From the Department of Pharmacology, the
** Department of Biochemistry and Biophysics, and the
§ Lineberger Comprehensive Cancer Center, The University of
North Carolina, Chapel Hill, North Carolina 27599 and the
Department of Microbiology and Molecular Genetics, University of
Medicine and Dentistry of New Jersey, New Jersey Medical School,
Newark, New Jersey 07103
Received for publication, January 6, 2003, and in revised form, February 28, 2003
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ABSTRACT |
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Dbl family members are guanine nucleotide
exchange factors specific for Rho guanosine triphosphatases
(GTPases) and invariably possess tandem Dbl (DH) and pleckstrin
homology (PH) domains. Dbs, a Dbl family member specific for Cdc42 and
RhoA, exhibits transforming activity when overexpressed in NIH 3T3
mouse fibroblasts. In this study, the PH domain of Dbs was mutated to
impair selectively either guanine nucleotide exchange or
phosphoinositide binding in vitro and resulting
physiological alterations were assessed. As anticipated, substitution
of residues within the PH domain of Dbs integral to the interface with
GTPases reduced nucleotide exchange and eliminated the ability of Dbs
to transform NIH 3T3 cells. More interestingly, substitutions within
the PH domain that prevent interaction with phosphoinositides yet do
not alter in vitro activation of GTPases also do not
transform NIH 3T3 cell and fail to activate RhoA in vivo
despite proper subcellular localization. Therefore, the PH domain of
Dbs serves multiple roles in the activation of GTPases and cannot be
viewed as a simple membrane-anchoring device. In particular, the data
suggest that binding of phosphoinositides to the PH domain within
the context of membrane surfaces may direct orientations or
conformations of the linked DH and PH domains to regulate GTPases activation.
The Rho family GTPases1
are an essential subset of the Ras superfamily of small molecular
weight GTPases. Like Ras, Rho family GTPases cycle between GDP- and
GTP-bound forms. When GDP-bound, Rho proteins are inactive and do not
functionally couple to their downstream effectors. However, when
GTP-bound, Rho GTPases elicit profound effects on the organization of
the actin cytoskeleton, in addition to tightly regulating the
activation state of transcription factors such as the serum response
factor, c-Jun, and NF- Members of the Dbl family of oncoproteins act as GEFs exclusively for
Rho GTPases (RhoGEFs) and, like constitutively activated members of Rho
GTPases, can exhibit potent transformation potential within various
cell types upon overexpression or constitutive activation (4-6). Dbl
family members invariably contain a Dbl homology (DH) domain in tandem
with an adjacent, carboxyl-terminal pleckstrin homology (PH) domain.
However, outside of this region, Dbl-related proteins share little
sequence conservation and typically possess a variety of
protein-signaling domains, presumably reflecting diversity in
regulation and cellular function.
The invariant positioning of PH domains immediately carboxyl-terminal
to DH domains strongly implies a unique functional coupling. As
demonstrated by several complementary studies, DH-associated PH domains
are essential components of Dbl family activation of Rho family
GTPases. Truncation of all or part of the PH domains of Dbl (7), Dbs
(8), Lfc (9), and Lsc (10) results in the complete loss of cellular
transformation. Replacing the PH domains of Lfc and Dbs with a
membrane-targeting sequence restores at least partial transforming
activity, indicating a role in membrane targeting for these PH domains,
presumably through interaction with phosphoinositides, i.e.
low affinity (micromolar KD), nonspecific ligands
for the vast majority of PH domains (11). In addition to possibly
mediating the translocation of Dbl family proteins to cellular
membranes, phosphoinositides may also potentiate or suppress the
exchange activity of some Dbl family proteins in solution (12-14);
however, this does not appear to be a general mechanism of regulation
(15). Moreover, recent evidence indicates that PH domains associated
with DH domains can accelerate the catalytic exchange of nucleotides on
Rho family GTPases independent of phosphoinositides. For example, a
DH/PH fragment of Trio catalyzes nucleotide exchange within Rac
~200-fold better than the isolated DH domain (16).
The PH domain of Dbs makes direct contacts to Cdc42, and these
interactions are necessary for efficient guanine nucleotide exchange
in vitro (17). In this study, we define functional roles for
the PH domain of Dbs in directly mediating cellular transformation
associated with downstream signaling events, especially the in
vivo activation of GTPases. We found that mutations within the PH
domain of Dbs, previously shown to be necessary for exchange in
vitro, completely eliminated the ability of Dbs to transform NIH
3T3 cells. More interestingly, mutations within the PH domain that
prevent binding of phosphoinositides, but not in vitro
nucleotide exchange by Dbs, also prevent Dbs from activating RhoA
in vivo and do not allow the transformation of NIH 3T3 cells
by a normally highly oncogenic form of Dbs. These effects were not
caused by the mislocalization of Dbs, because none of the mutations
affected subcellular targeting as assessed by cellular fractionations
and indirect immunofluorescence. Therefore, the PH domain of Dbs must be functioning in some capacity other than a simple membrane anchor dependent upon binding phosphoinositides to alter the gross subcellular distribution of Dbs leading to GTPase activation. Indeed, the data are
more consistent with phosphoinositide binding being required to
direct orientation or conformations of the invariantly associated DH
and PH domains with respect to cellular membranes and membrane-resident GTPases for regulated guanine nucleotide exchange.
