From the Laboratorio di Biologia Molecolare, Istituto
G. Gaslini, Largo G. Gaslini 5, 16147 Genova, Italy, the
§ Department of Molecular Sciences, University of Tennessee,
Memphis, Tennessee 38163, the ¶ Dipartimento di Medicina
Sperimentale e Patologia, Università di Roma "La Sapienza,"
00161 Roma, and
Dipartimento di Oncologia e Neuroscienza,
Cattedra di Oncologia Medica, Università di Chieti,
66100 Chieti, Italy
Received for publication, October 25, 2000, and in revised form, February 23, 2001
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ABSTRACT |
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The Dbl family guanine nucleotide exchange
factors (GEFs) contain a region of sequence similarity consisting of a
catalytic Dbl homology (DH) domain in tandem with a pleckstrin homology (PH) domain. PH domains are involved in the regulated targeting of
signaling molecules to plasma membranes by protein-protein and/or
protein-lipid interactions. Here we show that Dbl PH domain binding to
phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-triphosphate results in the inhibition of Dbl GEF activity on Rho
family GTPase Cdc42. Phosphatidylinositol 4,5-bisphosphate binding to
the PH domain significantly inhibits the Cdc42 interactive activity of
the DH domain suggesting that the DH domain is subjected to the PH
domain modulation under the influence of phosphoinositides (PIPs). We
generated Dbl mutants unable to interact with PIPs. These
mutants retained GEF activity on Cdc42 in the presence of PIPs and
showed a markedly enhanced activating potential for both Cdc42 and RhoA
in vivo while displaying decreased cellular transforming activity. Immunofluorescence analysis of NIH3T3 transfectants revealed
that whereas the PH domain localizes to actin stress fibers and plasma
membrane, the PH mutants are no longer detectable on the plasma
membrane. These results suggest that modulation of PIPs in both the GEF
catalytic activity and the targeting to plasma membrane determines the
outcome of the biologic activity of Dbl.
The Rho family GTPases are tightly regulated molecular switches
that modulate several important cellular functions. They have a central
role in the regulation of actin-based cytoskeletal organization and
mediate signal transduction pathways leading to transcriptional control
and cell growth regulation (1). The Rho GTPases are regulated by three
different classes of proteins as follows: the guanine nucleotide
exchange factors (GEFs)1 that
activate Rho GTPases by stimulating the GTP-GDP exchange reaction;
the GTPases-activating proteins (GAPs) that stimulate the
intrinsic GTPase activity of G proteins; and the guanine nucleotide dissociation inhibitors (GDIs) that antagonize GEFs and GAPs activities (2).
GEFs for Rho family of GTPases are composed of a large family of
proteins all characterized by a region of sequence similarity consisting of the Dbl homology (DH) domain in tandem with a pleckstrin homology (PH) domain. Whereas the DH domain is the minimal region required to bind Rho GTPases, both domains are necessary for the biologic activity of these proteins in vivo (2, 3).
The pleckstrin homology domains are protein motifs of about 100 amino
acids that have been identified in more than 100 molecules involved in
signal transduction, including serine/threonine and tyrosine kinases,
phospholipases, cytoskeletal proteins, and regulators of small GTPases
(4, 5). Several studies have indicated that the PH domain can bind to
specific phosphoinositides (PIPs) as well as to All the PH domains have a core The presence of PH domain in GEFs for the Rho family of GTPases
suggests that PIPs may regulate these protein functions. Indeed PI3,4,5P3 was shown to bind to Vav PH domain and to enhance
Vav GEF activity on Rac1 GTPase. On the other hand binding of
PI4,5P2 to Vav PH domain causes inhibition of GEF activity,
suggesting that the PH domain of Vav regulates GEF activity through
binding to PI3K substrate and products (20, 21). In agreement with this
hypothesis Das et al. (22) have recently reported that PI4,5P2 promotes inhibitory intramolecular interactions
between Vav DH and PH domains, whereas PI3,4,5P3 activates
Vav GEF activity by disrupting these interactions. Similarly, it
appears that intramolecular interactions between DH and PH domains
regulate Sos GEF activity and that these interactions are mediated by
PIPs binding to Sos PH domain (22, 23).
The Dbl oncoprotein (24) is the putative exchange factor for the small
GTPases RhoA and Cdc42 (25). We have previously reported that both the
DH and the PH domains are required for the cellular functions of Dbl.
The minimal structural unit of oncogenic Dbl conferring transforming
activity just encompasses the DH and the PH domains. Moreover, we have
reported that the Dbl PH domain targets the Dbl DH domain to the Triton
X-100-insoluble fraction of the cell (26).
In light of the recent reports (20-23) indicating that Vav and Sos PH
domains regulate these protein GEF activities through interactions with
PIPs, we sought to investigate if PIPs regulate Dbl oncogene functions.
Here we report that Dbl PH domain interacts in vitro with
PI4,5P2 and PI3,4,5P3 and that these
interactions modulate Dbl GEF activity both in vitro and
in vivo. Moreover, we show evidence that Dbl PH domain
localizes to the plasma membrane and that this localization is
dependent on its interactions with PIPs. This dual modulation of PH
domain functions by PIPs appears to result in the efficient outcome of
the biologic activity of Dbl.
Expression and Purification of Dbl Fusion
Proteins--
Production and purification of Sf9 insect
cell-expressed His6-Dbl proteins was performed as
described (27).
