©2006 L

ANDES BIOSCI

ENCE.

DO NOT DIST

RIBUTE.Cristina Vanni1

Vincenzo Visco2,3

Patrizia Mancini2

Alessia Parodi1,6

Catherine Ottaviano1 Marzia Ognibene1 Andrea D. Manazza4

Saverio Francesco Retta5 Luigi Varesio1 Maria Rosaria Torrisi2,3 Alessandra Eva1,*1Laboratorio di Biologia Molecolare; Istituto G. Gaslini; Genova Italy

2Dipartimento di Medicina Sperimentale; Università di Roma; Roma Italy

3Azienda Ospedaliera Sant’Andrea; Roma Italy

4Dipartimento di Patologia e CeRMS; Università di Torino; Torino Italy

5Dipartimento di Genetica; Biologia e Biochimica; Università di Torino; Torino Italy

6Present address: Centro Cellule staminali; Azienda Ospedaliera Universitaria S. Martino; Genova Italy

*Correspondence to: Alessandra Eva; Laboratorio di Biologia Molecolare; Istituto G. Gaslini; Largo Gaslini 5; 16147 Genova Italy; Tel.: +39.010.5636633; Fax: +39.010.3733346; Email: alessandraeva@ospedale-gaslini.ge.it

Original manuscript submitted: 09/13/06Manuscript accepted: 09/28/06

Previously published online as a Cell Cycle E-publication:http://www.landesbioscience.com/journals/cc/abstract.php?id=3445

KEy wORDS

proto-Dbl, GEF, GTPase, MAPK, transfor-mation, proliferation, motility

ACKnOwLEDgEMEnTS

We are grateful to Drs. T. Balla, J. Downward, and J. S. Gutkind for providing reagents. This work was supported by grants from the Italian Association for Cancer Research (AIRC), from the Compagnia S. Paolo, Torino, from the Italian Health Ministry and from MIUR.

Report

Inhibition of PI3K induces Rac Activation and Membrane Ruffling in Proto-Dbl Expressing Cells

AbSTRACTProto‑Dbl protein, a guanine nucleotide exchange factor (GEF) for Rho GTPases, is

tightly regulated by a combination of mechanisms that involve intra‑ and intermolecular interaction and N‑ and C‑terminal domain‑dependent turnover of the protein. Moreover, the interaction of the PH domain of proto‑Dbl with phosphoinositides regulates its subcellular localization and biological activity. Here we show that inhibition of the phosphatidylinositol 3‑kinase (PI3K) by molecular and pharmacological inhibitors causes a strong inhibition of proto‑Dbl‑induced cell proliferation and transformation. Conversely, inhibition of PI3K results in the translocation of proto‑Dbl to the plasma membrane, Rac activation and increased membrane ruffles and cell motility. Furthermore, we investigated the signaling molecules involved in proto‑Dbl‑induced cell transformation and motility and observed that inhibition of PI3K in proto‑Dbl expressing cells induces an increase in p38 activity and a decrease in ERK phosphorylation. Our results suggest that proto‑Dbl activates distinct downstream effectors to induce morphological changes and cell transformation.

InTRODuCTIOnThe proto-Dbl protein is the prototype of the Dbl family of guanine nucleotide

exchange factors (GEFs) for Rho family of G proteins. These proteins are all characterized by a region of homology sequence in which a Dbl homology domain (DH), responsible for catalyzing the nucleotide exchange on Rho GTPases, is located just upstream of the pleckstrin homology domain (PH), responsible for the proper subcellular localization of the protein.1-3 Proto-Dbl is a cytoplasmic phosphoprotein of 115 kDa that contains a long stretch of amino acids within its NH2-terminal half that negatively regulate its transforming activity 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.4 We have previously shown that the interaction of proto-Dbl PH domain with phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-triphosphate (PIP3) modulates its intracellular localization and transforming activity.5 In fact, proto-Dbl protein that bears mutations in the PH domain, which affect the binding to phosphoinositides (PIPs), shows the loss of plasma membrane targeting ability and transforming activity.

