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Originally published In Press as doi:10.1074/jbc.M111025200 on March 20, 2002

J. Biol. Chem., Vol. 277, Issue 22, 19745-19753, May 31, 2002
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Regulation of Proto-Dbl by Intracellular Membrane Targeting and Protein Stability*

Cristina VanniDagger , Patrizia Mancini§, Yuan Gao||, Catherine OttavianoDagger , Fukun Guo||, Barbara SalaniDagger , Maria Rosaria Torrisi§, Yi Zheng||, and Alessandra EvaDagger **

From the Dagger  Laboratorio di Biologia Molecolare, Istituto G. Gaslini, Largo Gaslini 5, 16147 Genova, Italy, § Dipartimento di Medicina Sperimentale e Patologia, Università di Roma "La Sapienza," 00161 Roma, Italy, and the  Department of Molecular Sciences, University of Tennessee, Memphis, Tennessee 38163

Received for publication, November 16, 2001, and in revised form, March 13, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The pleckstrin homology (PH) domain of onco-Dbl, a guanine nucleotide exchange factor (GEF) for Cdc42 and RhoA GTPases, interacts with phosphoinositides (PIPs). This interaction modulates both the GEF activity and the targeting to the plasma membrane of onco-Dbl. Conversely, we have previously shown that in proto-Dbl an intramolecular interaction between the N-terminal domain and the PH domain imposes a negative regulation on both the DH and PH functions, suppressing its transforming activity. Here we have further investigated the mode of regulation of proto-Dbl by generating proto-Dbl mutants deleted of the last C-terminal 50 amino acids, which contain a PEST motif, and/or unable to bind to PIPs due to substitutions of the positively charged residues of the PH domain. The PH mutants of proto-Dbl retained a relative weak GEF activity toward Cdc42 and RhoA in vitro, but their RhoA activating potential was impaired in vivo. Further, these mutants lost both the plasma membrane targeting and the transforming activities, contrary to the PH mutants of onco-Dbl that retained the exchange activity both in vitro and in vivo and showed significant, but partially, reduced transforming activity. Deletion of the C-terminal sequences from onco-Dbl did not affect its function, whereas similar deletion of proto-Dbl led to an increase of transforming activity. Analysis of the half-life of the proto-Dbl mutants revealed that deletion of the C-terminal sequences increases the stability of the protein. Overall, the transformation potential of proto-Dbl mutants was associated with an augmented localization of the protein to the plasma membrane and a strong activation of Jun N-terminal kinase activity and transcription of cyclin D1. Together with previous observations, these data suggest that the biological activity of proto-Dbl is tightly regulated by a combination of mechanisms that involve intramolecular interaction, PH binding to PIPs, and the N- and C-terminal domain-dependent turnover of the protein.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Small GTP-binding proteins of the Rho family belong to a group of signaling molecules involved in a wide spectrum of biological processes, including actin cytoskeleton reorganization, transcriptional regulation, membrane trafficking, and cell growth control and development (1, 2). These proteins function as molecular switches, cycling between a GDP-bound, inactive form and a GTP-bound, active form. The nucleotide-bound state of Rho family GTPases is controlled by three classes of proteins: the guanine nucleotide exchange factors (GEFs)1 that stimulate the dissociation of the tightly bound GDP nucleotide in response to upstream signals; the GTPase-activating proteins that promote the intrinsic GTPase activity of Rho proteins, leading to the inactive GDP-bound form; and the guanine nucleotide dissociation inhibitors that act by preventing spontaneous and GEF-catalyzed release of nucleotide, thereby maintaining the GTPases in the inactive state (3).

The GEFs for Rho GTPases are composed of a large family of proteins all characterized by a region of homology sequences in which a pleckstrin homology (PH) domain, responsible for proper cellular localization of the protein (4-6), is located immediately C-terminal to the Dbl homology (DH) domain, responsible for catalyzing the nucleotide exchange on Rho GTPases (3). Several lines of evidence suggest that the biological functions of DH-containing GEFs, such as actin cytoskeleton regulation, cell growth stimulation, and transformation, are intimately dependent upon their ability to interact with and activate Rho GTPases.

The Dbl family GEF proteins seem to exist as inactive or partially active molecules until specific intracellular stimuli lead to their activation. In some cases, the inactive basal state is maintained by a self-regulatory mechanism that involves an intramolecular interaction between different domains of GEFs themselves. Proto-Vav protein regulation, for example, involves an intramolecular interaction between the N-terminal sequences and the Rho GTPase binding site of the DH domain (7).

The signaling mechanisms mediating the GEF activation have been investigated intensively. Phosphorylation of Tyr174 of proto-Vav by Src-like kinases, for example, results in its activation (7), while phosphorylation of Tiam1 by protein kinase C II causes the activation of the protein by inducing its translocation to the plasma membrane (8). Upstream signals such as heterotrimeric G-proteins may also contribute in varying degrees to the GEF activation, as in the case of p115RhoGEF, which utilizes its N terminus to couple directly to Galpha 12/Galpha 13, resulting in an enhanced GEF catalytic activity and membrane translocation (9). Moreover, the recruitment of Cdc24p, a yeast Dbl family member, to the plasma membrane requires Far1p that recruits in turn Cdc24p to activated Gbeta gamma during mating response of Saccharomyces cerevisiae (10-12). In addition, inositol phospholipids binding to the PH domain has been reported to regulate GEF activity. Vav is regulated through binding of PH domain to phosphoinositide (PI) 3-kinase substrates and products (13, 14). PI4,5P2 promotes inhibitory intramolecular interactions between DH and PH domains, whereas PI3,4,5P3 activates Vav GEF activity by disrupting these interaction. Similarly, PIP binding to Sos PH domain mediates the interactions between DH and PH regulating Sos GEF activity (15).