The coding regions encompassing Dbl PH domain (residues 703-813) and
Dbl DH/PH domains (residues 497-875) were amplified by the polymerase
chain reaction from Dbl cDNA. Mutations within the PH domain,
i.e. substitution of Arg724 to Gly (PH-s)
and substitutions of Lys712 to Ala, Lys714 to
Ala, and Arg724 to Gly (PH-t), were introduced by "Quick
Change Site-directed Mutagenesis Kit" (Stratagene). All the
polymerase chain reaction products were sequenced by T7 Sequenase
version 2.0 kit (Amersham Pharmacia Biotech) before subcloning them.
The PH domain was subcloned into the BamHI and
EcoRI sites of pGEX-2TK (Amersham Pharmacia Biotech) to
generate GST fusion proteins for the lipid dot-blot assays and into the
pCEFL-GST vector (kindly provided by S. Gudkind) for the localization
experiments. The DH/PH domains were subcloned into pCEFL-GST vector for
the GDP dissociation and GTPase activation assays. GST fusion proteins
were expressed and purified using glutathione-agarose beads as
described (28).
32P Labeling of GST-PH Fusion Protein--
GST-PH
fusion proteins expressed by pGEX-2TK were labeled with 32P
as described (19). Briefly about 70 µg of purified GST-PH proteins
were incubated with 25 µCi of [ Lipid Dot-blot Assay--
PIPs (Echelon) were dissolved in
chloroform/methanol/water, 20:9:2, v/v/v, at a final concentration of
0.5, 0.25, and 0.125 µg/µl. Diluitions were made in the same
solution. 2 µl of each PIPs dilution were spotted onto Immobilon P
membranes (Millipore). The membranes were incubated with 1 × 107 cpm of 32P-labeled fusion protein in 25 ml
of Tris-buffered saline containing 3% of BSA for 30 min at room
temperature. Membranes were washed with Tris-buffered saline five
times, and bound radioactivity was visualized by autoradiography.
Cells Culture and Transfection--
COS-7 cells were obtained
form ATCC and were cultured in DMEM supplemented with 10% fetal calf
serum. NIH3T3 fibroblasts were cultured in Dulbecco's modified
Eagle's medium (DMEM) supplemented with 10% calf serum. For
transfection COS-7 cells were grown to 80% confluence in 100-mm tissue
culture dishes and transiently transfected with 8 µg of plasmid DNA
using LipofectAMINE PLUS method as described by the manufacturer (Life
Technologies, Inc.). Twenty hours after transfection the medium was
changed to DMEM containing 0.5% calf serum, and cells were incubated
for 24 h before lysis. NIH3T3 cells were transfected with 5 ng of
plasmid DNA by the calcium phosphate coprecipitation method and
cultured in the presence of 375 µg/ml of G418 (29).
GDP Dissociation and GDP/GTP Exchange Assay--
GDP
dissociation and GDP/GTP exchange assays were carried out similarly as
described before (30). 2 µg of Cdc42 loaded with [3H]GDP was incubated with buffer mixtures containing 100 mM NaCl, 50 mM HEPES (pH 7.6), 5 mM
MgCl2 (buffer A) with various lipids or Dbl proteins for
the indicated time at 25 °C. Dissociation reaction was stopped by
dilution of aliquots of a 20-µl sample into 10 ml of ice-cold buffer
A, and the protein-bound nucleotide was trapped by filtration onto
nitrocellulose filters. For GTP/GDP exchange assays, 100 µM GTP was also included in the reaction buffer.
Interaction of DH/PH Domain with Cdc42--
The GST, GST
DH/PH-wt, and GST DH/PH-t were expressed in COS-7 cells and purified to
homogeneity by glutathione-agarose chromatography. 2 µg of
immobilized GST, GST DH/PH-wt, or GST DH/PH-t were preincubated in a
buffer containing 50 mM Tris-HCl (pH 7.6), 5 mM
MgCl2, 1 mM DTT, 100 µg/ml BSA, and 30 µM PI4,5P2 for 30 min at room temperature (total volume of 200 µl). 10 µg of His6-Cdc42 was then
added to binding reaction and further incubated at room temperature for 2 h. After the incubation, the GST-agarose beads were extensively washed with buffer A supplemented with 1% Triton X-100, and the bound
Cdc42 protein was detected by anti Cdc42 Western blotting.
In Vivo Rho GTPases Activation Assay--
The GST-PAK-CRIB
domain fusion protein (residues 56-141, kindly provided by J. Collard)
containing the Cdc42 and Rac binding region of human PAK1 and the
GST-mDIA fusion protein (residues
The lysate-incubated beads were then washed three times with lysis
buffer, eluted in Laemmli sample buffer, and subjected to
SDS-polyacrylamide gel electrophoresis on a 10% gel. Bound Cdc42 and
RhoA were detected by Western blot using polyclonal antibody against
Cdc42 and monoclonal antibody anti RhoA (Santa Cruz Biotechnology).
Cells and Immunofluorescence--
Stable NIH3T3 transfectants
expressing Dbl PH-wt, PH-s, or PH-t were plated onto glass coverslips,
previously coated with 10 µg/ml fibronectin (Sigma), fixed with 4%
paraformaldehyde in PBS for 30 min at 25 °C, and permeabilized with
0.1% Triton X-100 in PBS for 5 min. Cells were subsequently stained
with anti-GST polyclonal antibodies (Molecular Probes), followed by
incubation with FITC-conjugated goat anti-rabbit IgG (Cappel, Organon
Teknika Corp.). Filamentous actin was visualized by incubating with
TRITC-labeled phalloidin 10 µg/ml in PBS (Sigma) for 30 min at
25 °C. Cells were observed with a Zeiss Axioplan fluorescence
microscope using specific filters to abolish cross-talk in
double-labeled experiments. The FITC and TRITC fluorescence signals
were obtained using excitation filters at 490 and 550 nm, respectively,
and emission filters at 525 and 580 nm, respectively. Cells were
analyzed by recording and merging single images using a cooled CCD
color digital camera SPOT-2 (Diagnostic Instruments) and FISH 2000/HI
software (Delta Sistema). Colocalization of the two signals appears in
yellow in the merged images.