It has been shown that PIPs, and in particular PIP2, have a pivotal role in cytoskeletal organization during diverse cellular functions such as vesicle trafficking, focal adhesion formation and cell migration.6,7 In mammalian cells PIP2 not only serves as a substrate for PLC and PI3K, but it can directly interact with and affect the localization, the catalytic activity or the functionality of many intracellular proteins, such as actin-binding and actin-remodeling proteins.7

Small GTPases of the Rho family (Rho, Rac and Cdc42) regulate a variety of signal transduction pathways that have important effects on the actin cytoskeleton, gene expres-sion and cell proliferation.8,9 RhoA activity regulates the formation of stress fibers and focal adhesions, while Rac regulates lamellipodium production and membrane ruffling. The formation and turnover of these structures are important in cell migration.10 Since Rho GTPases and PIP2 are involved in the regulation of actin dynamics, it was hypoth-esized that Rho proteins might exert their effects by modulating PIPs metabolism.11 Some of the actin signaling pathways depend on intact PIP2 rather than on the product of its hydrolysis.12,13 In particular, it has been shown that PIP2 is required for the effects of Rho family proteins on the formation and stabilization of actin filaments.10

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In light of these studies, we attempted to determine whether and how the binding to PIPs could affect the proto-Dbl-mediated biolog-ical processes including focus forming activity, cell morphology and motility. We show that treatment of cells expressing proto-Dbl with pharmacological and molecular inhibitors of PI3K results in the translocation of proto-Dbl to the plasma membrane, increase in Rac activation and formation of membrane ruffles and lamellipodia. These morphological changes are associated with the stimulation of the migration ability of proto-Dbl expressing cells. On the other hand, the treatment of proto-Dbl-expressing cells with the PI3K inhibitors causes a strong reduction in cell proliferation and trans-formation. We also show that the treatment of proto-Dbl expressing cells with the PI3K inhibitor LY294002 causes an increase in p38 activity, and a decrease in ERK phosphorylation, while JNK kinase activity is not affected. Our results suggest that proto-Dbl activate different signaling pathways to induce cell transformation and cell motility.

MATERIALS AnD METhODSReagents and plasmids. LY294002 was purchased from SIGMA

Aldrich (St. Louis, MO, USA) . Wortmannin was purchased from Calbiochem (Merck KgaA, Darmstad, Germany). Monoclonal anti-body against Rac was purchased from Upstate Biotecnology (Lake Placid, NY, USA). Monoclonal antibody anti HA was purchased from Babco (Richmond, CA, USA). Anti phospho-ERK and phospho-Akt antibodies were purchased from New England Biolabs (Beverly, MA, USA). Polyclonal antibodies against ERK1/2 and Akt were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti GST was purchased from Molecular Probes (Inc., Eugene, OR). Anti His was from Invitrogen (Carlsbad, CA, USA). pCEFL-GST-proto-Dbl construct was previously described.5 pEF-His-proto-Dbl construct was obtained by subcloning proto-Dbl cDNA into the BamHI cloning site of pEF4C vector (Invitrogen). pCDNA3 HA-JNK and pCDNA3 HA-p38 were kindly provided by S. Gutkind. p85aDinter-SH2N and Akt-PH constructs, kindly provided by J. Downward and T. Balla, respectively, were subcloned into the BamHI/EcoRI restriction sites of pCEFL-GST vector.

Cell culture and transfection. NIH3T3 fibroblasts were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% calf serum. Mass cultures of stable transfected cell lines were generated by transfecting NIH3T3 cells with 100 ng of plasmid DNA by the calcium phosphate coprecipitation method and culturing them in DMEM supplemented with 375 mg/ml of G418.14 Focus forming assay was performed by transfecting NIH3T3 cells with 300 ng of plasmid DNA by the calcium phosphate coprecipitation method and culturing them in DMEM supplemented with 5% calf serum in the presence or absence of 10 mM LY294002. Cotransfection experiments were performed utilizing 5-fold excess (pmoles) of pCEFL-GST vector, pCEFL-GST-Dp85a or pCEFL-GST-Akt-PH as indicated. Foci of transformed cells and colonies of G418-resistant cells were scored 15–21 days after transfection.

COS cells were obtained from ATCC (Manassas, VA, USA) and cultured in DMEM supplemented with 10% fetal calf serum. For kinase assays and pulldown experiments cells were grown to 80% confluence in 100-mm tissue culture dishes and transiently trans-fected with 4 mg of the indicated plasmids using LipofectAMINE PLUS as described by the manufacturer (Invitrogen). Twenty-four hours after transfection the medium was changed to DMEM containing 0.5% fetal calf serum, the cells were incubated for another 18 hours and treated with 20 mM LY294002 for 30 min at 37˚C

before lysis.Cell growth curve. Confluent cells were detached from culture

dishes, resuspended in DMEM containing 10% calf serum and counted. Twenty-five x 103 cells were plated per well of a 24 well microtiter plate. Cells were cultured for 24, 48, 72 and 96 hr in the presence or absence of LY294002 or wortmannin as indicated and growth was determined by counting cells.