Molecular Constructs--
pAX142-dbs-HA6 and
pCTV3H-dbs-HA6 contain cDNAs that encode
fragments of murine Dbs fused to an HA epitope tag and include the Dbs DH/PH domain along with amino- and carboxyl-terminal flanking regions (residues 525-1097) (18). pCTV3H-dbs-HA8 encodes an HA epitope-tagged Dbs DH domain (residues 525-833), whereas
pCTV3P-dbs-HA7 encodes the Dbs DH domain fused to the plasma
membrane-targeting sequence (GCMSCKCVLS) of H-Ras (Dbs DH plus
CAAX).
GST-C21 contains the Rho·GTP binding domain of Rhotekin (19).
pEGFP (Clontech) used for immunofluorescence
studies contains GFP under the control of a cytomegalovirus promoter.
The Dbs DH/PH domain (murine, residues 623-967), fused to a
carboxyl-terminal hexa-histidine tag, was expressed in
Escherichia coli from pET-28a (Novagen) (17). A pET-21a
(Novagen) expression construct was used to bacterially express human
placental Cdc42 (residues 1-188, C188S) (17). Human RhoA (residues
1-190, C190S), fused to an amino-terminal hexa-histidine tag, was
expressed from pProEX-HT (Invitrogen). The PH domain of
PLC- Protein Expression and Purification--
Protein expression in
stably transfected NIH 3T3 cells was determined by Western blot
analysis as described previously (9). Protein was visualized with
Enhanced Chemiluminescence reagents (Amersham Biosciences). Protein
expression and purification of Dbs DH/PH domains, Cdc42(C188S)
and RhoA(C190S) from E. coli, were performed as described
previously (17, 20).
Guanine Nucleotide Exchange Assays--
Fluorescence
spectroscopic analysis of N-methylanthraniloyl (mant)-GTP
incorporation into bacterially purified Cdc42 and RhoA was carried out
using a PerkinElmer Life Sciences LS 50B spectrometer at 25 °C.
Exchange reaction assay mixtures, containing 20 mM Tris, pH
7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM dithiothreitol, and 100 µM mant-GTP
(BIOMOL), and either 1 or 2 µM (as indicated) of either Cdc42 or RhoA protein, were prepared and allowed to equilibrate with
continuous stirring. After equilibration, Dbs DH/PH domain proteins
were added at 200 nM, and the rates of nucleotide loading (kobs) of Rho GTPases were determined by
monitoring the decrease in Cdc42 or RhoA tryptophan fluorescence
( Cell Culture, Transfection, and Transformation Assays--
NIH
3T3 cells were maintained in Dulbecco's modified Eagle medium (high
glucose) supplemented with 10% bovine calf serum (NIH 3T3, JRH,
Lenexa, KS). Primary focus formation assays were performed in NIH
3T3 cells exactly as described previously (21). Briefly, NIH 3T3 cells
were transfected by calcium phosphate coprecipitation in conjunction
with a glycerol shock. Focus formation was scored at 14 days. NIH 3T3
cells that stably express pCTV3H, pCTV3H-dbs-HA6, or the
pCTV3H versions of Dbs mutants were generated by calcium phosphate
coprecipitation followed by selection for 14 days in growth medium
supplemented with hygromycin B (200 µg/ml). Multiple drug-resistant
colonies (>100) were pooled together to establish stable cell lines.
All assays for transformation were performed in triplicate.
Docking of Ins(4,5)P2 onto the PH Domain of
Dbs--
Potential lipid binding pockets on the surface of the Dbs PH
domain were predicted using the SiteID option of SYBYL (Tripos Inc.). A
coordinate file for the phosphoinositol ring of phosphatidylinositol 4,5-bisphosphate (Ins(4,5)P2) was created using SYBYL. All
docking procedures utilized the DOCK suite of programs (version 4.0, I. D Kuntz, University of California at San Francisco) and were carried out in a manner previously reported (22). Briefly, a molecular surface
for each potential binding pocket was generated using the surface
calculation algorithm MS (23) and used as an input for generating
space-filling spheres using Sphgen. A scoring grid encompassing
each pocket plus an additional radius of ~8 Å was calculated using
the grid. Parameters used for docking Ins(4,5)P2 into the
Dbs PH domain were standard DOCK parameters for the single anchor
search method using torsion drive and torsion minimization. Residues
846-852 in the disordered Phosphoinositide Binding Assays--
The ability of
His6-tagged Dbs proteins to bind to phosphatidylinositol
4,5-bisphosphate was assessed by an enzyme linked immunosorbent assay
(ELISA) utilizing a 96-well microtiter plate containing a various
amounts of diC6-modified phosphatidylinositol 4,5-bisphosphate (Echelon Biosciences Inc.). Protein stock solutions were diluted to 10 µM in buffer C (20 mM
Tris, pH 7.5, 100 mM NaCl, and 5% glycerol) before adding
50 µl of each protein to the wells. 100 µl of an anti-His antibody
(Santa Cruz Biotechnology) diluted 1:500 in buffer C was then incubated
with each well, followed by 100 µl of an horseradish
peroxidase-conjugated sheep anti-mouse IgG (Amersham Biosciences)
diluted 1:1000 in buffer C. All incubations were performed for 1 h
at 18 °C, and the wells were extensively washed with buffer C after
each incubation step. Protein-lipid interactions were detected with the
horseradish peroxidase substrate o-phenylenediamine (Sigma),
and absorbances were measured at 490 nm.