Ultrathin Cryosections--
Cells were fixed with a mixture of
2% paraformaldehyde and 0.2% glutaraldehyde in 0.1 M
phosphate buffer for 30 min at room temperature. Fixed cells were
scraped with a rubber policeman, washed with PBS, and embedded in 12%
gelatin (Sigma) that was solidified on ice. Gelatin blocks were infused
overnight with 2.3 M sucrose at 4 °C, frozen in liquid
nitrogen, and cryosectioned. Ultrathin cryosections were collected
using sucrose and methylcellulose on Formvar carbon-coated grids,
incubated with anti-GST polyclonal antibodies (Molecular Probes), 1:10
in phosphate buffer for 1 h at 25 °C and subsequently with 18 nm colloidal gold (prepared by the citrate method), conjugated with
protein A (Amersham Pharmacia Biotech), 1:10 in phosphate buffer for 30 min at 25 °C. Finally, ultrathin cryosections were stained with a
solution of 2% methylcellulose and 0.4% uranyl acetate before EM examination.
Dbl PH Domain Binds to PIPs--
The GEF proteins for the Rho
family of GTPases, Vav, Sos, and Tiam1 have been shown to bind PIPs
through their PH domain (18, 20, 33, 34). Binding of PI3K products to
the PH domain activates the GEF activity of Vav, whereas
PI4,5P2 inhibits Vav GEF activity (20). To evaluate if Dbl
PH domain specifically recognizes PIPs we used a qualitative lipid
dot-blot assay to assess apparent binding specificity. The Dbl PH
domain was subcloned into the pGEX-2TK vector for the expression of a
GST fusion protein with a site for phosphorylation by
cAMP-dependent protein kinase between GST and PH domain.
The fusion protein GST-PH domain was purified by agarose-GSH beads and
labeled by protein kinase A in the presence of
[ PI4,5P2 and PI3,4,5P3 Inhibit the Ability
of Dbl to Stimulate GDP Dissociation from Cdc42--
It has been shown
that the binding of PH domains to PIPs can regulate protein catalytic
activity (20). We have shown previously that the Dbl DH domain alone is
sufficient for GEF activity on Cdc42 in vitro, whereas the
PH domain is essential for Dbl activity in vivo (25, 35). To
evaluate whether Dbl GEF activity could be affected by binding of its
PH domain to PIPs, we examined the ability of Dbl to stimulate the
release of GDP from its substrate, Cdc42, in the presence of
PIPs.
Insect cell-expressed and purified His6-Dbl protein was
analyzed for its ability to stimulate [3H]GDP
dissociation from Cdc42 in the presence of PIPs. As shown in Fig.
2, incubation of the Dbl protein for 5 min in the GEF reaction mixture in the presence of 10 µM
PI4,5P2 or PI3,4,5P3 resulted in up to 45%
inhibition of Dbl GEF activity, whereas the presence of
phosphatidylinositol or PI3P did not have a significant effect.
We then measured the [3H]GDP released from Cdc42
stimulated by His6-Dbl protein at the 5-min time point in
the presence of increasing concentrations of PI4,5P2. Fig.
2B shows that over 80% of inhibition of
[3H]GDP release could be reached in the presence of
PI4,5P2 at a concentration of 20 µM, whereas
under the same conditions, PI4,5P2 had a minimum effect on
the intrinsic GDP dissociation from Cdc42 (data not shown). These data
suggest that binding to PI4,5P2 and PI3,4,5P3
can inhibit Dbl GEF activity and that this inhibition is directly
dependent on PIPs concentration.
Dbl PH Mutants Are Defective in Binding PIPs--
The tertiary
structures of several PH domains have been explained (14-17).
It has been demonstrated that positively charged amino acids in a loop
between GEF Activity of Dbl DH/PH Mutants Is Not Affected by PIPs--
To
examine the effect of the PH mutations on Dbl GEF activity, we
generated two mutant proteins in the DH/PH backbone containing the PH-s
(DH/PH-s) or the PH-t (DH/PH-t) mutation. The cDNAs encoding the
wild type Dbl DH/PH (DH/PH-wt) and each of the Dbl DH/PH mutants were
then subcloned into pCEFL-GST vector and transiently transfected into
COS-7 cells. Each protein was purified from COS-7 cell lysates by
glutathione-agarose beads and tested in vitro for GEF
activity on Cdc42. Equal amounts of purified Dbl GST-DH/PH-wt,
GST-DH/PH-s, and GST-DH/PH-t proteins were incubated with 2 µg of
purified Cdc42, and the release of [3H]GDP was measured
at 5 min in the absence or in the presence of 10 µM PIPs.
No significant differences were observed for the abilities of these
three proteins to stimulate [3H]GDP dissociation from
Cdc42 in the absence of PIPs (Fig.
4A), whereas the inhibitory
effect of PI4,5P2 or PI3,4,5P3 on the DH/PH wt
was significantly weakened for mutant proteins (Fig. 4B).