Immunoblot analysis. Cells lysates were made by washing the cells with cold phosphate-buffered saline (PBS) on ice and then lysed in a lysis buffer containing 1% Triton X-100, 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM b-glicerolphosphate, 1 mM sodium orthovanadate, 1 mM AEBSF, 10 mg/ml each of aprotinin and leupeptin. Protein concentration was determined with the Bradford method (Biorad). Lysates were subjected to SDS-PAGE and transferred to Immobilon-P membranes (Millipore Corp.).

Proteins were detected by Western Blot analysis with specific antibodies, according to protocols provided by the manufacturer.

Kinase assay. pCDNA3 HA-JNK or pCDNA3 HA-p38 constructs were cotransfected in COS cells with pCEFL-GST vector or pCEFL-GST proto-Dbl constructs. JNK kinase activity was deter-mined as described15 using purified bacterially expressed GST-ATF2 fusion protein as substrate. p38 kinase activity was determined in the same way by using myelin basic protein (MBP) (Sigma) as substrate. The HA-tagged proteins were immunoprecipitated by using an anti HA antibody, separated by SDS-PAGE, transferred to Immobilon-P membranes 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 chemioluminescent detection (Pierce) using Protein G horseradish peroxidase conjugated (Pierce).

In vivo Rho GTPase activation assay. The GST-PAK-CRIBThe GST-PAK-CRIB domain fusion protein (residues 56-141) containing the Cdc42 and Rac binding region of human PAK1, was expressed and purified as described previously.16 To evaluate Rac activation, transfected cells were washed with ice-cold PBS 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 mg/ml each of aprotinin and leupeptin, 2 mM AEBSF and 40 mg of GST-PAK. Lysates were incubated with 60 ml of glutathione-coupled Sepharose beads (Amersham Pharmacia Biotech) for 1 hour at 4˚C. Bound Rac was detected in Western Blot by using monoclonal antibody against Rac. The expression of proto-Dbl protein in transfected cells was confirmed by Western blot analysis. Aliquots of cell lysates were subjected to SDS-PAGE and immunoblotting using a specific polyclonal antibody against Dbl.17

Immunofluorescence. NIH3T3 transfectants expressing GST- proto-Dbl protein were plated onto glass coverslips, previously coated with 10 mg/ml fibronectin (Sigma) and treated with 10 mM LY294002 for 30 min. at 37˚C. Alternatively, NIH3T3 cells were transiently cotransfected with pEF4C-proto-Dbl and pCEFL-GST-Akt-PH or pCEFL-GST vector and plated onto glass coverslips, previously coated with 10 mg/ml fibronectin. Cells were fixed in 4% paraformal-dehyde in phosphate-buffered saline (PBS) for 30 min at 25˚C, and permeabilized with 0.1% Triton X-100 in PBS for 5 min. The fusion proteins were visualized with anti GST polyclonal antibodies or anti His followed by incubation with FITC-conjugated goat anti rabbit IgG (Cappel, Organon Teknica Corp.) or with TRITC-conjugated goat anti mouse IgG. Filamentous actin was visualized by incubating the cells with 10 mg/ml TRITC-labeled phalloidin (Sigma) in PBS for 30 min at 25˚C. Fluorescence signals were visualized either by

Role of PI3K in Proto-Dbl Activities

recording stained images using a cooled CCD color digital camera SPOT-2 (Diagnostic Instruments Incorporated, MI) and FISH 2000/H1 software (Delta Sistemi, Roma, Italy) or by confocal vertical 8x-z sections (interval: 0.5 mm) obtained with a Zeiss Confocal Laser Scan Microscope (Zeiss, Oberkochen, Germany). Quantitative analysis of the extension of ruffles on the cell plasma membrane was performed evaluating for each single cell the ratio between the length of the plasma membrane occupied by ruffles and the total length of the plasma membrane. The percentage of cells with different lengths of ruffling was assessed by counting 100 cells for five different micro-scopic fields randomly taken from two different experiments.

Cell migration. Cell migration was assayed in Boyden cham-bers (8.0 mm pore size, FALCON cell culture insert). NIH3T3 stably transfected with pCEFL-GST and pCEFL-GST proto-Dbl were trypsinized and counted. One x 105 cells were resuspended in serum-free medium, added to the upper chamber and treated with 20 mM LY294002. Inserts, previously coated with fibronectin on the lower side, were incubated for 4 h at 37˚C with 700 ml of 8% calf serum in DMEM in the lower chamber. Cells on the inside of the inserts were removed with a cotton swab, and cells on the underside of the insert were fixed and stained with May-Grunwald/Giemsa. The number of cells were counted and the average number of the cells that had transmigrated was determined.