Dbs binding to small unilamellar vesicles (SUVs) containing
phosphatidylinositol 4,5-bisphosphate was measured by surface plasmon
resonance using a BIAcore 2000 instrument. SUVs (containing by molar
fraction 80% dipalmitoyl phosphatidylcholine, 17% dipalmitoyl phosphatidylserine, 3% phosphatidylinositol 4,5-bisphosphate, and
0.1% N-biotinylated dipalmitoyl phosphatidylethanolamine) were prepared by sonicating lipids into 20 mM Hepes (pH
7.5) and 150 mM NaCl. SUVs were then captured on an SA5
chip (BIAcore) via a biotin-streptavidin interaction. Equal amounts of
SUVs were immobilized on each respective flow cell, whereas an empty
flow cell was maintained to control for nonspecific binding. 25 µl of
various Dbs proteins in 20 mM Hepes (pH 7.5) and 150 mM NaCl were injected onto the flow cells at 50 µM and followed by 100 µl of buffer. Experiments were
performed at 25 °C with a flow rate of 100 µl/min. Raw sensorgrams
from each experiment were aligned, and the signal due to binding the
empty flow cell was subtracted from each curve using the software
BIAevaluation 3.0 (24).
RhoA Activation Assays--
The Rho binding domain of Rhotekin
(GST-C21) (19) was expressed as a GST fusion in BL21(DE3) cells and
immobilized by binding to glutathione-coupled Sepharose 4B beads
(Amersham Biosciences). The immobilized GST-C21 was then used to
precipitate GTP-bound RhoA from NIH 3T3 cell lysates. Cells were washed
in cold phosphate-buffered saline and then lysed in 50 mM
Tris-HCl, pH 7.4, 2 mM MgCl2, 100 mM NaCl, 10% glycerol, 1% Nonidet P-40, 1 µg/ml
leupeptin, 1 µg/ml pepstatin, 1 µg/ml aprotinin, and 1 µg/ml
phenylmethylsulfonyl fluoride. Cell lysates were then cleared by
centrifugation at 10,000 × g for 5 min at 4 °C.
Lysates used for affinity purification were normalized for endogenous
Rho levels. Affinity purifications were carried out at 4 °C for
1 h, washed three times in an excess of lysis buffer, and then
analyzed by Western blot. RhoA was detected by a monoclonal antibody
(sc-418, Santa Cruz Biotechnology).
Immunofluorescence--
NIH 3T3 cells were plated on coverslips
and then transiently cotransfected with 1 µg of pEGFP
(Clontech) and 3 µg of either pAX142 or
pAX142-dbs-HA6 harboring the PH domains mutations by calcium
phosphate precipitation. Cells were fixed with 3.7% formaldehyde (in
PBS) for 10 min, permeabilized, and blocked in 0.1% Triton X-100, 3%
BSA in PBS for 30 min. Coverslips were then incubated in a humidity
chamber with an anti-HA mouse monoclonal antibody (BAbCO) for 1 h
in 0.1% Triton X-100 with 0.1% BSA, washed in PBS, and then incubated
with red-fluorescent Alexa Fluor 488-conjugated goat anti-mouse IgG
(0.1% Triton X-100, 0.1% BSA, Molecular Probes) for 30 min in the
dark. Coverslips were washed in PBS and mounted on glass slides using
FluorSave reagent (Calbiochem). Cells were viewed with an Olympus IX50
inverted confocal microscope, and images were captured using the
optronics digital charge-coupled device camera system.
Membrane Fractionation Analyses--
Mass populations of NIH 3T3
cells stably expressing Dbs-HA6 or Dbs-HA6 harboring a PH domain
mutation were washed with ice-cold PBS and resuspended in cold HYPO
buffer (10 mM Tris, pH 7.4, 1 mM
MgCl2, 5 µg/ml leupeptin, 5 µg/ml aprotinin, 5 µg/ml
pepstatin, 1 mM phenylmethylsulfonyl fluoride). Lysates
were then homogenized, harvested with HYPO buffer supplemented with
0.15 M NaCl, and then centrifuged at 38,000 rpm for 30 min
at 4 °C. The supernatant (cytosolic fraction) was removed, and the
pellet (particulate fraction) was resuspended in HYPO buffer
supplemented with 0.15 M NaCl. The protein
concentrations of the total, cytosolic, and membrane fractions were
determined with a bicinchoninic acid protein assay kit (Pierce). 30 µg of protein for each fraction was resolved by SDS-PAGE, transferred
to Immobilon-P membranes (Millipore), and probed with anti-HA epitope
antibody (BAbCO).
The PH Domain of Dbs Is Integral to the Interface with Rho GTPases
Necessary for Guanine Nucleotide Exchange and Cellular
Transformation--
The recently reported structure of a fragment of
Dbs in complex with Cdc42 highlights a
crucial role for the PH domain in promoting an extensive set of interactions between Dbs and Cdc42 necessary for efficient guanine nucleotide exchange in vitro
(Fig. 1A) (17). In particular, Tyr-889 within
The in vitro exchange assays utilizing mutants of Dbs and
Cdc42 strongly suggest that the PH domain of Dbs, acting in concert with the DH domain, is necessary to fully activate Rho family GTPases
within cells. To further assess the physiological function of the PH
domain of Dbs in activating Rho GTPases, we introduced a subset of
the previously studied mutations into a transforming version of Dbs
(Dbs-HA6) (8) and measured the capacities of the mutated proteins to
transform NIH 3T3 cells (Fig. 1C). Dramatically, whereas
Dbs-HA6 possesses potent transformation activity, either H814A or Y889F
completely eliminates the focus-forming activity of Dbs-HA6 in NIH 3T3
cells. Similarly, K885A, which is also within the PH domain,
significantly reduces transformation by Dbs. Because transformation of
Dbs depends upon activation of RhoA in NIH 3T3 cells (25), these data
suggest that the PH domain of Dbs is required for direct activation of
Rho family proteins, irrespective of membrane targeting. These effects
are not due to loss of affinity for phosphoinositides by Dbs, because
Dbs(H814A), Dbs(Y889F), and Dbs(K885A) bind to phosphatidylinositol
4,5-bisphosphate similar to wild-type Dbs (Fig.