The prototype member of Dbl family GEFs is the proto-Dbl protein, a cytoplasmic phosphoprotein of 115 kDa containing a long stretch of amino acids within its NH2-terminal half with the potential to form an alpha -helical coiled-coil (16, 17). This region negatively regulates the transforming activity of proto-Dbl through direct interaction with the PH domain. This interaction limits the access of Rho GTPases to the DH domain and masks the intracellular targeting function of the PH domain (18). Consistent with this mechanism of inhibition, oncogenic activation of proto-Dbl occurs by truncation of the amino-terminal 497 residues (16, 19).

In the present work, we have further investigated the mechanism of proto-Dbl regulation. We approached this in two ways. First, given our findings demonstrating that the PH domain of onco-Dbl binds to PI(4,5)P2 and PI(3,4,5)P3 and that this interaction modulates onco-Dbl protein transforming activity and intracellular localization (20), we analyzed the effects of PIPs binding to PH domain on proto-Dbl GEF activity and examined how the lipid binding might influence the cellular location, transcription stimulation, and transformation activities. Second, we studied the contribution of the C terminus 50 amino acids of proto-Dbl to proto-Dbl regulation. Although this region contains PEST-like sequences (21), its influence on proto-Dbl function has not been established. The effects of proto-Dbl mutations made at the PH domain and/or C terminus on their response to PIPs, GEF activity, subcellular localization, transcription regulation, protein stability, and transformation are examined in detail. The results shed new light on the complex regulatory mechanism of proto-Dbl that might be employed in a similar context by other Dbl family members.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Culture and Transfection-- NIH3T3 fibroblasts were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% calf serum. Mass cultures of stable transfected cell lines were generated by transfecting NIH3T3 cells with 300 ng of plasmid DNA by the calcium phosphate coprecipitation method and cultured in DMEM supplemented with 375 µg/ml G418 (22)

COS-7 cells were obtained from ATCC and were cultured in DMEM supplemented with 10% fetal calf serum. For kinase assays, cells were grown to 80% confluence in 100-mm tissue culture dishes and transiently transfected with 8 µg of the indicated plasmids using LipofectAMINE PLUS as described by the manufacturer (Invitrogen). Twenty hours after transfection the medium was changed to DMEM containing 0.5% fetal calf serum, and the cells were incubated for another 24 h before lysis.

Generation of Proto-Dbl Mutants-- The proto-Dbl Delta C mutant, which lacks the last C-terminal 50 amino acids, was generated by substituting the C terminus region (residues 554-925) with the C terminus region (residues 554-875) derived from onco-Dbl PMA3 deletion mutant (23). The proto-Dbl Delta C PH-t was obtained in a similar way by substituting the C-terminal region of proto-Dbl with the C-terminal region of DH/PH-t (residues 554-875), which carries substitutions of Lys712 to Ala, Lys714 to Ala, and Arg724 to Gly (PH-t) (20).

Mutations within the PH domain of proto-Dbl full-length (i.e. substitutions of Lys712 to Ala, Lys714 to Ala, and Arg724 to Gly (PH-t)) were introduced by the QuikChange site-directed mutagenesis kit (Stratagene). The products were sequence-proofed by the T7 Sequenase version 2.0 kit (Amersham Biosciences).

All proto-Dbl mutant cDNAs were subcloned into the pCefl-GST vector (kindly provided by S. Gutkind). To assay and compare the in vitro GEF activities, the GST fusions of proto-Dbl mutants were expressed in COS-7 cells and purified using glutathione-agarose affinity beads as described (24).

GDP/GTP Exchange Assay-- GDP/GTP exchange assays were carried out similarly as described before (25). 2 µg of Cdc42 loaded with [3H]GDP were incubated with a buffer mixture containing 100 mM NaCl, 50 mM HEPES (pH 7.6), 100 µM GTP, 5 mM MgCl2 (buffer A) with proto-Dbl proteins in the presence or absence of various lipids for the indicated time at 25 °C. The exchange reaction was stopped by dilution into 10 ml of ice-cold buffer A without excess GTP, and the protein-bound nucleotide was trapped by filtration onto nitrocellulose filters.

In Vivo Rho GTPase 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 -2 to 304, kindly provided by S. Narumiya), containing the Rho-binding domain, were expressed and purified as described previously (26, 27). The expression of proto-Dbl mutants in stably transfected cells was confirmed by Western blot analysis. Aliquots of cell lysates were subjected to SDS-PAGE and immunoblotting by using a specific polyclonal antibody against Dbl (16). To evaluate Cdc42 activation, the stably transfected cell lines were washed with ice-cold phosphate-buffered saline buffer 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 aprotinin and leupeptin, 2 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, and 40 µg of GST-PAK. Lysates were incubated with 60 µl of glutathione-coupled Sepharose beads (Amersham Biosciences) for 1 h at 4 °C under constant rotation. 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 4-(2-aminoethyl)benzenesulfonyl fluoride, 10 µg/ml each of aprotinin and leupeptin. The lysates were then clarified and incubated with 30 µg of GST-RBD fusion protein conjugated with glutathione beads for 2 h at 4 °C. The beads were then washed three times with lysis buffer, eluted in Laemmli sample buffer, and subjected to SDS-PAGE. Bound Cdc42 and RhoA were detected in Western blot by using polyclonal antibody against Cdc42 and monoclonal antibody anti-RhoA (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

Kinase Assays-- pCDNA3 HA-JNK or pCDNA3 HA-ROCK constructs were cotransfected in COS cells with pCefl-GST vector, pCefl GST-proto-Dbl, GST-proto-Dbl PH-t, GST-proto-Dbl Delta C, and GST-proto-Dbl Delta C PH-t constructs. JNK kinase activity was determined as described (28), using purified bacterially expressed GST-ATF2 fusion protein as substrate. ROCK kinase activity was determined in the same way by using myelin basic protein (Sigma) as substrate. The HA-tagged proteins were immunoprecipitated by using an anti-HA antibody (Babco), separated by SDS-PAGE, transferred to Immobilon-P membranes (Millipore Corp.), which were used for both autoradiography and Western blot analysis to evaluate kinase and proto-Dbl protein expression level. Immunocomplexes were visualized by West Dura extended chemiluminescent detection (Pierce) using protein G horseradish peroxidase-conjugated (Pierce).