RESuLTSFocus formation activity of Proto‑Dbl is dependent on PI3K

activity. We have previously shown that proto-Dbl is tightly regulated by a combination of mechanisms that involve intra- and inter-mo-lecular interactions, PH binding to PIPs and N- and C-terminal domain-dependent turnover of the protein.

The association of the PH domain with PIPs 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 Dbl protein. In fact mutations in the PH domain that inhibit binding to PIPs result in the inhibition of membrane localization of proto-Dbl and loss of its transforming activity. On this basis, we further investigated the involvement of PIPs in the regulation of proto-Dbl activity.

NIH3T3 cells were transfected with the proto-Dbl cDNA cloned into the mammalian pCEFL-GST expression vector. As negative control, pCEFL-GST vector alone was tested in parallel. Transfected cells were treated with molecular and specific pharmacological inhib-itors of PI3K. As shown in Figure 1A, the treatment of proto-Dbl expressing cells with increasing amounts of the PI3K inhibitor LY294002 resulted in a dose dependent inhibition of proto-Dbl focus forming activity. Moreover, cotransfection of p85aDinter-SH2 (pCEFL-GST-Dp85a), a mutant construct of the PI3K regulatory subunit p85a lacking the minimal binding site for the catalytic subunit p110, that has been used to block PI3K signaling in a domi-nant negative fashion18 caused about 50% inhibition of proto-Dbl transforming activity (Fig. 1B). Finally, coexpression of the PH domain of Akt (pCEFL-GST-Akt-PH), described previously to have a strong inhibitory effect on Akt activation and thus to function as a negative regulator of PI3K signaling,19 also resulted in inhibition of focus formation (Fig. 1B). Cotransfection of proto-Dbl with pCEFL-GST vector did not cause a significant reduction of focus formation (Fig. 1B). These results suggest an involvement of PI3K in the focus forming activity of proto-Dbl.

To further analyze the role of PI3K signaling on proto-Dbl function, we evaluated the activation state of the serine/threonine kinase Akt in proto-Dbl transfected cells. Akt is a major downstream effector of PI3K20 and its function contributes to cell growth and

Figure 1. Proto‑Dbl induced focus formation requires PI3K. A.Transfection assays were done by transfecting pCEFL‑GST‑proto‑Dbl on recipient NIH3T3 cells. Transfected cells were subjected to focus assay with or without treat‑ment with 10, 5 or 2.5 mM LY294002. Foci were scored at 15–21 days post transfection. The results shown are the average of four independent experiments. B. NIH3T3 cells were cotransfected with pEF4C‑proto‑Dbl and pCEFL‑GST, pCEFL‑GST‑Dp85a or pCEFL‑GST‑Akt‑PH. To ensure that the majority of the transfectable cells would acquire and express both proto‑Dbl and the cotransfected plasmids a ratio of 1:5 (pmoles of proto‑Dbl:pmoles of pCEFL‑GST, pCEFL‑GST‑Dp85a or pCEFL‑GST‑Akt‑PH) was utilized. Foci of proto‑Dbl transformed cells were scored 15–21 days after transfection. The results shown are the average of three independent experiments.

Figure 2. Inhibitory effect of LY294002 on proto‑Dbl‑induced Akt activation. COS cells were transfected with the indicated plasmids and total cell lysates were analyzed. The inhibitory efficiency of LY294002 was assessed by Western blotting using anti P‑Akt antibody to detect the activation level of the kinase. The same membrane was probed with anti Akt and anti Dbl for protein level control. Data shown are representative of three independent experiments.

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survival. As shown in Figure 2, proto-Dbl activates Akt, while the treatment with 10 mM LY294002 completely inhibited Akt activity, as reflected by the lack of detection of the Akt phosphorylated form. Similarly, LY294002 completely inhibited Akt phosphorylation in mock transfected cells. These data confirm that LY294002 effectively inhibits PI3K and further support the concept that PI3K has an important role in proto-Dbl-mediated focus formation.