Thus, our results suggest that PI4,5P2 and
PI3,4,5P3 binding to the PH domain negatively regulates Dbl
GEF activity in the cells, and substitutions of positively charged
amino acids to neutral ones in the PH domain
To determine whether the inhibitory effects of PIPs on the GEF activity
of Dbl is due to an interference with the DH domain interaction with
Rho GTPase substrate, we compared the complex formation pattern of
purified Dbl GST-DH/PH-wt and GST-DH/PH-t proteins with Cdc42 in the
presence or absence of PI4,5P2. As shown in Fig.
5, Dbl DH/PH-wt was inhibited in its
ability to complex with Cdc42 in the presence of PI4,5P2,
whereas the DH/PH-t capability to associate with Cdc42 remained
unaffected by PI4,5P2. These data indicate that the
inhibition of GEF activity of Dbl for Cdc42 we observed in the presence
of PIPs is likely caused by the lipid-mediated regulation of the
ability of Dbl catalytic domain to complex with its substrate.
Effect of the PH Mutations on the Cdc42- and RhoA-activating
Potential of Dbl in Cells--
To evaluate how the mutations in Dbl PH
domain could affect Dbl GEF activity in vivo, NIH3T3 cells
were stably transfected with Dbl DH/PH-wt, DH/PH-s, and DH/PH-t
cDNAs, and the activated Cdc42 was collected on GST-PAK-CRIB domain
fusion protein and analyzed by Western blot. As shown in Fig.
6A, expression of either DH/PH-s or DH/PH-t induced a strong activation of endogenous Cdc42 in
NIH3T3 cells. Densitometric analysis revealed that the expression of
Dbl DH/PH-s and DH/PH-t mutants increased activated Cdc42 levels by
2.5- and 6-fold, respectively, in comparison with Dbl DH/PH wt. We also
evaluated the ability of the mutants to activate endogenous RhoA, the
other GTPase substrate of Dbl. NIH3T3 cells stable transfected with Dbl
DH/PH wt, DH/PH-s, and DH/PH-t cDNAs were lysed, and the activated
RhoA was collected on GST-mDIA fusion protein and analyzed by Western
blot. As shown in Fig. 6B, expression of DH/PH-s and DH/PH-t
also induced an activation of endogenous RhoA in NIH3T3 cells.
Densitometric analysis revealed that expression of DH/PH-s and DH/PH-t
increased activated RhoA levels by 1.8-4.5-fold, respectively, in
comparison with DH/PH wt.
Thus, consistent with the in vitro data, loss of DH/PH
binding to PIPs results in enhanced GEF activity of Dbl in cells.
Previous mutagenesis studies of the DH domain have indicated that the
cellular transforming activity of Dbl is intimately dependent upon its
GEF catalytic capability (25, 35). However, the enhanced activating
potential for Cdc42 and RhoA by DH/PH-s and DH/PH-t mutants does not
appear to correlate with their transforming activity. In fact, as shown
in Table I, DH/PH-s displayed a
focus forming activity just comparable to that of DH/PH-wt, and
the transforming activity of DH/PH-t was almost 3-fold lower than that
of DH/PH-wt. These results indicate that the inhibition of the GEF
activity induced by PIPs through its binding to Dbl PH domain is
necessary for Dbl efficient transforming activity and raises the
possibility that modulation of the GEF activity by PIPs binding to PH
domain reflects only one aspect of the functional effects of the
lipid-PH interaction.
PIPs Mediate Subcellular Localization of Dbl to Plasma
Membrane--
We have demonstrated previously that a significant
fraction of the PH domain of Dbl localizes to the Triton
X-100-insoluble fraction of the cells (26). On the other hand PH
domains have been implicated in transient localization of proteins to
the plasma membrane. To evaluate whether Dbl PH domain translocates to
the plasma membrane, we subcloned the cDNAs of Dbl PH-wt, PH-s, and PH-t domains into pCEFL-GST vector and analyzed stable NIH3T3 transfectants expressing Dbl GST-PH-wt, GST-PH-s, and GST-PH-t domain
fusion proteins for intracellular localization by using anti-GST
antibodies and FITC-labeled secondary antibodies. The staining of the
GST-PH-wt domain appeared diffuse all over the cell cytoplasm (Fig.
7) and associated with the plasma
membrane (Fig. 7, arrowheads and inset). In cells
transfected with the mutant GST-PH-s or GST-PH-t, the cytoplasmic
pattern of staining was similar to that observed for the wild type, but
no signal was detected on the plasma membrane (Fig. 7). A careful
analysis of the pattern of the cytoplasmic signal revealed an array
distribution that resembled the actin stress fiber organization.
Therefore, to investigate the possible co-distribution of Dbl PH domain
with cytoskeleton actin stress fibers, we double-labeled the cells with
phalloidin-TRITC. The single images, obtained using specific filters to
abolish possible crossover of the two signals, were recorded separately
and then merged to assess the extent of co-localization (in
yellow). NIH3T3 cells expressing either the wild type or the mutated forms of Dbl PH domain showed a flat and elongated shape and
the actin cytoskeleton organized in stress fibers. All the PH domains
used, either wild type or mutated forms, partially localized along
stress fibers. We performed similar experiments utilizing NIH3T3 cells
transformed with Dbl DH/PH-wt, DH/PH-s, and DH/PH-t. Similar to what we
had observed with PH domains, all the Dbl DH/PH proteins localized
along stress fibers, whereas no localization at the plasma membrane was
detected for the DH/PH-s and the DH/PH-t proteins (data not shown).