2, B and C).
Mapping the Interface within the PH Domain Necessary for Binding
Phosphoinositides--
Typically, the majority of pleckstrin homology
domains bind phosphoinositide headgroups with low affinity and
specificity. For instance, the PH domain of Dbs promiscuously binds
several multiphosphorylated phosphoinositides with low affinity,
including phosphatidylinositol 3,4-bisphosphate and
phosphatidylinositol 4,5-bisphosphate (15). Primary sequence analysis
and structural homology comparisons suggest Dbs binds phosphoinositides
similarly to other PH domains largely through residues in and
surrounding the
To determine if regions comprising RhoA Activation and Cellular Transformation Require Engagement of
Phosphoinositides by the PH Domain--
To assess if phosphoinositide
binding to the PH domain is critical for Dbs transformation of NIH 3T3
cells, we placed each mutation into the Dbs-HA6 construct and compared
transformation potentials in primary focus formation assays (Fig.
3A). Strikingly, except for
the low level of foci formation exhibited by Dbs(K874E), transformation
was completely eliminated by all other mutations within the pocket of
the PH domain responsible for binding phosphoinositides. Although exact
identification of the endogenous phosphoinositide targeted by Dbs
awaits more detailed analyses, these data resoundingly highlight that
phosphoinositide binding by the PH domain is essential for Dbs-mediated
transformation.
Dbs transformation of NIH 3T3 cells is closely correlated to the
activation of endogenous pools of RhoA and not Cdc42 (25). Because the
mutations within the PH domain destroy transforming activity in primary
focus assays, an associated decrease in the levels of activated RhoA in
these cells is expected. To measure endogenous levels of activated
RhoA, the Rho-binding domain of Rhotekin was used to precipitate
GTP-bound RhoA from lysates of NIH 3T3 cells expressing either
wild-type Dbs-HA6 or Dbs-HA6 harboring the previously characterized
mutations within the PH domain (Fig. 3B). Although Dbs-HA6
exhibited robust activation of RhoA, activation of RhoA was not
detectable in cells expressing the mutant versions of Dbs that were
previously shown to be defective in binding phosphatidylinositol 4,5-bisphosphate and transformation. The loss of transformation and
RhoA activation was not a result of poor expression or stability of the
various Dbs mutants, because the expression levels for all Dbs proteins
were found to be equivalent (Fig. 3B, lower
panel). These data demonstrate that cellular transformation of NIH
3T3 cells is dependent upon RhoA activation and both processes require Dbs to functionally engage phosphoinositides.
However, the formal possibility exists that the observed loss of
in vivo transforming activity and RhoA activation results from reduction of the inherent GEF activity of Dbs and not the loss of
phosphoinositide binding upon introducing substitutions within the Dbs
PH domain. To determine the functional consequences of substitutions
within the lipid binding site of Dbs upon guanine nucleotide exchange,
we analyzed the ability of each mutant to exchange Cdc42 and RhoA
in vitro (Fig. 4 and Table
II). As expected, none of the mutations
within the PH domain of Dbs were detrimental to Dbs-stimulated exchange
of Cdc42 or RhoA, and both the K874E and K892E mutations within Dbs
actually resulted in a modest, but reproducible, increase in exchange
activity of the GEF (Fig. 4, B and D, and Table
II). In summary, removing the ability of Dbs to interact with
phosphoinositides results in the complete loss of both transforming
ability as well as the ability of Dbs to activate endogenous pools of
RhoA.
Proper Subcellular Localization of Dbs Does Not Require the PH
Domain to Bind Phosphoinositides--
It is widely assumed that PH
domains generally promote association of their host proteins to
membranes dependent upon specific phosphoinositides. However, only
high affinity interactions of PH domains with phosphoinositides are
capable of inducing efficient membrane localization (11), and the
relatively low affinity between the PH domain of Dbs and
phosphatidylinositol 4,5-bisphosphate (15) argues against a critical
role for phosphoinositides in localizing Dbs to cellular
membranes. To assess potential linkage between the binding of
phosphoinositides to the PH domain and the subcellular localization of
Dbs, the previously characterized mutations within the PH domain shown
to abrogate binding of phosphoinositides were assessed for affects on
the cellular distribution of Dbs determined by membrane fractionation
(Fig. 5A). Contrary to the straightforward idea of the PH domain as a simple membrane-tethering device, none of the mutations within the PH domain grossly altered the
subcellular distribution of an extended fragment of Dbs (Dbs-HA6) stably expressed in NIH 3T3 cells and containing both DH and PH domains
as well as flanking sequences. The lack of observable changes in
membrane localization of the Dbs mutants were not due to the
insensitivity of our assay, because a membrane-targeted version of the
Dbs DH domain (DH-CAAX) was nearly entirely associated with
the insoluble, membrane fraction. In contrast, a non-targeted DH domain
is distributed largely within the cytoplasm of NIH 3T3 cells as
previously reported (18). These data demonstrate that, although the PH
domain and surrounding regions are necessary for the recruitment of Dbs
to cellular membranes, this recruitment is independent of the need for
the PH domain to bind phosphoinositides.