PI3K inhibitors affect Proto‑Dbl‑induced cell proliferation. To evaluate the effects of the treatment with LY294002 on cell prolifera-tion we analyzed and compared growth of proto-Dbl transfectants treated with increasing amounts of LY294002 versus untreated transfectants for different length of time. As shown in Figure 3, while proto-Dbl transfected cells showed an increased growth rate (Fig. 3B), as compared with control cells (Fig. 3A), the growth pattern of proto-Dbl transfectants treated with the PI3K inhibitor was drasti-cally reduced to control levels. Specifically, NIH3T3 cells transfected with proto-Dbl and treated with 10 mM LY294002 grew on average to 39% of their untreated counterpart. The control NIH3T3 cells transfected with the empty vector and treated with 10 mM LY294002 grew to an average of 60% of the untreated control. We also evalu-ated the effects of the treatment with increasing amounts of another PI3K inhibitor, wortmannin on cell proliferation of transfectants for different length of time. As shown in (Fig. 3D), the growth pattern of proto-Dbl expressing cells treated with the PI3K inhibitor was again reduced to control levels (Fig. 3C). We further examined the role of

PI3K signalling on proto-Dbl-induced cell proliferation by evalu-ating G418 resistant colony formation of transfectants treated with increasing amounts of LY294002 versus untreated cells. Proto-Dbl and mock transfected cells were cultured in medium supplemented with 385 mg/ml of G418 for two weeks and colonies were counted. Figures 4A and 4B show the growth inhibitory activity of LY294002 on transfected cells as indicated by the dose-dependent decrease in number of G418-resistant colony formation. In untreated cells the stimulation of proliferation induced by proto-Dbl can be envisaged in the larger size of the colonies formed compared with the colonies of mock transfected cells (Fig. 4C). Therefore, PI3K signaling is essential for proto-Dbl induced cell proliferation.

Signaling pathways involved in Proto‑Dbl‑induced focus forma‑tion. It has been shown that onco-Dbl transformed cells have significantly elevated JNK activity and that this activity is related to the transforming capacity of onco-Dbl protein.5,15,21 Therefore we investigated the signaling pathway involved in proto-Dbl-induced focus formation and evaluated whether the inhibition of PI3K affects proto-Dbl-induced JNK activation. COS cells were transiently cotransfected with plasmids encoding for HA-JNK and proto-Dbl or the control vector and treated with 20 mM LY294002 for 30 min at 37˚C.22 (Fig. 5A-A') shows that JNK activity is not affected by the treatment with the inhibitor of the PI3K. In fact, the ability of JNK to phosphorylate the substrate is similar to that observed in untreated cells. This observations could explain why treatment with LY294002

Figure 3. Influence of pharmacological inhibitors of PI3K on growth rate of proto‑Dbl expressing cells. NIH3T3 cells stably expressing GST (A) or GST‑proto‑Dbl (B) were cultured in the presence of 10% calf serum and 2.5, 5, or 10 mM LY294002 and at the indicated times trypsinized and counted. Data are representative of three independent experiments. Similarly, NIH3T3 cells stably expressing GST (C) or GST‑proto‑Dbl (D) were cultured in the pres‑ence of 10% calf serum and 5, 10 or 20 nM Wortmannin (Wrt) and at the indicated times trypsinized and counted. The data shown are the average of three independent experiments.

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causes a strong reduction but not a complete inhibition of proto-Dbl capability to transform cells.

In an attempt to further understand the molecular mechanisms that are responsible for the inhibiting effect of LY294002 on focus forming ability of proto-Dbl we analyzed the activity of two other MAPKs involved in regulating cell differentiation and proliferation, p38 and ERK. COS cells were transiently cotransfected with plas-mids encoding for HA-p38 and proto-Dbl cDNA construct or the control vector and treated with LY294002 as previously described. The activity of p38 was then assessed in a kinase assay using the MBP protein as a substrate. As shown in Figure 5B the treatment of proto-Dbl expressing cells with the PI3K inhibitor causes an increase in the activity of p38. p38 activity was also stimulated in control cells but the fold of induction was 2.5 versus 3.5 in proto-Dbl (Fig. 5B'). We then examined the effect of LY294002 on ERK acti-vation by analyzing the phosphorylation levels of the endogenous kinase in COS cells expressing proto-Dbl. As shown in (Figs. 5C-C'), the results obtained indicated that treatment with LY294002 consid-erably inhibited ERK activation in proto-Dbl transfectants. These results suggest that the PI3K-ERK pathway is important to sustain the full transforming potential of proto-Dbl.

