,
comparison of the abilities of oncogenic Dbl (
) and the DH domain of
Dbl (
) to stimulate [3H]GDP dissociation of RhoA. The
dissociation of [3H]GDP from RhoA, alone, is depicted by
(
). B, effect of the PH domain on the kinetics of
Dbl-stimulated [3H]GDP dissociation from RhoA. represents Dbl-stimulated GDP dissociation in the absence of the PH
domain and 
represents Dbl-stimulated GDP dissociation in the
presence of the PH domain. and 
represent the corresponding
controls for RhoA in the absence of Dbl.
ARK and spectrin has been attributed to
their PH domains (24, 25). The PH domains of ARK, BTK, PLC
,
IRS-1, and Ras-GRF have been shown to bind to plasma
membrane-associated subunits of the heterotrimeric G-proteins
(26, 27), and they all behave as antagonists of G-mediated
signaling (28). Recent evidence also suggests that PH domains from many
signaling molecules including ARK and Ras-GAP can bind to specific
phospholipids, namely phosphatidylinositol 4,5-bisphosphate
(PIP2) (29). These findings raised the possibility that the PH
domain mediates the membrane targeting of oncogenic Dbl. It has been
shown that the introduction of a membrane-targeting sequence into the
Ras GEFs, Cdc25 and Sos (30, 31), was sufficient to activate Ras, and
more recently, that the addition of a membrane-targeting sequence in
place of the PH domain of the Lfc oncoprotein was able to restore full
transformation activity (32). Thus, we examined whether the
substitution of the dPH domain with a membrane-targeting sequence
enabled the DH domain of Dbl to induce transformation. A chimeric
molecule containing the DH domain (residues 498-756) fused to the Ras
membrane-targeting farnesylation signal sequence (designated
pdHF in Fig. 1) was constructed and assayed for
focus-forming activity in NIH 3T3 cells. This chimera was expressed at
a comparable level to oncogenic Dbl and a percentage (10-20%) of the
total chimeric molecules was targeted to the membrane surface
(i.e. the Triton X-100 solubilized fraction (T)
in Fig. 4A). However, this did not restore
transforming activity to the DH domain (Fig. 1). Although, one possible
explanation is that the amount of the chimera expressed at the membrane
surface was not sufficient to stimulate a transforming signal, this
does not seem likely based on what we have observed regarding the range
of expression of oncogenic Dbl that will yield cellular transformation
(34).
Fig. 4.
The PH domain mediates the cytoskeletal
association of the Dbl oncoprotein. A, the membrane
attachment signal from Ha-Ras targets the DH domain to the Triton
X-100-soluble fraction from cell membranes. pZIPneo/DHF (see Fig. 1)
represents the construct encoding the DH domain of Dbl and the
carboxyl-terminal 16 amino acids of Ha-Ras, including the palmitylation
and farnesylation sites. pZIPneo/DHF* represents the construct encoding
the DH domain of Dbl and the carboxyl-terminal 16 amino acids of Ha-Ras
(except that the cysteine which serves as the farnesylation site has
been changed to serine). S represents the soluble fraction,
T is the Triton X-100-soluble fraction from membranes, and
I is the Triton X-100-insoluble fraction. B,
oncogenic Dbl is associated with the Triton X-100-insoluble fraction of
cells. C, fractionations of the pMA4 and DH domain
transfectants. D, fractionation of cells expressing the Dbl
PH domain. The data shown in A-C were obtained by
immunoprecipitating the Dbl proteins with the anti-Dbl antibody from
cells that were labeled with [35S]methionine and
[35S]cysteine. The data shown in D represent
immunoblots using the anti-Flag M5 antibody.
§
Present address: Dept. of Biochemistry, University of Tennessee,
Memphis, TN 38163.
To whom correspondence should be addressed.
''
Present address: Laboratory of Molecular Biology, Istituto G. Gaslini, via 5 Maggio, Genova Quarto, Italy.
1
The abbreviations used are: Bcr, break point
cluster region protein; DH, Dbl homology; PH, pleckstrin homology;
GAPs, GTPase-activating proteins for low molecular mass GTP-binding
proteins; GRF, guanine nucleotide-releasing factor; GEFs, guanine
nucleotide-exchange factor for low molecular mass GTP-binding proteins;
GST, glutathione S-transferase.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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K. M. Kriauciunas, M. G. Myers Jr., and C. R. Kahn Cellular Compartmentalization in Insulin Action: Altered Signaling by a Lipid-Modified IRS-1 Mol. Cell. Biol., September 15, 2000; 20(18): 6849 - 6859. [Abstract] [Full Text]
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 D. D. Billadeau, S. M. Mackie, R. A. Schoon, and P. J. Leibson The Rho Family Guanine Nucleotide Exchange Factor Vav-2 Regulates the Development of Cell-mediated Cytotoxicity J. Exp. Med., August 8, 2000; 192(3): 381 - 392. [Abstract] [Full Text] [PDF]
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 E. Liao, B. Paw, L. Peters, A Zapata, S. Pratt, C. Do, G Lieschke, and L. Zon Hereditary spherocytosis in zebrafish riesling illustrates evolution of erythroid beta-spectrin structure, and function in red cell morphogenesis and membrane stability Development, January 12, 2000; 127(23): 5123 - 5132. [Abstract] [PDF]
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 J.-P. Mira, V. Benard, J. Groffen, L. C. Sanders, and U. G. Knaus Endogenous, hyperactive Rac3 controls proliferation of breast cancer cells by a p21-activated kinase-dependent pathway PNAS, January 4, 2000; 97(1): 185 - 189. [Abstract] [Full Text] [PDF]
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R. Arudchandran, M. J. Brown, M. J. Peirce, J. S. Song, J. Zhang, R. P. Siraganian, U. Blank, and J. Rivera The Src Homology 2 Domain of Vav Is Required for Its Compartmentation to the Plasma Membrane and Activation of c-Jun NH2-terminal Kinase 1 J. Exp. Med., January 3, 2000; 191(1): 47 - 60. [Abstract] [Full Text] [PDF]
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I. P. Whitehead, Q. T. Lambert, J. A. Glaven, K. Abe, K. L. Rossman, G. M. Mahon, J. M. Trzaskos, R. Kay, S. L. Campbell, and C. J. Der Dependence of Dbl and Dbs Transformation on MEK and NF-kappa B Activation Mol. Cell. Biol., November 1, 1999; 19(11): 7759 - 7770. [Abstract] [Full Text] [PDF]
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N. Movilla and X. R. Bustelo Biological and Regulatory Properties of Vav-3, a New Member of the Vav Family of Oncoproteins Mol. Cell. Biol., November 1, 1999; 19(11): 7870 - 7885. [Abstract] [Full Text] [PDF]
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A. D. Ma and C. S. Abrams Pleckstrin Induces Cytoskeletal Reorganization via a Rac-dependent Pathway J. Biol. Chem., October 1, 1999; 274(40): 28730 - 28735. [Abstract] [Full Text] [PDF]
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S. N. Prokopenko, A. Brumby, L. O'Keefe, L. Prior, Y. He, R. Saint, and H. J. Bellen A putative exchange factor for Rho1 GTPase is required for initiation of cytokinesis in Drosophila Genes & Dev., September 1, 1999; 13(17): 2301 - 2314. [Abstract] [Full Text]
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R. H. Daniels, F. T. Zenke, and G. M. Bokoch alpha Pix Stimula |