Transient Luciferase Reporter Gene Induction Assays-- For determination of NF-kappa B- and cyclin D1-dependent gene expression, the NF-kappa B- and cyclin D1-luciferase reporter plasmids (Stratagene) that contain the promoter response elements of NF-kappa B or cyclin D1 were used in transient co-transfections. Transfection into NIH3T3 cells was performed using LipofectAMINE reagents (Invitrogen) according to the manufacturer's protocols. Analysis of luciferase expression in the cotransfected cells was carried out by using a luciferase assay kit from Promega

Biosynthetic Labeling-- Subconfluent cultures containing 5 × 106 cells were starved for 1 h at 37 °C in DMEM without serum, cysteine, and methionine. Thus, cells were labeled with [35S]methionine and [35S]cystine (500 µCi/plate of Pro-mix L-35S; Amersham Biosciences). 30 min later, medium was removed and replaced with DMEM containing an excess of cold methionine and cysteine followed by incubation in the same medium for the chase period.

Cells and Immunofluorescence-- Stable NIH3T3 transfectants expressing GST-proto-Dbl, GST-proto-Dbl PH-t, GST-proto-Dbl Delta C, or GST-proto-Dbl Delta C PH-t proteins were plated onto glass coverslips, previously coated with 10 µg/ml fibronectin (Sigma), fixed with 4% paraformaldehyde in phosphate-buffered saline for 30 min at 25 °C, and permeabilized with 0.1% Triton X-100 in phosphate-buffered saline for 5 min. The fusion proteins were visualized with anti-GST polyclonal antibodies (Molecular Probes, Inc., Eugene, OR), followed by incubation with fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (Cappel; Organon Teknika Corp.). Coverslips were mounted onto slides in 60% glycerol-Tris-buffered saline, and cells were observed by a Zeiss confocal microscope (Zeiss, Oberkochen, Germany).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Effect of PH Mutations and C-terminal Deletion on the Catalytic GEF Activity of Proto-Dbl in Vitro-- To investigate the mechanisms of regulation of proto-Dbl activity, we generated three mutants carrying alterations in the coding sequences. We have previously demonstrated that positively charged amino acids, located in the first loop of the Dbl PH domain, are necessary for binding to PIPs and for the efficient outcome of Dbl transforming activity (20). To evaluate the possible effect of PIP binding on proto-Dbl function and to make a comparison with that on onco-Dbl, we mutagenized the PH domain in proto-Dbl by substituting Lys712 to Ala, Lys714 to Ala, and Arg724 to Gly (PH-t). To examine whether the C-terminal region of proto-Dbl protein is involved in the regulation of this protein function, we generated a truncated form of the protein in which the C-terminal 50 amino acids were deleted (proto-Dbl Delta C). Further, we generated a double mutant in which the PH-t was inserted in the proto-Dbl Delta C cDNA (proto-Dbl Delta C PH-t) (Fig. 1). The previously described (20) C-terminal truncated mutant of oncogenic Dbl (DH/PH) and its corresponding PH mutant (DH/PH-t) (Fig. 1) were also used for comparison.


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  		Fig. 1.   Schematic presentation of proto-Dbl mutants and their parental structures. Numbering of residues corresponds to the amino acids of proto-Dbl protein. Each deletion (Delta C) or amino acid substitution (PH-t) is indicated.

To determine the effects of the PH domain mutations and C-terminal deletion on proto-Dbl GEF activity, we transiently transfected wild type GST-proto-Dbl and each of the GST-proto-Dbl mutants in COS cells. Dbl DH/PH and Dbl DH/PH-t were used as control. Each protein was purified from COS cell lysates by glutathione-agarose affinity chromatography and tested in vitro for GEF activity on Cdc42. Equal amounts of purified GST-fused proto-Dbl, proto-Dbl Delta C, proto-Dbl PH-t, proto-Dbl Delta C PH-t, Dbl DH/PH, and DH/PH-t proteins (Fig. 2A) were assayed for their ability to release [3H]GDP from 2 µg of Cdc42-[3H]GDP. As previously reported (18), we observed that while DH/PH was very efficient in stimulating GDP dissociation such that over 80% of bound GDP was dissociated within 10 min, proto-Dbl displayed a rather weak activity catalyzing only 30% of GDP dissociation from Cdc42 within the same time period (Fig. 2B). When the abilities of proto-Dbl and onco-Dbl PH mutants to release [3H]GDP from purified Cdc42 were compared with their wild type counterparts at 10 min in the same assay conditions, no significant differences were observed (Fig. 2C). The proto-Dbl and its mutants contained significantly less GEF activity than that observed for onco-Dbl DH/PH and DH/PH-t (Fig. 2C).


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  		Fig. 2.   Effect of mutations within proto-Dbl coding sequences on proto-Dbl GEF activity in vitro. A, GST-tagged onco-Dbl and proto-Dbl proteins were purified from COS-7 cells by affinity chromatography. After elution with 5 mM reduced glutathione, the purified proteins were analyzed by Western blot with anti-Dbl antibody. B, 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 GST proto-Dbl or GST DH/PH. The reactions were terminated by nitrocellulose filtration at the indicated times, and the amount of radioactivity at time 0 was taken as 100%. C, [3H]GDP/GTP exchange on Cdc42 was measured in the same buffer described above in the presence or absence of 50 ng of GST-proto-Dbl, GST-proto-Dbl PH-t, GST-proto-Dbl Delta C, GST-proto-Dbl Delta C PH-t, GST-DH/PH, and DH/PH-t. The reaction was terminated by nitrocellulose filtration at the 10-min time point, and the amount of radioactivity at time 0 was taken as 100%. The results shown are representative of three independent experiments.