PI3K inhibitors induce proto‑Dbl localization on the plasma membrane. The data presented above demonstrate the importance of PI3K in proto-Dbl-induced proliferation and focus formation. Proto-Dbl activity is dependent on its localization at the plasma

membrane. In fact, the loss of trans-forming activity of proto-Dbl bearing PH domain mutations that inhibit its interaction with PIPs correlates with the loss of plasma membrane targeting ability.5 Thus, we next investigated the role of PI3K pathway in proto-Dbl subcellular localiza-tion and induction of morphological changes. We performed immunoflu-orescence analysis on NIH3T3 cells transfected with GST-proto-Dbl and treated with LY294002 using anti GST polyclonal antibodies and FITC-labeled secondary antibodies. As previously described,4,5 cells expressing the proto-Dbl protein displayed an elongated shape with only slight enlargement of the cell body. Moreover, in agreement with the previously published informa-tion, staining of proto-Dbl protein showed a diffuse cytosolic pattern with a predominant perinuclear distribution and a limited protein localization on the plasma membrane at the ruffling areas (Fig. 6A, arrows and Table 1). However, LY294002 treatment induced evident cell body enlargement and an increase in the extension of the ruffles, with a parallel increased localization of the proto-Dbl protein along the plasma membrane at the ruffling sites (Fig. 6A). In addition, to investigate

the possible colocalization of proto-Dbl with the cytoskeleton actin, we double labeled the cells with TRITC-phalloidin. Confocal analysis showed that in all cells, either treated with LY294002 or untreated, the proto-Dbl protein colocalized with actin at the plasma membrane on the ruffling areas) (Fig. 6, arrows). Similar results were obtained by treating the cells with the PI3K inhibitor wortmannin (data not shown). Furthermore, coexpression of Akt PH domain also induced marked cell body enlargement and an increase in the extension of the ruffles, with a parallel increased localization of the proto-Dbl protein along the plasma membrane at the ruffling sites (Fig. 6B). These data indicate that in cells transformed with proto-Dbl the block of the PI3K can activate pathways that influence cell morphology, stimu-lating proto-Dbl translocation to the plasma membrane.

Inhibition of PI3K induces Rac activation. The formation of membrane ruffles requires the regulated remodeling of actin filaments and the localized activation of Rac1 and downstream effectors for regulation of actin assembly.23 Studies with pharmacological inhibi-tors of PI3K, LY294002 and wortmannin have demonstrated that PI3K is necessary for activation of Rac by many stimuli,24,25 while in other conditions the activation of Rac1 is completely insensitive to LY294002.26

Dbl protein acts as a GEF for the Rho GTPase Rac.27 Thus, to assess the Rac involvement in the morphological changes of proto-Dbl transfectants treated with the PI3K inhibitor, we performed a pull down assay on lysates obtained from cells transfected with proto-Dbl

Figure 4. G418 resistant colony formation is inhibited by LY294002. NIH3T3 cells were transfected with pCEFL‑GST (A) or pCEFL‑GST‑proto‑Dbl (B) and cultured in DMEM supplemented with 10% calf serum and 375 mg/ml of G418. G418 resistant colony formation was evaluated after two weeks of culture in selection medium. Results shown are the average of three independent experiments. (C) A representative photomicrograph of the G418‑resistant colony obtained is shown.

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or the control vector and treated with 20 mM LY294002 for 30 min at 37˚C. As shown in Figure 7A and B, the level of the GTP-bound form of Rac increases in proto-Dbl transfected cells treated with the PI3K inhibitor. Moreover, the same effect was observed when Akt PH was coexpressed in proto-Dbl transfectants where a three fold increase in Rac activation was observed. Rac activity in untransfected cells can be visualized only with longer exposure of the membrane and was not significantly affected by LY294002 treatment or Akt PH coexpression. These data indicate that Rac activation correlates with the morphological changes observed in proto-Dbl transfectants treated with the PI3K inhibitor.

LY294002 treatment leads to an increase in cell migration. Rac activation leads to a remodeling of cell morphology through the regulation of lamellipodium production and membrane ruffling.8 Increased level of PIP2 affects the activity of several actin cytoskel-eton-associated proteins leading to the formation of cytoskeletal structures such as membrane ruffles and lamellipodia, required for cell migration. Therefore, we investigated whether the increase in the formation of ruffles and activation of Rac in cells transformed with proto-Dbl and treated with LY294002 correlated with an increase in cell migration. As shown in Figure 8, we found that the treatment of proto-Dbl expressing cells with LY294002 caused an enhanced cell migration, while in control cells the migration capability was reduced