Results arising from the cellular fractionations are
supported by more detailed immunofluorescence studies (Fig.
5B). When transiently expressed in NIH 3T3 cells, Dbs-HA6
localizes predominantly in membrane structures that coincide with
lamellipodia and dense cytoplasmic pockets of indeterminate identity.
Importantly, the entire panel of mutations within the PH domain
previously tested by cellular fractionations similarly have no effect
on subcellular localization; Dbs-HA6(K892E) is shown as a
representative example. Similarly, stable expression of the panel of
mutant proteins shows no alterations in subcellular distribution
relative to wild-type Dbs, although staining is weaker and more diffuse
(data not shown). Fragments of Dbs, truncated before the PH
domain (wild-type Dbs DH, Fig. 5A) as well as the isolated
PH domain,2 are predominantly
cytosolic; therefore, regions carboxyl-terminal of the PH domain are
likely to direct subcellular localization of Dbs. Future studies will
test this idea. Given that the entire panel of mutants is defective in
binding phosphoinositides through the PH domain yet retains wild-type
ability to localize within cells as determined by both subcellular
fractionation and immunofluorescence, it seems unequivocal that
localization of Dbs to cellular compartments is independent of any
requirement of the PH domain to bind phosphoinositides.
The invariant positioning of PH domains immediately
carboxyl-terminal to DH domains strongly implies a unique functional
interrelationship. Recent structures of several DH/PH fragments bound
to Rho GTPases highlight a highly conserved mechanism of exchange by DH
domains (15, 17, 26). However, there is mounting evidence that PH domains associated with DH domains can cooperate in the exchange process. For example, relative to the isolated DH domain, the DH/PH
fragment of Trio is more efficient at catalyzing nucleotide exchange
upon Rac1 in vitro (16). Similarly, DH/PH fragments of p115
RhoGEF, Dbl, and Dbs are more proficient GEFs relative to their
isolated DH domains (17, 27, 28).
Importantly for Dbs, the underlying mechanism for the PH
domain-associated increase in the rate of nucleotide exchange has been
revealed within the structure of Dbs·Cdc42. The structure shows that
the PH domain of Dbs interacts with Cdc42 through interactions within
Pleckstrin homology domains typically associate with phosphoinositides,
and the lipid binding sites are predominantly composed of residues in
and around the The binding of phosphoinositides to PH domains is often
invoked to explain the translocation of cytosolic proteins to membrane surfaces. However, for Dbs, this function does not appear to be the
main role for the PH domain. For instance, the PH domain of Dbs binds
phosphatidylinositol 4,5-bisphosphate with relatively low affinity
(KD of ~10 µM) (15) typically
considered insufficient to recruit proteins to plasma membranes (11).
Therefore, not surprisingly, mutants of Dbs deficient in binding
phosphoinositides through the PH domain continue to associate with
distinct cellular compartments similar to wild-type Dbs as assessed by
membrane fractionation and immunofluorescence studies. Other Dbl family proteins behave similarly. For example, the affinity of
phosphatidylinositol 4,5-bisphosphate binding to the PH domain of Sos1
is ~2 µM (30, 31) and while the PH domain of Sos1
translocates to plasma membranes in response to serum-treatment of
cells, this recruitment is not due to association with
phosphoinositides but likely involves association with other ligands
(31). In fact, whenever it has been measured (12, 15, 32, 33)
affinities between DH-associated PH domains and phosphoinositides are
sufficiently low as to suggest a role other than membrane targeting as
the principal function for these PH domains.
An alternative model consistent with the available data
postulates that binding of phosphoinositides by the PH domain reorients the DH and PH domains to engage effectively Rho GTPases (Fig. 6). In the simplest scenario, binding of
phosphoinositides to the PH domain results in the en bloc
reorientation of the DH and PH domains relative to membrane surfaces.
However, a plethora of structural data (17, 20, 26, 34, 35) highlights
a large degree of conformational flexibility between PH domains and
associated DH domains. In the extreme case of Sos1, the PH domain must
necessarily move out of the way to allow access of Rho GTPases to the
major binding determinant within the DH domain subsequent to Rac
activation dependent upon phosphoinositide 3-kinase (36). Therefore,
perhaps it is more likely that binding of membrane-resident phosphoinositides to PH domains serves to alter the relative
orientation of DH and PH domains to engage effectively Rho GTPases.