We next analyzed proto-Dbl proteins for their ability to stimulate [3H]GDP/GTP exchange from Cdc42 in the presence of PIPs by incubating proto-Dbl proteins for 10 min in the GEF reaction mixture in the presence of 10 µM PI4,5P2 or PI3,4,5P3. As previously reported (20), Dbl DH/PH protein GEF activity was inhibited by PIPs, whereas the protein carrying the mutant PH domain, which is unable to bind PIPs, was not affected by them (Fig. 3). Similarly, the inhibitory effect of PI4,5P2 and PI3,4,5P3 was evident on proto-Dbl and proto-Dbl Delta C (Fig. 3), whereas proto-Dbl PH-t and proto-Dbl Delta C PH-t were not affected by PIPs in their GEF capacity (Fig. 3). Thus, these results suggest that similar to the onco-Dbl case, PI4,5P2 or PI3,4,5P3 binding to the PH domain negatively regulates proto-Dbl GEF activity in vitro, and substitutions of positively charged amino acids to neutral ones in the PH domain beta 1-beta 2 loop do not affect the intrinsic catalytic activity but rather the PIP-elicited inhibitory response.


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  		Fig. 3.   Histograms show the effects of PI4,5P2 and PI3,4,5P3 on the GEF activity of proto-Dbl mutants. The GEF reactions on Cdc42 were carried out with proto-Dbl or onco-Dbl DH/PH proteins in the presence or absence of 10 µM PI4,5P2 and PI3,4,5P3. The results shown are representative of three independent experiments.

Effects of PH Domain Mutations and C-terminal Deletion on the GEF Activity of Proto-Dbl in Vivo-- To determine whether the GEF activity of the proto-Dbl protein in vivo is affected by binding to PIPs or by the presence of the C-terminal sequences, COS cells were transiently transfected with proto-Dbl, proto-Dbl Delta C, proto-Dbl PH-t, or proto-Dbl Delta C PH-t constructs, and the active Cdc42-GTP and RhoA-GTP were collected on GST-PAK-CRIB and GST-mDIA fusion proteins, respectively. As shown in Fig. 4A, expression of proto-Dbl mutants effectively induced activation of endogenous Cdc42 in these cells. Densitometric analysis revealed that the expression of proto-Dbl Delta C, proto-Dbl PH-t, and proto-Dbl Delta C PH-t mutants increased the active Cdc42-GTP level by 5-fold in comparison with proto-Dbl WT. On the other hand, the expression of proto-Dbl Delta C increases RhoA activation by ~3-fold, whereas both PH-t mutants, with or without the C terminus sequences, were not able to activate Rho A GTPase above background level (Fig. 4B). The differences observed in GTPases activation were not due to differences in onco-Dbl and proto-Dbl expression levels, since no significant variations in protein amounts were detected by immunoblotting with specific anti-Dbl antibody (Fig. 4). Moreover, as previously reported (20), DH/PH and DH/PH-t both effectively induced activation of endogenous Cdc42 and RhoA (Fig. 4, A and B, respectively). Thus, loss of PH binding to PIPs results in different effects on proto-Dbl and Dbl mutants' ability to activate RhoA; DH/PH-t is still able to activate RhoA, while the PH mutants of proto-Dbl have lost this ability. Interestingly, deletion of the C-terminal sequences further enhance the RhoA-specific GEF activity, suggesting a negative regulatory action of these sequences on proto-Dbl catalytic activity in cells.


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  		Fig. 4.   Comparison of the in vivo GEF activity of proto-Dbl mutants. 0.05 mg of pCEFL-GST plasmid expressing proto-Dbl, proto-Dbl PH-t, proto-Dbl Delta C, proto-Dbl Delta C PH-t, Dbl DH/PH, and DH/PH-t were stable transfected into NIH3T3 cells. Specific Dbl and proto-Dbl products were detected by immunoblotting using an anti-Dbl antibody (16). Three weeks after transfection, cells were lysed, and pull-down assays were performed. A, cell lysates were subjected to GST-PAK 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.

Effects of PH Domain Mutations and C-terminal Deletion of Proto-Dbl on JNK Activation-- Rho family GTPases exert their effect at least in part through the activation of serine-threonine kinase signaling pathways. It has been shown that onco-Dbl transformed cells show significantly elevated JNK activity (28, 29). To explore how the mutations in the PH domain and the C-terminal deletion of proto- and onco-Dbl affect JNK activation, we transiently transfected COS cells with plasmids encoding for HA-JNK in the absence or presence of proto-Dbl or onco-Dbl cDNA constructs. Cells were harvested 24 h after incubation in serum-free medium. As shown in Fig. 5A, the expression of proto-Dbl Delta C increased JNK activity by 5-fold compared with WT proto-Dbl. In fact, the activity of JNK in proto-Dbl Delta C transfectants was comparable with that detected in cells transfected with onco-Dbl DH/PH (Fig. 5B). The expression level of each proto-Dbl and onco-Dbl product was comparable in all of the samples analyzed, as shown by immunoblotting analysis of total cell lysates with anti-Dbl antibody (Fig. 5). These results indicate that the presence of the C terminus 50 amino acids greatly affects the ability of proto-Dbl to activate JNK. In contrast, a basal JNK kinase activity was detected in proto-Dbl PH-t- and proto-Dbl Delta C PH-t-transfected cells, whereas DH/PH-t displayed a reduced capability of activating JNK compared with its wild type counterpart. Thus, mutations in the PH domain seem to affect the ability of both proto-Dbl and onco-Dbl to activate JNK.


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  		Fig. 5.   Activation of JNK kinase activity by proto-Dbl and onco-Dbl DH/PH proteins. COS cells were transiently cotransfected with pcDNA3-HA-JNK and pCefl-GST plasmid expressing proto-Dbl and DH/PH cDNAs. Kinase was immunoprecipitated, and its activity was assayed on GST-ATF2. Phosphorylated substrate was detected by autoradiography and quantitated by densitometric analysis. Western blot analysis was performed using total cell lysates, and proto-Dbl protein expression levels were evaluated with anti-proto-Dbl antibodies. The amount of the kinase was determined by Western blot with specific antibody. Results shown are representative of three independent experiments.

