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J. Biol. Chem., Vol. 278, Issue 16, 14414-14419, April 18, 2003

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p19^Arf-p53 Tumor Suppressor Pathway Regulates Cell Motility by Suppression of Phosphoinositide 3-Kinase and Rac1 GTPase Activities^*

Fukun Guo, Yuan Gao, Lei Wang, and Yi Zheng

From the Division of Experimental Hematology, Children's Hospital Research Foundation, University of Cincinnati, Cincinnati, Ohio 45229

Received for publication, January 13, 2003, and in revised form, February 4, 2003

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

The p19^Arf-p53 tumor suppressor pathway plays a critical role in cell-cycle checkpoint control and apoptosis, whereas Rho family small GTPases are key regulators of actin structure and cell motility. By using primary mouse embryonic fibroblasts that lack Arf, p53, or both, we studied the involvement of the p19^Arf-p53 pathway in the regulation of cell motility and its relationship with Rho GTPases. Deletion of Arf and/or p53 led to actin cytoskeleton reorganization and a significant increase in cell motility. The endogenous phosphoinositide (PI) 3- kinase and Rac1 activities were elevated in Arf^/ and p53^/ cells, and these activities are required for p19^Arf- and p53-regulated migration. Reintroduction of the wild type Arf or p53 genes into Arf^/ or p53^/ cells reversed the PI 3-kinase and Rho GTPase activities as well as the migration phenotype. These results suggest a functional relationship between an established tumor suppressor pathway and a signaling module that controls actin structure and cell motility and show that p19^Arf and p53 negatively regulate cell migration by suppression of PI 3-kinase and Rac1 activities.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

The p53 and pRb cell cycle inhibitors and their regulators, including p19^Arf and p27^Kip1, are well established tumor suppressors that are components of a complex signaling network central to tumor suppression (1). Deletion or mutation in these tumor suppressors or their regulators occurs in the majority of tumor cases and correlates with the onset of a wide spectrum of cancer (2). Specifically the p19^Arf-p53 tumor suppressor pathway is thought to be involved primarily in monitoring proliferation signals to prevent cells from uncontrolled growth (3). Rho family small GTPases, on the other hand, are molecular switches that transduce diverse intracellular signals leading to cell proliferation, gene induction, apoptosis, and in particular, cytoskeleton remodeling and migration (4). Accumulating evidence has implicated the Rho GTPases, RhoA, Rac1, and Cdc42, in many aspects of tumor development, including tumor cell migration and proliferation (5). Many mitogenic signals, including those from growth factor receptors and integrins, can promote the exchange of GDP for GTP on Rho GTPases, enabling them to interact with an array of effector targets to elicit cytoskeletal changes (6, 7).

Although much of the attention in tumor suppressor studies has been focused on their roles in cell cycle control and/or apoptosis, preliminary evidence has become available suggesting a potential functional linkage between p53 and cell morphological effect/cell motility. For example, p53 has been shown to regulate the transcription of a number of genes involved in cell morphology and/or movement, including that of smooth muscle -actin (8), collagens II1 and VI1 (9), vascular epidermal growth factor (10), metalloproteinase-1 (11), and fibronectin (12). Moreover, overexpression of p53 seems to cause a partial reversion of Ras-induced morphological transformation (13) and to inhibit cell migration (14). A recent study further suggests that p53 may have a role in Cdc42-mediated filopodia formation and cell polarization (15).

To investigate the functional connection between the tumor suppressors and Rho GTPase-regulated cytoskeleton events, we have examined the actin cytoskeletal structure and migration capability of p19^Arf-, p53-, p27^Kip1-, or pRb-deficient primary mouse embryonic fibroblast (MEF)¹ cells. We report that the p19^Arf-p53 tumor suppressor pathway, but not the related p27^Kip1-pRb pathway, negatively regulates cell motility through suppression of the PI 3-kinase-Rac1 signaling module.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

DNA Constructs-- Site-directed mutagenesis of Rac1, Cdc42, RhoA, and p53 was performed by the polymerase chain reaction-based, oligonucleotide-mediated method (16). The retroviral constructs expressing p19^Arf, p53 wild type or mutant, and dominant negative Rho GTPases (Rac1N17, Cdc42N17, and RhoAN19) were generated by ligating the corresponding cDNA fragments into the BamHI and EcoRI sites of the retroviral GFP bicistronic vector, MIEG3 (17).