These conformational rearrangements could relieve inhibitory
constraints in the case of Sos1 and has been proposed for Vav (12) and
Dbl (14, 37) or promote activation as has been suggested for Tiam1 (13,
38), Vav (12), and P-Rex1 (39). As previously discussed, Dbs·Cdc42 is
organized to allow simultaneous engagement of membranes by the PH
domain of Dbs and the geranylgeranyl group of Cdc42 (17). The structure
of the complex further suggests that membrane binding by the PH domain
would not preclude and, indeed, may favor concomitant interactions
between GTPases and the PH domain. Therefore, the PH domain of Dbs, in
concert with the DH domain, possibly cooperates to integrate
information regarding local fluctuations in both Rho GTPase
concentrations and the phosphoinositide composition of nearby
membranes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B (1-3). GDP/GTP cycling within Rho proteins
is primarily accomplished through the actions of two classes of
regulatory proteins. GTPase activating proteins promote the inactive,
GDP-bound form of Rho proteins by enhancing their intrinsic GTPase
ability to convert bound GTP to GDP. In contrast, the actions of
guanine nucleotide exchange factors (GEFs) upon Rho GTPases results in
Rho activation by exchanging their bound GDP for GTP.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 (residues 11-140), fused to an amino-terminal
hexa-histidine tag, was also expressed from the pProEX-HT vector for
bacterial expression. Site-directed substitutions placed within
all Dbs DH/PH domains and Cdc42 expression constructs were prepared
using the QuikChange site-directed mutagenesis kit (Stratagene) as per
the manufacturer's instructions and were verified by automated sequencing.
ex = 295 nm,
em = 335 nm) in response
to binding mant-GTP. The rates (kobs) of guanine nucleotide exchange were determined by fitting the data as
single-exponential decays utilizing the program GraphPad Prism.
Data were normalized to wild-type curves to yield the percent GDP released.
1/
2 loop region were modeled in and
minimized for some docking procedures. To validate our docking
procedure, inositol 1,4,5-trisphosphate was successfully docked into
the previously determined structure of the PH domain of PLC-
bound
to inositol 1,4,5-trisphosphate (RCSB accession number 1MAI).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
4 of
the PH domain is critical for exchange because its substitution to
phenylalanine dramatically decreases Dbs-catalyzed exchange of both
Cdc42 and RhoA without affecting the overall arrangement of domains
within the Dbs·Cdc42 complex (17). To clarify the structural role of the PH domain in contributing productively to the interface between Dbs
and GTPases, a series of substitutions within the interface were
assessed for functional importance. In particular, Arg-66 of Cdc42
interacts with multiple residues within the PH domain of Dbs, including
Tyr-889. Yet substitution of Arg-66 to alanine does not affect
nucleotide exchange (Fig. 1B, Table
I, and see Ref. 17), suggesting that
Tyr-889 likely stabilizes the electronic configuration of His-814 to
promote interaction with Asp-65 of Cdc42. In support of the importance
of Asp-65 to nucleotide exchange, amino acid substitutions placed at
Asp-65 (D65A, D65E, and D65N) severely limit the ability of Dbs
to activate Cdc42 (Fig. 1B and Table I). Of particular note,
the isosteric substitution D65N in Cdc42 should not disrupt
interactions with Asn-810 of the DH domain but will disrupt hydrogen
bonding with His-814 of Dbs. Consistent with an "uncoupling" of
Asp-65 from Tyr-889, Cdc42 (D65N) suffers a reduction of ~7-fold in
the activation by Dbs, on par with the effect of Dbs(Y889F) on
wild-type Cdc42 (Table I).
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Fig. 1.
Interactions between the PH domain of Dbs and
Rho GTPases are critical for transformation. A, as
previously reported (17), the PH domain (blue,
disorder region in gray) of Dbs is an integral part of the
interface with Cdc42 (green, switch regions are
red) and the DH domain (yellow) necessary for
robust GEF activity in vitro. The inset
highlights specific interactions with dashed lines
indicating potential hydrogen bonds. B, within Cdc42,
Asp-65, but not Arg-66, is critical for exchange supported by the PH
domain. Asp-65 of Cdc42 interacts primarily with His-814 of the DH
domain buttressed by Gln-834 and Tyr-889 of the PH domain. Guanine
nucleotide exchange reactions were performed by stimulating 2 µM of wild-type or mutant Cdc42 by 0.2 µM
of the Dbs DH/PH domain. The intrinsic exchange data for the D65N,
D65E, and D65A Cdc42 mutants are not shown. C, residues
within the PH domain and integral to the interface with Cdc42 are
necessary for transformation of NIH 3T3 cells mediated by Dbs (3 µg
of plasmid). Data shown are representative of three assays performed on
triplicate plates. The error bars indicate standard
deviations.
Rate constants of Dbs catalyzed guanine nucleotide exchange of mutant
Cdc42 proteins
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Fig. 2.
Mutational analysis of the
phosphoinositide-binding pocket. A, model of the
headgroup of phosphatidylinositol 4,5-bisphosphate docked onto the PH
domain of Dbs. The inositol ring of phosphatidylinositol
4,5-bisphosphate was docked onto the crystal structure of the PH domain
of Dbs (PDB code 1KZ7) using the program DOCK (22). The electropositive
surface (blue) was used during docking, and the top-scoring
complex is shown with residues that impair binding of phosphoinositides
upon mutation labeled. B, all substitutions within the
putative phosphoinositide-binding pocket of Dbs DH/PH abrogate binding
of phosphatidylinositol 4,5-bisphosphate as measured by
microtiter-based ELISA. Conversely, mutations within the DH domain
(H814A) or the 3/
4 loop of the PH domain have insignificant
effects on phosphatidylinositol 4,5-bisphosphate binding. Also shown is
the binding profile for the PH domain of PLC-
1 known to bind
phosphatidylinositol 4,5-bisphosphate with high affinity (40) as well
as the isolated DH domain of Dbs. C, similar binding results
were obtained using small unilamellar vesicles doped with
phosphatidylinositol 4,5-bisphosphate at 3% mol fraction and measuring
interactions by surface plasmon resonance. Raw data were normalized to
the signal achieved from nonspecific binding to an empty flow cell
surface.