These results provide evidence that Dbl PH domain associates with both
the actin stress fibers and the plasma membrane and indicate that PIPs
modulate the targeting of the Dbl proteins to the plasma membrane.
To analyze at the ultrastructural level the localization of Dbl PH
domain, we performed immunoelectron microscopy on ultrathin cryosections. Frozen sections were incubated with anti-GST polyclonal antibodies followed by colloidal gold protein A conjugates. Immune gold
labeling of both wild type Dbl PH domain or Dbl PH mutants appeared
distributed on the cell cytoplasm (Fig.
8). However, in wild type Dbl
PH-expressing cells, a significant portion of the gold particles was
associated with the plasma membranes (Fig. 8, arrowheads),
whereas no immunolabeling was detected on the plasma membranes of cells
expressing the mutated Dbl PH forms, PH-s or PH-t (Fig. 8).
It has been demonstrated that the interaction of PH domains with
specific lipids can regulate the activity of associated catalytic domains. PH-containing proteins, like dynamin, Bruton's tyrosine kinase, protein kinase B, and ARNO activities are regulated by PIPs (36-38). Moreover, GEF activity of exchange factors for the Rho
family GTPases can be modulated by PIPs via binding to their PH domains
(18, 20, 23, 34) which, in turn, seem to promote activation of GTPases
by providing proper localization of these proteins within the cell
(39-40).
Here we have investigated whether Dbl PH can associate with
phosphatidylinositols, and we have examined the functional consequences of such associations. Our results show that purified Dbl PH domain associates with PIPs in a lipid dot-blot assay and provide evidence that interaction of PIPs with PH domain modulates both the GEF catalytic activity and the intracellular localization of Dbl. The lipid
dot-blot assay does not allow a quantitative comparison of
phosphoinositide binding specificity but rather it is a valuable way to
assess qualitative specificity of ligand recognition by PH domains
(19). Although 32P-labeled GST alone gave no signals above
background, we found that the Dbl PH domain showed clear selectivity
and bound strongly to PI4,5P2 and PI3,4,5P3. We
have occasionally observed a very weak binding of Dbl PH domain to
PI3,5P2 (see Figs. 1 and 3). However, we did not consider
this signal indicative of a real interaction between Dbl PH domain and
PI3,5P2. The strong difference in Dbl PH binding
specificity to PI3,5P2 versus
PI4,5P2 and PI3,4,5P3 and the
non-reproducibility of these data suggest a nonspecific type of
interaction, possibly due to the presence in the PIP preparations of
contaminants like PI4,5P2.
We have previously demonstrated that in the Dbl oncogene the PH domain
mediates the protein localization to the Triton X-100-insoluble component of the cell, suggesting a protein-protein interaction (26).
On the other hand, binding of PH domains with PIPs is thought to
determine the translocation of proteins to the plasma membrane where
they may be activated by membrane components or have improved access to
substrates. Indeed several proteins containing a PH domain translocate
to plasma membrane upon receptor stimulation. Likewise, the PH domain
of the GEF Lfc mediates the protein membrane localization (41), and the
interaction of the GEF Tiam1 N-terminal PH domain with
PI3,4,5P3 and PI4,5P2 seems to be responsible
for the regulated translocation of Tiam1 to the membrane (33, 42). Our
results show that the Dbl PH domain both localizes to plasma membrane
and actin stress fibers. Whereas the actin stress fiber location
confirms our previous cell fractionation results, the association with
the plasma membrane raises new possibilities in Dbl PH regulatory
functions. Positively charged amino acids located in a loop between
Several PH domains were shown to bind to F-actin (43). In particular
basic residues toward the ends of a short sequence around the
Analysis of the crystal structure of the DH/PH domains of Sos (17), the
discovery of PH domain capability to enhance DH domain catalytic
activity of Trio (44), and the report of a possible interaction between
DH and PH domains in Sos (45) suggest a direct association between
these two domains. More recent studies (20) have shown that Vav GEF
activity is regulated by binding of the PH domain to substrates and
products of PI3-kinase that promote or disrupt the intramolecular
interaction between the DH and the PH domains (21). We have previously
shown that Dbl GEF activity in vitro is not influenced by
the PH domain, whereas its integrity is indispensable for Dbl activity
in vivo (25, 46). Binding studies indicate that the Dbl PH
domain does not interact in vitro with the DH domain in the
absence of PIPs.2 We show
here that interaction of PH domain with PIPs interferes with complex
formation between Dbl DH domain and its substrate Cdc42. These results
suggest that DH domain function is subjected to the PH domain
modulation under the influence of PIPs, possibly by induction of a
direct intramolecular interaction between these two domains.
The transforming activity of Dbl mutant proteins does not appear to
correlate with their ability to stimulate activation of GTPases in the
cells. In fact, whereas GTP-bound Cdc42 and RhoA increase from about
2-fold up to 6-fold in NIH3T3 cells transformed with DH/PH-s and
DH/PH-t, respectively, the focus forming activity of the DH/PH-s
protein does not increase proportionally and that of the DH/PH-t mutant
even decreases by almost 3-fold in comparison with wild type Dbl
protein. Thus, Dbl mutant proteins are fully active in terms of their
GEF activity, but their ability to stimulate GTPases remains somehow
ineffective. There appears to be a lack of direct correlation between
inhibition of GEF activity by PIPs, as we noticed in the in
vitro exchange assays, and the reduction of transforming activity
that we observed in vivo with Dbl PH mutants. One possible
explanation is that the site of activation, not occurring at the plasma
membrane, does not correlate with an efficient interaction of Cdc42
and/or RhoA with their substrates. Alternatively, GTPases may be
activated in a region of the cell where they cannot interact with GAPs.