Cell Culture, Retroviral Infection, and Cell Imaging-- Primary MEFs from p19Arf^/, p53^/, p19Arf^//p53^/, p27Kip1^/, and pRb^/ mice (all of the C57BL/6 × 129/sv background) were generous gifts from Dr. Martine Roussel (St. Jude, Memphis, TN) and were generated as described previously (18). Recombinant retroviruses were produced using the ecotropic Phoenix packaging cell system (17). The primary MEFs were infected with the retroviruses and harvested 48-72 h after infection. GFP-positive cells were isolated by fluorescence-activated cell sorting.

Primary MEFs were seeded onto coverslips at 3 × 10⁴ cells/slip density. The cells were serum-starved for 12 h prior to fixation. The F-actin structure of the cells was visualized by rhodamine-phalloidin staining, and the stained cells were analyzed by using a conventional fluorescence microscope (Zeiss).

Cell Motility and Migration Assays-- Single cell motility assays using the colloidal gold particles were carried out as described (19). The wound healing assays to semi-quantitatively determine the migration distance were performed following a scratch of the confluent monolayer of cells as described (20). The migration distances of cells were monitored at various times and were measured at 9 h after the introduction of the wound under low serum (0.5%) conditions.

PI 3-kinase Assay, Chemical Inhibitors, and Immunoblotting-- The endogenous PI 3-kinase activities of Arf^/, p53^/, Arf^//p53^/, and Arf^/ or p53^/ cells reconstituted with Arf or p53 were assayed according to a described protocol (21). Briefly the cells were lysed in buffer A containing 20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.3 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 1% Triton X-100, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 0.1 mM sodium orthovanadate and 25 mM NaF. After centrifugation at 4 °C for 30 min at 14,000 rpm, the protein contents in the supernatant of cell lysates were measured by the Bradford method. An equal amount of proteins was incubated with anti-p85 polyclonal antibody coupled to protein A-agarose (Upstate Biotechnology) overnight or subjected to anti-p85 or anti-p110 Western blot analysis. The immunoprecipitates were washed twice with buffer C containing 20 mM Tris-HCl, pH 8.0, 100 mM NaCl, and 10 mM MgCl₂ and washed once with a kinase assay buffer (20 mM Tris-HCl, pH 7.6, 10 mM MgCl₂). 5 µg of sonicated phosphatidylinositol together with [-³²P]ATP (200 µCi/ml) in 45 µl of the kinase assay buffer were incubated with the washed beads at 25 °C for 10 min. The reactions were terminated by the addition of 100 µl of 1 N HCl. The reaction products were extracted by 200 µl of CHCl₃/MeOH (1:1) and resolved on a TLC silica plate coated with potassium oxalate in a solvent containing CHCl₃/MeOH/NH₄OH, 4 M (9:7:2). The PI 3-kinase reactions were analyzed by autoradiography.

Prior to Western blotting, the protein contents of the cell lysates were normalized by the Bradford method. Anti-phospho-Akt(Ser⁴⁷³) and anti-PTEN antibodies were obtained from Cell Signaling, Inc., anti-p85 and the anti-p110 of PI 3-kinase antibodies were obtained from Upstate Biotechnology, Inc., and the PI 3-kinase inhibitors wortmannin and LY294002 were obtained from Sigma.

Endogenous Rho GTPases Activity Assay-- GST-PAK1, GST-Rhotekin, and GST-WASP, which contain Rac1, RhoA, and the Cdc42- interactive domains of PAK1, Rhotekin, and WASP, respectively, were used to probe the endogenous Rac1-GTP, RhoA-GTP, and Cdc42-GTP activities as described (20).

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

p19^Arf, p53, p27^Kip1, and pRb are well recognized cell cycle regulators that are critical to checkpoint control. p19^Arf, in response to oncogenic signals, stabilizes p53 by sequestering Mdm2, a negative regulator of p53 (3), whereas p27^Kip1, on its way through pRb, regulates G₁ transition by modulating growth-essential E2F transcription factors (1). All four are important components of a complex tumor suppressor network. To examine their potential involvement in the regulation of cell actin structure and motility, we have generated the p19^Arf-, p53-, p27^Kip1-, or pRb-deficient primary MEFs from the respective tumor suppressor knock-out mice. To avoid possible alteration of cell properties under tissue culture conditions, only low passages (<6 passages) of the primary cells were used and at least two independent p53- or p19^Arf-deficient MEF preparations were examined.