Stimulation of NF-kappa B and Cyclin D1 Expression by Proto-Dbl Mutants-- It has been recently reported that both Dbl and Rho family members can stimulate transcription from NF-kappa B-responsive elements (30, 31). It has also been suggested that transformation of NIH3T3 cells by onco-Dbl may require NF-kappa B activation (32). We therefore examined how the PH mutations and C-terminal deletion affect NF-kappa B activation by proto-Dbl and Dbl expression. NIH3T3 cells were transiently transfected with each of the proto-Dbl and onco-Dbl constructs in the presence of the NF-kappa B-luciferase reporter plasmid that contains the promoter response elements of NF-kappa B. We observed a significant activation of luciferase activity in all of the samples analyzed in comparison with the activity of the vector alone (Fig. 6A), suggesting that the PH mutations as well as the deletion of the C-terminal 50 amino acids do not interfere with NF-kappa B activation in the transfected cells.


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  		Fig. 6.   Activation of NF-kappa B and cyclin D1 promoters by proto-Dbl mutants. 0.5 µg of indicated proto-Dbl mutant or the empty vector pCEFL was cotransfected with the NF-kappa B-luciferase (50 ng) (A) or cyclin D1-luciferase (1 µg) (B) reporter plasmid into the cultured NIH3T3 cells. The cells were allowed to recover for 30 h, followed by starvation in DMEM supplemented with 0.5% calf serum for another 16 h. The luciferase activities in the cell lysates were assayed by using a luciferase substrate and are indicated as the -fold activation relative to the level of the empty vector.

It has been demonstrated that stimulation of cyclin D1 transcription correlates with Dbl activity in vivo (33). We next analyzed cyclin D1 promoter stimulation by proto-Dbl proteins in cotransfected NIH3T3 cells. Proto-Dbl, proto-Dbl Delta C, and Dbl DH/PH proteins all stimulated cyclin D1 promoter expression about 3-fold in comparison with control vector (Fig. 6B). In contrast, proto-Dbl PH mutant proteins did not cause elevated cyclin D1 expression, whereas onco-Dbl DH/PH-t remained capable of stimulating cyclin D1 (Fig. 6B). These data suggest that mutations in the PH domain interfere with the ability of proto-Dbl to stimulate transcription from cyclin D1 promoter but not that of onco-Dbl.

The C-terminal Truncation Is Associated with Increased Membrane Localization of the Proto-Dbl Protein-- We have previously demonstrated that both Dbl PH domain and the oncogenic protein localize to the plasma membrane and that mutations of the positively charged amino acids located in the loop between beta -sheet one and beta -sheet two of the PH domain impaired membrane localization (20). We have also demonstrated that the N-terminal sequences of proto-Dbl contain elements that impose the intracellular distribution of proto-Dbl by interfering with the PH domain targeting function such that proto-Dbl displays a mostly perinuclear distribution pattern and a rather limited membrane localization (18). Thus, to evaluate the subcellular localization of the proto-Dbl mutants, we performed immunofluorescence followed by confocal analysis in NIH3T3 fibroblasts expressing proto-Dbl, proto-Dbl PH-t, proto-Dbl Delta C, or proto-Dbl Delta C PH-t. For comparison, we also analyzed cells transfected with Dbl DH/PH and Dbl DH/PH-t. Cells were stained with anti-GST polyclonal antibodies, followed by fluorescein isothiocyanate-conjugated secondary antibodies, and observed with a Zeiss confocal microscope.

In agreement with our earlier results (20), staining of Dbl DH/PH appeared diffuse all over the cytoplasm and associated with the plasma membrane, while in cells transfected with the mutant DH/PH-t, no signal was detected on the plasma membrane (Fig. 7). Moreover, cells expressing Dbl DH/PH displayed a typical Dbl-transformed phenotype, appearing rather enlarged and multinucleated (23, 34) and characterized by a polygonal shape and pronounced membrane ruffling (arrows in the DH/PH image, Fig. 7) or occasional lamellipodia at the cell surface (short arrows in the DH-PH image, Fig. 7). Cells transfected with Dbl DH/PH-t, on the other hand, were elongated (Fig. 7), showing a typical fibroblastic shape and a single nucleus.


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  		Fig. 7.   Immunofluorescence analysis of the distribution of GST-proto-Dbl fusion proteins. NIH3T3 fibroblasts transfected with GST-proto-Dbl, GST-proto-Dbl PH-t, GST-proto-Dbl Delta C, GST-proto-Dbl Delta C PH-t, GST-DH/PH, and DH/PH-t were stained with anti-GST polyclonal antibodies and fluorescein isothiocyanate-conjugated secondary antibodies and analyzed with a Zeiss confocal microscope. Staining appears mostly diffused in the cytoplasm of the cells and is localized on the plasma membranes of cells expressing the proto-Dbl (arrows) and, to a larger extent, on the plasma membranes of cells expressing the proto-Dbl Delta C (arrows). Bar, 10 µm.

Staining of the WT proto-Dbl product was cytoplasmic with a perinuclear distribution and some membrane localization, whereas, similarly to what observed for Dbl DH/PH-t, no staining was detected on the plasma membrane of cells expressing proto-Dbl PH-t or proto-Dbl Delta C PH-t proteins (Fig. 7). Cells expressing proto-Dbl were flat, showing a slight enlargement of the cell body and membrane ruffles at the cell surface (arrows in Fig. 7), while the morphology of the cells expressing proto-Dbl PH-t or proto-Dbl Delta C PH-t (Fig. 7) was similar to that of cells transfected with Dbl DH/PH-t. Interestingly, removal of the C-terminal sequences induced a more extensive localization of the proto-Dbl Delta C protein along the plasma membrane (Fig. 7), and part of the cells displayed both a more evident enlargement of the cell body and membrane ruffling (arrows in Fig. 7).