GTP-bound GTPases will thus accumulate because of an improper cycling
of GTP binding and hydrolysis. In fact, both published (47) and our
unpublished observations3
indicate that constitutively activated, GTPase-defective Cdc42 expressed in NIH3T3 cells is not transforming but rather has a detrimental effect on cell growth.
To improve the sensitivity of our transformation assay, we have used a
truncated form of Dbl protein (residues 497-875) rather than the
full-length clone. Our previous studies have demonstrated that removal
of the N terminus induces activation of Dbl protein and that the
removal of the C-terminal 50 residues does not affect Dbl
oncogene-transforming efficiency. Nevertheless, it is possible that the
full-length proto-oncogene product reacts differently to binding to
PIPs. Future studies will determine the biochemical and biological
responses of proto-Dbl protein to PIP interaction.
In conclusion, we show here that inhibition of binding of Dbl PH domain
to PIPs causes an increase in activation of Cdc42 and RhoA in NIH3T3
cells, prevents Dbl protein association with plasma membrane, and
induces a decreased cellular transforming activity. These data suggest
that PIPs may regulate DH catalytic activity toward its substrates
through PH binding to plasma membrane and support a model whereby a
fine balance between modulation of GEF activity and regulated targeting
to plasma membrane of Dbl protein by PIPs may determine the efficient
outcome of the Dbl biologic activity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunits of
heterotrimeric G proteins and protein kinase C (6-13).
Three-dimensional structures of several PH domains have been solved
(14-17). Despite the fact that the sequence homologies among PH
domains are very low, it was found that their tertiary structures are
very similar.
-sandwich formed by two antiparallel
-sheets of three and four strands, respectively, and capped at one
corner by a C-terminal
- helix. The three loops between the
-strands are very different in length and sequence and form a
positively charged surface that has been shown to represent the
ligand-binding site to phosphoinositides (8). These observations lead
to the hypothesis that PH domains are implicated in the transient localization of proteins to the plasma membrane. Further studies on the
specificity of binding between PH domain and PIPs have demonstrated
that different PH domains bind distinct PIPs probably to allow
regulation by specific extracellular signals (18, 19).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP (Amersham
Pharmacia Biotech) and 20 units of cAMP-dependent protein
kinase (Sigma) for 30 min at room temperature in a buffer containing 50 mM potassium phosphate buffer (pH 7.15), 10 mM
MgCl2, 5 mM NaF, 4.5 mM DTT in a
reaction volume of 160 µl. Beads were washed extensively with
phosphate-buffered saline (PBS). 32P-Labeled fusion
proteins were eluted from beads with 400 µl of a buffer containing 15 mM reduced glutathione, 50 mM Tris-HCl (pH
8.0), 5 mM NaF, 1 mM EDTA, 0.005% Nonidet
P-40, 0.5% BSA, 1 mM DTT, 1 mM AEBSF. The
concentration and integrity of the purified proteins were estimated by
Coomassie Blue staining following SDS-polyacrylamide gel
electrophoresis using BSA as a standard.
2 to 304, kindly provided by S. Narumiya) containing Rho-binding domain (RBD) were expressed and
purified as described previously (31, 32). NIH3T3 cells were
transfected with 5 ng of plasmid DNA by the calcium phosphate
coprecipitation method and cultured in DMEM supplemented with 10% calf
serum and 375 µg/ml G418. To evaluate Cdc42 activation stably
transfected cell lines were washed with ice-cold PBS buffer once before
lysis on the dish in a buffer containing 50 mM Tris-HCl (pH
7.4), 100 mM NaCl, 2 mM MgCl2, 1% Nonidet P-40, 10% glycerol, 10 µg/ml each of leupeptin and
aprotinin, 2 mM AEBSF, and 40 µg of GST-PAK. Lysates were
incubated with 60 µl of glutathione-coupled Sepharose beads (Amersham
Pharmacia Biotech) for 1 h at 4 °C under constant agitation. To
evaluate RhoA activation, cells were lysed in 50 mM
Tris-HCl (pH 7.5) containing 100 mM NaCl, 1 mM
EDTA, 5 mM MgCl2, 10% glycerol, 50 mM NaF, 1 mM Na3VO4, 1 mM dithiothreitol, 0.1% Nonidet P-40, 1 mM
AEBSF, 10 µg/ml each of leupeptin and aprotinin. The clarified
lysates were then incubated with 30 µg of GST-RBD fusion protein
conjugated with glutathione beads for 2 h at 4 °C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP. The labeled fusion protein was then used to
probe PVDF membranes on which 1 µg of phosphatidylinositol, PI3P,
PI3,5P2, PI4,5P2, and PI3,4,5P3 had
been spotted. GST and GST-PH proteins were labeled uniformly with a
specific activity of about ~7 × 104 cpm/pmol. As
shown in Fig. 1A, GST protein
did not react with PIPs, whereas the Dbl GST-PH domain specifically
bound to PI4,5P2 and PI3,4,5P3 (Fig.
1B). Thus, as reported for the PH domain of a few other
GEFs, Dbl PH can interact with PI4,5P2 and
PI3,4,5P3, the substrate, and product of PI3K,
respectively.
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Fig. 1.
Interaction of Dbl PH domain with PIPs.