1/
2 and
3/
4 loops of the PH domain.
Like most PH domains, the
1/
2 and
3/
4 loop region of Dbs
exhibits a large envelope of positive electrostatic potential,
compatible with binding the electronegative headgroups of
phosphoinositides (17). To further define the lipid-binding pocket
within the Dbs PH domain, we used a computational approach to dock
various inositol phosphates onto the surface of the protein (Fig.
2A). These analyses suggest that residues within the PH
domain of Dbs that are predicted to contact phosphoinositides within
this site, and/or contribute to the positive electrostatic potential
include: Lys-849, Lys-851, Arg-855, and Lys-857 (within the
1/
2
loop); Arg-861 (at the base of
2); Lys-874 (within
3); and
Lys-892 (within
4).
1/
2 and
3/
4 of the PH
domain of Dbs are responsible for mediating binding to
phosphoinositides, we substituted the identified lysine and arginine
residues with glutamate and assessed the effect of each mutation upon
the ability of Dbs to bind phosphatidylinositol 4,5-bisphosphate.
Initially, an enzyme-liked immunosorbent assay (ELISA) was used to
assess binding to phosphatidylinositol 4,5-bisphosphate in the absence of secondary lipids (Fig. 2B). These results were supported
by a more detailed analysis using surface plasmon resonance and small unilamellar vesicles containing a low molar fraction of
phosphatidylinositol 4,5-bisphosphate within a background of negatively
charged lipids (Fig. 2C). As we observed previously (15),
wild-type Dbs DH/PH domain binds significantly to phosphatidylinositol
4,5-bisphosphate, whereas the isolated DH domain of Dbs shows no
binding. In comparison, Dbs proteins harboring either single or double
glutamate substitutions within the putative phosphoinositide
binding site of the PH domain do not significantly bind
phosphatidylinositol 4,5-bisphosphate in either assay. In contrast,
other mutations within the extended
3/
4 loop (K885A) or
4 loop (Y889F) of the PH domain, at sites distinct from the
phosphoinositide-binding pocket, do not significantly affect binding of
phosphoinositides. Therefore, as observed within the structures of
other PH domains bound to inositol phosphates, the PH domain of Dbs
binds lipids through portions of
1/
2 and
3/
4, and mutations
within this pocket selectively abolish lipid binding.
View larger version (25K):
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Fig. 3.
In vivo activation of RhoA and
associated transformation require phosphoinositide binding to the PH
domain. A, mutations within the PH domain of Dbs that
destroy binding to phosphoinositides also prevent transformation of NIH
3T3 cells as measured in primary focus formation assays. Data shown are
representative of three independent assays performed on triplicate
plates. B, the identical panel of mutants also fails to
activate RhoA in NIH 3T3 cells. Lysates were prepared from cell lines
stably expressing either Dbs-HA6, the various mutations within a
Dbs-HA6 background, or the cognate pCTV3H vector and examined by
Western blot for expression of RhoA (Total-RhoA) and the
HA-tagged Dbs proteins (anti-HA). Each lysate was
subsequently normalized for expression of total RhoA and subjected to
affinity purification using immobilized GST-C21 to isolate RhoA-GTP
also visualized by Western blot. All experiments were performed a
minimum of three times, and data shown constitute a representative
set.
View larger version (32K):
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Fig. 4.
In vitro GEF activity of Dbs is
unaffected by loss of phosphoinositide binding to the PH domain.
In vitro guanine nucleotide exchange of Cdc42 (A
and B) or RhoA (C and D) by each
phosphoinositide binding site mutant of Dbs was carried out as
described under "Experimental Procedures." Guanine nucleotide
exchange reactions were performed by stimulating 1 µM of
GTPase by 0.2 µM of wild-type or mutant Dbs DH/PH domain.
"None" refers to the intrinsic exchange data of
wild-type Cdc42 or RhoA in the absence of GEF.
Rate constants of guanine nucleotide exchange reactions catalyzed by
wild-type and mutant Dbs proteins on Cdc42 and RhoA
View larger version (60K):
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Fig. 5.
Mutations within the PH domain of Dbs that
destroy lipid binding do not affect subcellular localization.
A, NIH 3T3 cells that stably express Dbs-HA6, Dbs-HA8 (DH),
Dbs-HA7 (DH plus CAAX), or the various lipid binding mutants
were lysed and fractionated as described under "Experimental
Procedures." 30 µg of protein from total (T), soluble
(S), and particulate (P) fractions were resolved
by SDS-PAGE. Protein expression was determined with an anti-HA epitope
antibody (BAbCO). Data shown are representative of three independent
assays. B, NIH 3T3 cells, which were transiently
cotransfected with pEGFP and either Dbs-HA6 or Dbs-HA6 harboring
mutants defective in lipid binding, were examined for cellular
distribution of Dbs. GFP was used as both a counterstain and a marker
for transfected cells. Cells expressing only GFP were examined for
expression of the HA epitope. GFP and HA images were merged
(merge) to orient the Dbs staining with respect to cellular
structures.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1,
4, and the
3/
4 loop of the PH domain to contact switch 2 and
3b within Cdc42, and biochemical studies show these interactions
are critical for efficient guanine nucleotide exchange in
vitro (17). In particular, mutation of Tyr-889 to phenylalanine within the Dbs PH domain was particularly detrimental to guanine nucleotide exchange. Here we show that Tyr-889 likely acts to encourage
interactions between Asp-65 within Cdc42 (Asp-67 in RhoA) and His-814
within the Dbs DH domain. Consistent with the related in
vitro results, Dbs harboring Y889F (or H814A) failed to transform
NIH 3T3 cells. These effects are not due to an inability to bind
phosphoinositides, because Dbs(Y889F) possesses wild-type affinity
toward phosphoinositides (Fig. 2) and maintains a three-dimensional structure essentially identical to wild-type Dbs (17). Therefore, both
the DH and PH domains of Dbs are critical for directly catalyzing the
activation of Rho GTPases in cells.