Figure 5. Proto‑Dbl focus forming activity requires JNK and ERK. COS cells were transiently transfected with pCEFL‑GST or pCEFL‑GST‑proto‑Dbl and HA‑JNK (A) or HA‑p38 (B). Cells were serum‑starved and treated with 20 mM LY294002 for 30 min at 37˚C. HA‑JNK and HA‑p38 kinases were immunoprecipitated with anti HA antibody and their activity was assayed on GST‑ATF2 (A) or MBP (B), respecitvely. Phosphorylated substrates were detected by autoradiography. The same membranes were probed with anti HA antibody for protein level control. (C) COS cells were transiently trans‑fected with pCEFL‑GST or pCEFL‑GST‑proto‑Dbl. Cells were serum‑starved and treated with 20 mM LY294002 for 30 min at 37˚C. Cells were lysed and subjected to Western blot analysis with anti phospho‑ERK (anti ERK‑P) antibody, followed by reprobing the membrane with anti ERK. Western blot was performed using total cell lysates. Proto‑Dbl protein expression level was evaluated with anti Dbl antibody (D). Results from one of three independent experiments is shown as a representative. The amount of phosphorylated ATF2 (A’), MBP (B’) and ERK (C’) was quantified. Bars represent the fold of activation over control determined by densitometric analysis from three independent experiments.

Figure 6. Inhibition of PI3K induces membrane localization of proto‑Dbl and ruffles. (A) NIH3T3 cells transfected with pCEFL‑GST‑proto‑Dbl or pCEFL‑GST were double immunolabeled with anti GST polyclonal antibodies, followed by FITC‑labeled secondary antibodies, and with TRITC‑phalloidin. In all cells, staining with anti GST antibodies appears diffuse in the cytoplasm and local‑ized on ruffles of the plasma membrane. In GST‑proto‑Dbl expressing cells both the localization of proto‑Dbl at the plasma membrane and the extension of the ruffling areas at the cell surface increases following treatment with 10 mM LY294002, where the signals of anti GST antibodies and phalloi‑din‑TRITC colocalize. The arrows indicate the ruffling areas. (B) NIH3T3 cells cotransfected with pEF4C‑proto‑Dbl and pCEFL‑GST or pEF4C‑proto‑Dbl and pCEFL‑GST‑AKT‑PH were double immunolabeled for confocal analysis, as described in Materials and Methods, with anti His monoclonal antibody followed by FITC‑labeled secondary antibodies and anti GST polyclonal antibodies, followed by TRITC ‑labeled secondary antibodies. Coexpression of Akt‑PH increases both the localization of proto‑Dbl at the plasma membrane and the extension of the ruffling areas at the cell surface (arrows). Bar 10 mm.

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by the presence of the PI3K inhibitor. The growth curve shown in Figure 3 indicates that the results obtained with the cell migration assay were due to a higher motility and not to a higher proliferation capability of the proto-Dbl expressing cells. These results suggest that the PI3K inhibition in proto-Dbl transfected cells is associated with an enhanced cell migratory behavior.

DISCuSSIOnHere we show that proto-Dbl protein may

activate distinct downstream effectors to induce cell transformation and morphological changes. We observed that the focus formation activity of proto-Dbl requires PI3K since the presence of pharmacological and molecular inhibitors of PI3K inhibited focus forming ability of the proto-Dbl protein. On the other hand, inhibition of PI3K causes relocalization of proto-Dbl to the plasma membrane, Rac activation and increased membrane ruffles and cell motility. These data are in agree-ment with the knowledge that PI3K/Akt signaling pathways regulate and promote cell growth, prolif-

eration and transformation.28

In this paper we also show that the transforming activity of proto-Dbl, strongly reduced by, but still detectable upon treatment with PI3K inhibitors, may be attributable to JNK kinase activity, whose activation is unaffected by the PI3K inhibitor. These data are in agreement with previously published observation reporting that onco-Dbl expression in cells stimulates the activation of JNK15,21 that directly correlates with the transforming potential of proto-Dbl.5 Finally, the results of this work suggest that the signaling pathway involved in proto-Dbl-induced cell transformation and proliferation requires an activated PI3K-ERK pathway. In fact we observed that the treatment of proto-Dbl expressing cells with the PI3K inhibitor LY294002 causes inhibition of Akt, a decrease in the phosphoryla-tion level of ERK, activation of p38 MAPK and a strong inhibition of cell proliferation.

ERK is known to be essential for proliferation in many cell types29-32 while activation of p38 inhibits cell proliferation.33 Constitutive activation of ERK pathway allows for G1-S phase transition and cell division34,35 and ERK/MAPK activation is both necessary and sufficient in transforming experimental NIH3T3 cells.36,37 On the other hand, high level of p38 activity has a nega-tive effect on growth regulation and may suppress cell proliferation by inhibiting ERK and inducing G0-G1 arrest.38,39 p38 MAPK is antagonistic to Ras activity, causing inhibition of Ras-induced prolif-eration in NIH3T3 cells and suppression of Ras transformation in RIE cells.40,41 There is also evidence that activation of p38 MAPK can reverse Ras induced fibroblasts transformation42 and that p38 inhibition by Ras is required for transformation.40 In addition, high p38/ERK ratio induces tumor growth arrest while high ERK/p38 ratio favors tumor growth.39 In agreement with these data, our results show that activation of p38 MAPK and inhibition of ERK activity upon treatment with LY294002 of proto-Dbl expressing cells hampers proto-Dbl-induced cell transformation and proliferation.