32P-Labeled GST protein (A) and Dbl GST-PH
domain fusion protein (B) were used to probe PVDF membranes
onto which specific phosphoinositides had been spotted as described
under "Experimental Procedures." GST alone did not give any signal
above background, whereas Dbl GST-PH domain shows specific recognition
for PI4,5P2 and PI3,4,5P3. The results shown
are representative of five independent experiments.
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Fig. 2.
Inhibition of the GEF activity of Dbl
oncoprotein toward Cdc42 by PIPs. A,
purified lipids at a concentration of 10 µM were added
into the reaction mixture in a buffer containing 50 mM
HEPES (pH 7.6), 100 mM NaCl, 5 mM
MgCl2, 100 µM GTP, and 2 µg of
[3H]GTP-Cdc42 in the presence or absence of 20 ng of
His6 Dbl protein. The reactions were terminated after 5 min
at 25 °C. B, PI4,5P2
dose-dependent effect on Dbl-elicited nucleotide exchange.
The Dbl oncoprotein-catalyzed GEF reactions of Cdc42 were
assayed in the conditions described above in the presence of increasing
concentrations of PI4,5P2. Experiments were done three
times with essentially identical results.
-sheet one and
-sheet two of distinct PH domains have
direct interaction with PIPs (8). Sequence alignment of the Dbl PH
domain with these PH domains indicates that the Dbl PH domain has
positively charged amino acids in positions corresponding to those
known to interact directly with PIPs (3). Therefore, we generated two
mutant proteins (PH-s and PH-t) containing a single substitution of
Arg724 to Gly (PH-s) or a triple substitution of
Lys712 to Ala, Lys714 to Ala, and
Arg724 to Gly (PH-t). PH domain mutants were expressed as
GST fusion proteins and were used in lipid dot-blot assays in order to
determine if PH-s and PH-t were still able to bind PIPs. As shown in
Fig. 3, PH-s domain bound
PI4,5P2 and PI3,4,5P3 much less efficiently than PH-wt, whereas PH-t completely lost its ability to bind PIPs. These results indicate that positively charged amino acids located in
the first loop of Dbl PH domain in positions 712, 714, and 724 are
necessary for binding PI4,5P2 and
PI3,4,5P3.
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Fig. 3.
Dbl PH domain mutants are defective in
binding PIPs. A, wild type Dbl GST-PH domain fusion
protein (PH-wt) and GST-PH domain fusion protein containing either
(B) single amino acid substitution of Arg724 to
Gly (PH-s) or (C) triple amino acid substitution of
Lys712 to Ala, Lys714 to Ala, and
Arg724 to Gly (PH-t) were labeled with 32P and
used to probe PVDF membranes onto which specific PIPs had been spotted.
PH-s could bind PI4,5P2 and PI3,4,5P3 with much
less affinity than PH-wt, whereas PH-t almost completely lost its
ability to bind to PIPs. The results shown are representative of three
independent experiments.
1-
2 loop do not
affect the intrinsic catalytic DH domain activity but rather the
PIPs-elicited inhibitory response.
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Fig. 4.
Effects of mutations within Dbl PH domain on
Dbl GEF activity. A, time course of
[3H]GDP/GTP exchange on Cdc42 was measured in a GEF
reaction buffer containing 50 mM HEPES (pH 7.6), 100 mM NaCl, 5 mM MgCl2, 100 µM GTP, and 2 µg of [3H]GDP loaded Cdc42
in the presence or absence of 50 ng of Dbl GST-DH/PH-wt,
GST-DH/PH-s, or GST-DH/PH-t. The reactions were terminated by
nitrocellulose filtration at the indicated times, and the amount of
radioactivity at time 0 was taken as 100%. B,
histograms show the effects of PI4,5P2 and
PI3,4,5P3 on the GEF activity of Dbl with mutated PH
domains. The GEF reactions on Cdc42 were carried out with Dbl
GST-DH/PH-wt, GST-DH/PH-s, or GST-DH/PH-t in the presence or absence of
10 µM of PI4,5P2 and PI3,4,5P3.
The reactions were terminated at the 5-min time point. The results
shown in A and B are representative of three
independent experiments.
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Fig. 5.
Regulation of the interaction of DH/PH domain
with Cdc42 by PIPs. The GST, Dbl-GST-DH/PH-wt, and GST-DH/PH-t
proteins were expressed in COS-7 cells. His6-Cdc42 was
incubated with purified GST, GST DH/PH-wt, or GST DH/PH-t immobilized
on glutathione beads in the presence or in the absence of 30 µM PI4,5P2. Bound proteins were detected by
immunoblotting with anti-Cdc42 antibody. The results shown are
representative of three independent experiments.
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Fig. 6.
Comparison of the in vivo
GEF activity of mutant Dbl proteins. 0.05 µg of pCEFL-GST
plasmid expressing Dbl DH/PH-wt, DH/PH-s, or DH/PH-t were stably
transfected into NIH3T3 cells. Three weeks after transfection, cells
were lysed, and pull-down assays were performed. A, cell
lysates were subjected to GST-PAK1 pull-down assay and anti-Cdc42
Western blot analysis. B, cell lysates were subjected to
GST-mDia pull-down assay, and bound RhoA was detected by Western blot
using a monoclonal antibody against RhoA. The results shown in
A and B are representative of three independent
experiments.
Transforming activity of Dbl PH mutants
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Fig. 7.