1/
2 and
3/
4 loops (11, 29). Based on docking
experiments between inositol 4,5-bisphosphate (the phosphoinositol ring
of phosphatidylinositol 4,5-bisphosphate) and the Dbs PH domain, basic
residues within the Dbs PH domain that participate in
phosphatidylinositide binding have been predicted. Consistent with
these predictions, substitutions to glutamate at these sites abolish
the ability of Dbs to productively bind to phosphatidylinositol
4,5-bisphosphate in vitro. When the mutants of Dbs that are
deficient in binding lipid are analyzed for in vivo
function, they are completely devoid of transformation potential and do
not detectably activate RhoA in NIH 3T3 cells, despite suffering no
loss in exchange activity in vitro. Therefore, lipid binding
by the PH domain of Dbs is critical to both the transformation potential of Dbs and the ability of Dbs to activate Rho GTPases in vivo.
View larger version (19K):
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Fig. 6.
Model linking the binding of
phosphoinositides to activation of Rho GTPases. Prior to GTPase
activation (left panel), local accumulation of specific
phosphoinositides and membrane-resident, inactive GTPases favor
interactions with GEFs located in close approximation to the relevant
cellular membranes. For wild-type GEFs (upper panel),
interactions with phosphoinositides and GTPases promote ejection of GDP
and the subsequent loading of GTP to activate GTPases. For Dbs,
exchange requires the direct interactions of both the DH and PH domains
with Rho GTPases at the membrane surfaces to allow both domains to
participate effectively in GTPase activation. Mutations (lower
panel) that destroy the ability of the Dbs PH domain to bind
either phosphoinositide (dimple in the PH domain) or GTPase
(bump) consequently destroy GTPase activation without
altering the sub-cellular localization of Dbs. Most significantly, this
model invokes the PH domain in directly regulating GTPase activation by
detecting both GTPases and phosphoinositides, making the PH domain an
active participant in the exchange process and not simply a
membrane-anchoring device.
In conclusion, we have used the recently determined structure of
Dbs·Cdc42 to design mutations within the PH domain of Dbs that
specifically impair either guanine nucleotide exchange or phospholipid
binding in vitro and assessed their functional effects in vivo. In agreement with both the structure and in
vitro biochemistry, the PH domain is an integral part of the
interface with GTPases required for efficient exchange in
vivo and mutations at sites within the PH domain that directly
perturb this interface severely impair cellular transformation by Dbs.
Similarly, cellular transformation and GTPase activation in
vivo require the PH domain of Dbs to properly engage
phosphoinositides, but this requirement manifests only in the presence
of cellular membranes and membrane-associated GTPases. Moreover,
the PH domain clearly does not serve as a simple membrane anchor or
subcellular trafficking device dependent upon binding
phosphoinositides, because a variety of forms of Dbs deficient in
binding phosphoinositides to the PH domain still localize correctly within cells. Although the details await clarification, it seems likely
that the invariant placement of PH domains downstream of DH domains
must serve an invariant function. One intriguing possibility consistent
with these data is that the two domains function cooperatively to
detect specific membrane-resident GTPases and phosphoinositides produced under controlled stimuli.
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ACKNOWLEDGEMENTS |
---|
We are grateful to M. Pham and S. Gershburg for technical assistance and T. Leisner for help with ELISAs.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants CA-77493 (to I. P. W.) and R01-GM62299 (to J. S.).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.
¶ Both authors contributed equally to this work.
To whom correspondence should be addressed: Dept. of
Pharmacology, University of North Carolina, CB #7365, 1106 M. E. J.
Bldg., Chapel Hill, NC 27599. Tel.: 919-966-7530; Fax: 919-966-5640; E-mail: sondek@med.unc.edu.
Published, JBC Papers in Press, March 13, 2003, DOI 10.1074/jbc.M300127200
2 K. L. Rossman, L. Cheng, G. M. Mahon, R. J. Rojas, J. T. Snyder, I. P. Whitehead, and J. Sondek, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: GTPase, guanosine triphosphatase; Dbl, diffuse B-cell lymphoma; Dbs, the big sister of Dbl; DH, Dbl homology; ELISA, enzyme-linked immunosorbent assay; GEF, guanine nucleotide exchange factor; GST, glutathione S-transferase; HA, hemagglutinin; Ins(4, 5)P2, inositol 4,5-bisphosphate; Lfc, the first cousin of LBC; Lsc, the second cousin of LBC; mant, N-methylanthraniloyl; PBS, phosphate-buffered saline; PH, pleckstrin homology; PtdIns(4, 5)P2, phosphatidylinositol 4,5-bisphosphate; SUVs, small unilamellar vesicles; Tiam1, T-cell lymphoma invasion and metastasis 1; Tris, tris(hydroxymethyl)aminomethane; wt, wild-type; GFP, green fluorescent protein; EGFP, enhanced GFP; BSA, bovine serum albumin.
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