In summary, these results indicate that removal of the C-terminal sequences from proto-Dbl is associated with a stronger localization of the protein to the plasma membrane and the acquisition of a morphology that resembles that of cells transformed by oncogenic Dbl. On the other hand, proto-Dbl proteins that carry a mutated PH domain do not translocate to the plasma membrane and show a morphology typical of untransformed NIH3T3 cells.

C-terminal Truncation Is Associated with Increased Stability of Proto-Dbl Protein-- We have previously demonstrated that sequences within the N-terminal half of proto-Dbl contribute to a rapid turnover of proto-Dbl (19) and that this instability is associated with a reduced transforming activity of this protein in comparison with the oncogenic Dbl. Likewise, the higher transforming activity of the proto-Dbl Delta C could be due to a greater stability of this protein. In fact, the C-terminal domain of proto-Dbl contains a stretch of proline, glutamic acid, serine, and threonine residues (PEST sequences) that have been implicated in the rapid turnover of proteins (35, 36). To examine if the PEST sequences in the C terminus are involved in the stability of proto-Dbl, NIH3T3 cells expressing proto-Dbl and proto-Dbl Delta C products were pulse-labeled with [35S]methionine and [35S]cystine for 30 min and chased for various time intervals. As shown in Fig. 8, the half-life of the proto-Dbl product was around 2 h, in agreement with our earlier observation (17, 19), while the Delta C mutant protein had a half-life of ~5 h, similar to onco-Dbl (17). Moreover, the C terminus deletion does not significantly modify the half-life of the DH/PH protein whose stability remains similar to that of the onco-Dbl. These data suggest that the C-terminal residues regulate the turnover rate of proto-Dbl but not that of onco-Dbl.


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  		Fig. 8.   Effect of C-terminal deletion on onco-Dbl and proto-Dbl half-life. NIH3T3 transfectants were labeled with [35S]methionine and [35S]cysteine for 30 min and then chased with cold DMEM containing an excess of cold methionine and cysteine. Prior to the immunoprecipitation, incorporation of labeled amino acids into trichloroacetic acid-precipitable counts and the amount of total cellular proteins were determined. The counts/mg of protein were similar in each cell line. Immunoprecipitation was performed at the indicated time intervals using anti-Dbl antibody. The results shown are representative of three independent experiments.

Effect of the PH Domain Mutations and the Deletion of C-terminal Region on Proto-Dbl Transforming Activity-- To estimate the transforming potential of proto-Dbl mutants, the respective cDNAs, cloned into the mammalian pCefl-GST expression vector, were transfected into NIH3T3 cells. For comparison, WT onco-Dbl, DH/PH, and DH/PH-t were also included in the same set of experiments. As negative control, pCefl-GST vector alone was tested in parallel.

Onco-Dbl displayed a transforming activity of 3.1 × 105 focus-forming units/µg (Table I). As previously reported (23), removal of the C-terminal 50 residues did not further modify the transforming activity of onco-Dbl, whereas the transforming activity of the DH/PH-t mutant was about 3-fold lower than that of onco-Dbl and the WT DH/PH module (Table I). In comparison, proto-Dbl transforming activity was reproducibly about 20-fold lower than that of onco-Dbl or DH/PH, in agreement with our previously published observations (16, 19). The presence of the mutations in the PH domain completely abolished the transforming activity of both proto-Dbl PH-t and proto-Dbl Delta C PH-t but not that of onco-Dbl or DH/PH (Table I). These results argue that the PH interaction with PIPs is critical for the transforming activity of proto-Dbl but not that of onco-Dbl. Moreover, removal of the C-terminal 50 residues from proto-Dbl increased the transforming activity of proto-Dbl by 3.8-fold, indicating that the C terminus sequences that control the protein stability contribute to the regulation of proto-Dbl activity.

                              
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Table I
Comparison of the focus forming activity of proto-Dbl and its mutants
Transforming activity of proto-Dbl mutants is shown. Transfection assays were done on duplicate cultures by titration of each DNA (1.0, 0.1, and 0.01 µg) on recipient NIH3T3 cells. One set of each transfectant was used to score foci at 10-21 days post-transfection. The second set was grown in the presence of 375 mg/ml G418. Colonies of G418-resistant cells were scored 14 days after transfection. The results shown are the summary of four independent experiments. FFU, focus-forming units.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The Dbl family GEFs for Rho GTPases constitute one of the largest known groups of transforming proteins. While each member contains diverse multifunctional motifs, they all share the structural array of a central DH catalytic domain in tandem with a regulatory PH domain. We have previously analyzed the involvement of PH domain in the regulation of onco-Dbl activity (20, 37). We found that oncogenic Dbl localizes both to the plasma membrane and to the actin structures and that the PH domain mediates this localization. The PH domain interacts with PIPs, and this interaction is necessary for targeting the DH domain to the plasma membrane, modulating the GEF activity of the DH domain and affecting the cellular activity of onco-Dbl or the DH/PH module. In fact, mutations in the PH domain of the onco-Dbl or DH/PH backbone that inhibit binding to PIPs result in the inhibition of membrane localization, a loss in the response to PIPs in GEF activity, and a ~3-fold reduction of the transforming activity. However, such mutations do not seem to interfere with the DH catalytic activity, since the DH/PH-t mutant remains capable of stimulating Cdc42 and RhoA activation to a similar extent as wild type DH/PH in vitro and to a higher extent in vivo (20). Further, the PH mutations do not alter the actin cytoskeleton association pattern of onco-Dbl (20).