Immunofluorescence localization of the
Dbl PH domains and their distribution along stress fibers. Cells,
expressing Dbl PH wt, PH-s, or PH-t, were double-stained with anti-GST
polyclonal antibodies, followed by FITC-conjugated secondary antibodies
(green signal) and with TRITC-labeled phalloidin (red
signal). Staining with anti-GST antibodies appears diffuse in the
cytoplasm in all cells and localized to the plasma membrane only in
cells expressing wild type Dbl PH domain (arrowheads and
inset with enlargement of the area in dotted white
box). The colocalization of the Dbl PH domains and phalloidin
signals appears in yellow in merged images. All the PH
domains used, PH-wt or PH-s or PH-t, are distributed along stress
fibers. Bar, 10 µm.
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Fig. 8.
Immunoelectron microscopic localization of
Dbl PH domain. Ultrathin frozen sections of cells expressing Dbl
GST fusion protein PH-wt, PH-s, or PH-t were incubated with anti-GST
polyclonal antibodies and colloidal gold-protein A conjugates. The gold
immunolabeling appears distributed in the cytoplasm of the cells. In
addition, gold particles are associated with the plasma membrane of the
cells expressing the wild type PH domain (arrowheads),
whereas they are virtually absent on the plasma membranes of cells
expressing PH-s or PH-t. On a total of 50 µm of membrane length
analyzed, randomly taken from 10 different cells for each Dbl PH
domain, 150 gold particles were counted in the cells expressing wild
type Dbl PH domain, 19 in the cells expressing Dbl PH-s domain, and 13 for cells expressing Dbl PH-t domain. PM, plasma membrane;
NM, nuclear membrane; Nu, nucleus; ER,
endoplasmic reticulum; G, Golgi. Bars, 0.2 µm.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1- and
2-sheets have direct interaction with PIPs (8). We show
here that three positively charged amino acids in Dbl PH domain,
Lys712, Lys714, and Arg724, mediate
the interaction with phosphatidylinositols since their substitution
with neutral amino acids blocks this interaction. The Dbl PH mutants,
unable to bind PIPs, fail to localize to plasma membrane but fully
retain their ability to co-localize with the actin cytoskeleton.
Finally, our studies indicate that the Dbl GEF activity in
vitro is not augmented by interaction with PIPs but rather is
inhibited by both PI3K substrate and product and that the exchange
activity of Dbl bearing mutations in these amino acids is no longer
affected by PI4,5P2 and PI3,4,5P3. Therefore, the PH interaction with PIPs may have dual effects on the DH domain as
well as on Dbl protein, localizing it to the plasma membrane and
meanwhile negatively regulating its enzymatic activity.
1 region of the Btk PH domain were shown to be sufficient for
binding to actin. Because this was proven to be correct for several
other PH domains containing basic residues as well, actin binding does
not seem to be dependent exclusively on the presence of basic residues.
In fact, the Vav PH domain, which contains a cluster of basic residues,
binds weakly to actin. Dbl PH domain localization to both plasma
membrane and actin stress fibers may be mediated by different residues
in its PH domain. This could explain why mutations in the positively
charged amino acids located in positions corresponding to those
affecting binding to PIPs interfere only with plasma membrane
localization. Two positively charged residues,
Arg718 and Lys720, are present in the
1 region of the Dbl PH domain. These residues do not align
with those known in other PH domains to interact with PIPs.
Nevertheless, mutagenesis analysis will clarify if these basic
residues, present in the
1 region of the PH domain, mediate
the association of Dbl protein to cytoskeleton actin.
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ACKNOWLEDGEMENTS |
---|
We thank Giuseppe Lucania and Catherine Ottaviano for excellent technical assistance. We are also grateful to Dr. S. Gudkind for providing pCEFL-GST vector, to Dr. J. Collard for providing GST-PAK, and Dr. S. Narumiya for providing GST-mDia.
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FOOTNOTES |
---|
* This work was supported by grants from the Italian Association for Cancer Research (IARC), the Ministero della Sanità Progetto Finalizzato N. ICSO70.2/RF95.221, from MURST, from CNR (Target Project on "Biotechnologies"), and by Grant GM53943 from the National Institutes of Health (to Y. Z.).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: Laboratorio di Biologia Molecolare, Istituto G. Gaslini, Largo G. Gaslini 5, 16147 Genova, Italy. Tel.: 001 39 010 5636 633; Fax: 001 39 010 3733346; E-mail: molbiol@tin.it.
Published, JBC Papers in Press, February 26, 2001, DOI 10.1074/jbc.M009742200
2 Y. Gao and Y. Zheng, unpublished information.
3 Y. Zheng, and A. Eva, unpublished information.
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ABBREVIATIONS |
---|
The abbreviations used are: GEF, guanine nucleotide exchange factor; DH, Dbl homology; PH, pleckstrin homology; PIPs, phosphatidylinositol phosphates; PI3P, phosphatidylinositol 3-phosphate; PI3, 5P2, phosphatidylinositol 3,5-bisphosphate; PI4, 5P2, phosphatidylinositol 4,5-bisphosphate; PI3, 4,5P3, phosphatidylinositol 3,4,5-triphosphate; GAPs, GTPases-activating proteins; PVDF, polyvinylidene difluoride; AEBSF, 4-(2-aminoethyl)benzenesulfonyl fluoride; PBS, phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium; FITC, fluorescein isothiocyanate; GST, glutathione S-transferase; wt, wild type; DTT, dithiothreitol; BSA, bovine serum albumin; PI3K, phosphatidylinositol 3-kinase; TRITC, tetramethylrhodamine B isothiocyanate.
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