It is known that increased level of PIP2 affects the activity of several actin cytoskeleton-associated proteins leading to the formation of cytoskeletal structures such as membrane ruffles and lamellipodia, morphological structures required for cell migration, and that the localized activation of Rac and downstream effectors leads to a remodeling of the cell morphology regulating lamellipo-dium production and membrane ruffling.8,23 Moreover, a potential balance of MAPK activities exists in which inhibition of p38 leads to an increase in ERK phosphorylation and cell proliferation while preventing cell migration. In contrast the activation of p38 leads to a decrease in ERK phosphorylation and cell proliferation while inducing cell migration.33 Accordingly, inhibition of ERK and

Figure 7. In vivo GEF activity of proto‑Dbl on small G protein Rac. (A) COS cells were transiently transfected with pCEFL‑GST or pCEFL‑GST‑proto‑Dbl, serum‑starved for 18 hr and treated for 30 min at 37˚C with 20 mM LY294002, as indicated. Alternatively, cells were cotransfected with pCEFL‑ GST and pCEFL‑GST‑Akt‑PH or pCEFL‑GST‑proto‑Dbl and pCEFL‑GST‑Akt‑PH. Cells were then lysed and subjected to the Rac activation assay. Eluates from GST‑PAK precipitates or total cell lysates were subjected to immunoblotting with anti Rac antibody. Specific proto‑Dbl product was detected using an anti Dbl antibody. Results from one of three independent experiments is shown as a representative. (B) The amount of activated Rac was quantified. Bars represent the fold of activation over GST expressing control cells as deter‑mined by densitometric analysis from three independent experiments.

Table 1 Extentofrufflinginproto-Dbltransformedcellstreated withLY294002

Transfectants % of cells with % of cells with % of cells with limited ruffling1 moderate ruffling2 extensive ruffling3

Proto‑Dbl 26.5±1.3 53.2±3.3 21.3±1.1Proto‑Dbl+LY294002 6.2±0.4 59.2±1.2 35.2±1.0GST 43.1±1.5 57.9±2.1 0GST+LY294002 33.0±1.1 67.0±3.5 0

Percentage of cells with different lengths of ruffling was assessed by counting 100 cells from five different microscopic fields randomly taken from two different experiments. Results represent the mean values ± standard deviations. 1Ruffling length corresponding to less than 5% of the cell plasma membrane; 2Ruffling length corresponding to 5-30% of the cell plasma membrane; 3Ruffling length corresponding to over 30% of the cell plasma membrane.

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Role of PI3K in Proto-Dbl Activities

activation of p38 MAPK induced by LY294002 caused an increased localization of proto-Dbl along the plasma membrane at the ruffling sites, a cell body enlargement and an increase in the extension of the ruffles. These effects are accompanied by activation of Rac and an augmented cell motility.

It has been reported that the activation of PI3K contributes to Rac activity that leads to membrane ruffles formation. Inhibition of PI3K by both molecular and pharmacological approaches blocks membrane ruffling and inhibits cell migration.11,22,24 Conversely, there is evidence that membrane ruffling induced by GTPase-defective RacV12 is insensitive to PI3K inhibition.43 Moreover the expression of another GEF for Rho GTPases, Vav3, was shown to be sufficient to cause membrane ruffling irrespective of the PI3K activation status.44 Similarly, our results indicate that the block of PI3K does not inhibit Rac activation. On the contrary the treatment with LY294002 of cells transfected with proto-Dbl causes an increase in GTP-bound Rac. Therefore, our data are in agreement with the possibility that Rac can be activated both in a PI3K-dependent and in a PI3K-independent manner.25

Taken together our results show that the PI3K-ERK pathway is required to sustain proto-Dbl-induced cell transformation, while the p38-Rac-PIP2 pathway is necessary to induce morphological changes and cell migration.

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Figure 8. Proto‑Dbl‑mediated cell motility is independent of PI3K activation NIH3T3 cells stably transfected with either control pCEFL‑GST vector or pCEFL‑GST‑proto‑Dbl were treated with 10 mM LY294002 for four hours and subjected to cell motility assay as described in “Materials and Methods”. The average results from three independent experiments were calculated and represented. Standard deviation was less than 10%.

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