The transforming potential of Dbl family proteins directly correlates with their GEF activity, which may stay at a basal, autoinhibitory state prior to activation upon alterations of the protein conformation in response to upstream signals (4). The GEF protein function may thus be modulated by diverse regulatory mechanisms, which involve intra- and/or intermolecular interactions between different protein domains (7, 18, 38), oligomerization (39), phosphorylation (7, 8), and interaction with different intracellular signaling molecules such as PIPs (13, 15, 20), heterotrimeric G-protein subunits (9, 40-42), and/or scaffolding proteins (10, 11). Our previous work has established that proto-Dbl activity is tightly regulated by its N terminus domain. Interaction of this domain with the C-terminal PH domain only partly restrains the membrane localization of the protein but completely prevents its cytoskeleton association and further inhibits the DH catalytic activity. Moreover, the N-terminal sequences of proto-Dbl appear to facilitate a rapid turnover of the protein, shortening the half-life of proto-Dbl in cells. Hence, deletion of the N terminus domain gives rise to the oncogenic form that displays a plasma membrane and actin cytoskeleton localization pattern, increased GEF potential, and enhanced stability of the protein. When overexpressed in NIH3T3 cells, however, proto-Dbl does show transforming activity, perhaps by titrating out a negative regulator, although it is about 20-100-fold lower than that of onco-Dbl.

In the present work, we sought to further characterize the mechanism of regulation of proto-Dbl. A summary of the results is outlined in Fig. 9. In particular, we have analyzed the effects of the PH domain binding to PIPs on proto-Dbl transforming activity and compared such effects with those observed with onco-Dbl. Furthermore, we examined the effects of deleting the carboxyl-terminal sequences of proto-Dbl. We found that, unlike the case for oncogenic Dbl, mutating the PH domain of proto-Dbl did not abolish its guanine nucleotide exchange factor activity toward Cdc42 in vitro, although it did impact its ability to activate RhoA in cells. These mutants also lost the ability to associate with the plasma membrane and no longer showed any transformation capability. Deleting the carboxyl-terminal domain from proto-Dbl led to an increase in its ability to transform cells that appeared to correlate with an increased association with the plasma membrane.


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  		Fig. 9.   Summary of proto-Dbl mutants' activities. Proto-Dbl protein contains different motifs that might be involved in the regulation of the DH catalytic domain. Results of the current studies of proto-Dbl are summarized together with previous published data on onco-Dbl.

We have observed a different effect of the PH mutations on proto-Dbl activity in comparison with onco-Dbl. Proto-Dbl transforming activity is completely abolished by the lack of association with PIPs, but similar PH mutations in onco-Dbl led to only ~3-fold reduction in transforming activity (Fig. 9). The difference of the effect on transformation by proto-Dbl and onco-Dbl induced by the PH mutations may be rationalized by the fact that the PH domain of onco-Dbl is fully exposed and therefore still able to interact with cytoskeleton components in the absence of lipid binding capacity. Conversely, the PH domain of proto-Dbl is partially buried through intramolecular interaction with the N-terminal domain, thus incapable of recognizing additional binding partners such as certain cytoskeleton components once the lipid binding capability is lost by the mutations.

Similarly to what we have shown for the DH/PH-t-expressing cells, GTP-bound Cdc42 also increases by severalfold in cells expressing proto-Dbl PH mutants, but this activation appears to be ineffective in inducing transformation. Rather, the transforming potential of the proto-Dbl mutants seems to correlate with their ability to activate RhoA in cells, not Cdc42 (Fig. 9). Thus, mutations in the PH domain do not interfere with proto-Dbl GEF activity per se, but rather they may affect the selectivity of Rho GTPase activation, probably through the alteration of the intracellular targeting function of the PH domain.

Deletion of the C-terminal 50 amino acids induces a partial activation of proto-Dbl transforming activity. Proto-Dbl Delta C protein localizes more extensively to the plasma membrane, as visualized by the widespread staining along the plasma membrane of the proto-Dbl Delta C mutant transformed cells, and it activates Cdc42 and RhoA more efficiently than wild type proto-Dbl. These cellular effects correlate with a 4-fold increase in transforming activity of the mutant. On the contrary, deletion of this region from onco-Dbl does not affect the oncoprotein transforming capability or GEF activity. We have previously demonstrated that proto-Dbl and onco-Dbl proteins differ by their half-life in cells and that the rapid turnover of proto-Dbl correlates with its lower transforming activity (19). Analysis of the half-life of the proto-Dbl Delta C product indicates that the stability of the protein is similar to that of onco-Dbl, which is about 21/2-fold longer than that of proto-Dbl, indicating that deletion of the N terminus or the C terminus or both induces a similar increase in the half-life of the protein. Thus, our results here suggest that the instability of proto-Dbl can be mediated by both the N- and the C-terminal sequences.

The C terminus region contains a PEST sequence motif (21), a hydrophilic stretch of 24 amino acids (residues 888-913) enriched in proline, glutamic acid, serine, and threonine. It has been reported that PEST sequences with PEST-FIND scores greater than +5 can be considered the best candidates for being degradation signals (43). Since proto-Dbl PEST sequences score +6.5 they probably represent one such degradation signal. At this stage, it is not clear why the proto-Dbl stability is affected by the presence of C-terminal PEST sequences but onco-Dbl is not. One possible explanation relies on the presence of the N terminus sequences. This region, by binding to the PH domain, strongly inhibits the localization of the protein to the plasma membrane, sensitizing the protein to proteolytic degradation. On the other hand, binding to the plasma membrane may elicit a conformational change that prevents recognition of the PEST sequences by the cellular proteolytic machinery. Mechanisms that involve PEST motif sequestration by protein/protein interaction and thus regulation of protein turnover have been described (36, 44). Since the transforming activity of proto-Dbl Delta C mutant is about 5-fold lower than that of the DH/PH module, it appears that the inhibitory effect of the N-terminal sequences is also effective in preventing the deregulation of the protein.

The Dbl family GEFs have been linked to the regulation of gene expression through Rho GTPase substrates. It has been reported that onco-Dbl expression in cells stimulates the activation of JNK (28, 29). We found that activation of JNK directly correlates with the transforming potential of proto-Dbl mutants. Proto-Dbl Delta C protein, which displayed a transforming capability 4-fold higher than that of its normal counterpart, induced ~5-fold activation of JNK. On the contrary, proto-Dbl PH mutants, which are completely impaired in their transforming capability, failed to stimulate JNK above basal level, whereas the DH/PH-t mutant, which has a decreased transforming activity, also displayed a weakened JNK-stimulating activity in comparison with DH/PH WT. Under these assay conditions, the level of JNK activity in proto-Dbl Delta C mutant-expressing cells is similar to that of the onco-Dbl cells even if the transforming activity of this mutant is about 5-fold lower than onco-Dbl. The discrepancy here may be attributed to difference in cell settings of the assays, i.e. the focus forming activity of each construct analyzed is evaluated in stable transfections, whereas the kinase assays utilize transient transfections that may not allow to discern differences of activation above a certain level.

Transforming Dbl family proteins have been found to efficiently activate transcription from NF-kappa B-responsive elements and cyclin D1 promoter (32, 33), but the pathways by which this activation occurs and whether these transcription events are essential for transformation remain unclear. Jun may induce stimulation of transcription from cyclin D1 promoter, but a direct correlation between JNK activation, cyclin D1 expression, and transformation efficiency by Dbl family proteins has not been established. In this respect, it has been suggested that activation of transcription from cyclin D1 promoter occurs through Dbl protein-mediated activation of NF-kappa B rather than JNK (33). In agreement with these reports, we show that activation of transcription from cyclin D1 promoter correlates with the transforming capability of onco-Dbl and proto-Dbl proteins. Conversely, we found that NF-kappa B is activated at similar levels by all the mutants analyzed, independently of their transforming capability. Thus, our results suggest that proto-Dbl and DH/PH proteins may activate cyclin D1 through activation of JNK, which in turn may activate ATF-2 and Jun nuclear transcription factors to stimulate transcription from cyclin D1 promoter. In our assays, proto-Dbl, proto-Dbl Delta C, and their oncogenic counterparts all activate cyclin D1 at similar levels, even if they significantly differ by their transforming capability. It is likely that this also reflects the differences in transient (cyclin D1 reporter assays) and stable (focus-forming assays) transfection methods. Overall, the pathway from proto-Dbl and its RhoA substrate to JNK and cyclin D1 appears to be a critical transcriptional event that correlates with cellular transformation (Fig. 9). Details of this signaling pathway regulated by Dbl and Dbl-like molecules remain to be defined.

The GEF activity of proto-Dbl in vitro is not affected by the integrity of the PH domain or the C-terminal sequences and thus does not correlate with the protein transforming activity. In fact the WT and mutants all show the similarly weak GEF activity on both Cdc42 and RhoA in vitro. Proto-Dbl shows measurable transforming and biochemical activities in vivo when overexpressed in the cells, although these activities remain significantly lower than those of the onco-Dbl (Fig. 9). Further, we found that the GEF activity of proto-Dbl on Cdc42 in vivo does not correlate with the gene transforming activity or with the gene's ability to activate transcription such that the PH mutants all strongly activate Cdc42 in cells without showing a comparable increase in transforming activity. Conversely, the in vivo GEF activity of proto-Dbl on RhoA appears to directly correlate with its signaling activities and its ability to transform cells and is dependent on the protein localization on the plasma membrane (Fig. 9).

In summary, we have observed that both the interaction of the PH domain with PIPs and the presence of the C terminus sequences affect transformation by proto-Dbl. The increased transforming activity of proto-Dbl mutant by deletion of the last 50 amino acids at the C terminus domain is accompanied by an increased stability of the protein, a stronger localization to the plasma membrane, the activation of RhoA GTPase, and JNK/cyclin D1 signaling. On the other hand, the loss of transforming activity of the proto-Dbl mutants bearing the PH domain mutations correlates with the loss of plasma membrane targeting ability, the loss of RhoA activating potential, and the dampened ability to stimulate JNK and cyclin D1 pathways. These observations, taken together with previous studies of onco-Dbl (18, 20), argue that regulation of proto-Dbl activity may be distinct from that of onco-Dbl or the DH/PH module in many aspects. Proto-Dbl is probably tightly regulated by a combination of mechanisms that involve intra- and intermolecular interactions, PH binding to PIPs, and N- and C-terminal domain-dependent turnover of the protein.

    ACKNOWLEDGEMENTS

We are grateful to Dr. S. Gutkind for providing pCEFL-GST vector, to Dr. J. Collard for providing GST-PAK, and to Dr. S. Narumiya for providing GST-mDia.

    FOOTNOTES

* This work was supported by grants from the Italian Association for Cancer Research, from Ministero Universitá Ricerca Scientifica Tecnologica, and from Consiglio Nazionale delle Ricerche (Target Project on "Biotechnologies") and by National Institutes of Health Grant GM53943 (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.

|| Present address: Division of Experimental Hematology, Children's Hospital Research Foundation, 3333 Burnet Ave., Cincinnati, OH 45229.

** To whom correspondence should be addressed: Laboratorio di Biologia Molecolare, Istituto G. Gaslini, Largo Gaslini 5, 16147 Genova, Italy. Tel.: 1-39-010-5636633; Fax: 1-39-010-3733346; E-mail: biolmolecolare@ospedale-gaslini.ge.it.

Published, JBC Papers in Press, March 20, 2002, DOI 10.1074/jbc.M111025200

    ABBREVIATIONS

The abbreviations used are: GEF, guanine nucleotide exchange factor; DH, Dbl homology; PH, pleckstrin homology; PI, phosphatidylinositol; PIP, phosphatidylinositol phosphate; PI4, 5P2, phosphatidylinositol 4,5-bisphosphate; PI3, 4,5P3, phosphatidylinositol 3,4,5-triphosphate; DMEM, Dulbecco's modified Eagle's medium; GST, glutathione S-transferase; JNK, c-Jun N-terminal kinase; HA, hemagglutinin; WT, wild type.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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