In subconfluent culture and serum-free conditions, a significant percentage of the p19^Arf- or p53-deficient cells appeared to be in a round shape and displayed intense cortical actin staining, whereas the p27^Kip1- or pRb-deficient cells were similar to wild type MEFs in morphology and actin structure (Fig. 1A). To determine whether the altered actin structures might be associated with cell motility modulation, the cells were plated onto colloidal gold-coated plates, and the motility of single cells was traced by the trails left behind their movement paths. As shown in Fig. 1B, in the absence of serum stimulation, both p19^Arf- and p53-deficient cells produced long trails, whereas the Kip1 and Rb knock-out cells were similar to the wild type cells, showing no sign of movement.

View larger version (74K): [in this window] [in a new window]   Fig. 1.   The effect of the deletion of p19Arf, p53, p27, or Rb on actin structure and cell motility of primary MEF cells. 2×104 of wild type (WT) or knock-out MEF cells (Arf /, p53/, p27 /, Rb/) were cultured overnight on coverglass. Subconfluent cells were starved in medium containing 0.5% serum for 12 h. After fixation in 3.7% formaldehyde, cells were permeabilized with 0.1% Triton X-100 and stained for F-actin by using rhodamine-conjugated phalloidin (A). Single cell-based motility was observed 9 h after the cells were plated on colloidal gold-coated culture dishes (B).

To further quantify cell migration rate, we measured the migration distance of these cells in a wound healing assay. The Arf^/ and p53^/ cells migrated about 2-fold faster than the wild type, Kip1^/, or Rb^/ cells at 9-h intervals under low serum (0.5% fetal bovine serum) conditions (Fig. 2, A and B), at which time the difference in cell growth was less than 10%. Similar results were also obtained when the cells were serum-starved over a 16-h period (data not shown). When p19^Arf and p53 were reintroduced into Arf^/ and p53^/ cells, respectively, by retroviral induction, the migration phenotype of these cells was fully reversed (Fig. 2, C and D). The expression levels of p19Arf or p53 in the reconstituted cells were comparable with or slightly higher than that of the respective wild type cells (Fig. 2E). Thus, p19^Arf and p53 negatively regulate cell migration.

View larger version (51K): [in this window] [in a new window]   Fig. 2.   MEFs deficient in p19Arf or p53, but not p27 or Rb, display significantly enhanced migration. A, equal numbers of wild type (WT) and knock-out cells were seeded on 35-mm dishes and cultured to confluency. Migration into the wound is shown 10 h after the wound was introduced under low serum conditions. B, migration distances were determined by taking three independent measurements from each dish. Each experiment was conducted in triplicate, and the mean ± S.D. was calculated. The relative migration distance is normalized to that of wild type. C and D, the effect of reintroducing p19Arf or p53 to Arf / or p53/ cells on migration. Arf / or p53/ cells were infected with retrovirus expressing GFP, Arf and GFP, or p53 and GFP as indicated. GFP-positive cells were isolated by fluorescence-activated cell sorting and assayed for their relative migration distances. E, the expression of p19Arf or p53 in the reconstituted Arf //Arf or p53//p53 cells was probed by anti-p53 or anti-p19Arf Western blot.

p19^Arf and p53 display both interdependent and independent activities in the regulation of cell cycle progression and in response to DNA damage (3). Deletion of both Arf and p53 alleles led to an equally mobile cell phenotype as p19Arf^/ or p53^/ cells in the single cell-based motility assay and in the wound healing assay (Fig. 3A). Furthermore, introduction of p19^Arf into the Arf^//p53^/ cells did not affect cell migration (Fig. 3A). It appears that p19^Arf acts upstream of p53 in the regulation of cell motility.

View larger version (28K): [in this window] [in a new window]   Fig. 3.   A, p19Arf acts upstream of p53 in the regulation of cell migration. Single cell motility and wound healing migration of p19Arf //p53/ cells transduced with GFP or GFP with p19Arf were compared with that of the wild type MEFs (WT). B, a specific transcriptional event(s) is required by p53 to modulate migration. Two mutant forms of p53, p53(Gln22/Ser23) and p53(His175), were compared for their ability to rescue the migration phenotype of p53/ cells with that of wild type p53.

p53 functions as a DNA-binding dependent transcription factor in mediating multiple gene activation that is critical to its tumor suppressor activity (22). In addition, p53 is also known for a role in negatively regulating the transcription of an array of genes by repression of their transcription. To begin to address the issue of what function of p53 is required for the regulation of cell motility, we examined the ability of two p53 mutants, Gln²²/Ser²³ and His¹⁷⁵, to suppress the migration phenotype of p53^/ cells. Although both mutants are defective in transcriptional regulation of p53-positive genes including that of p21^Waf1/Cip1 (23-25), the Gln²²/Ser²³ mutation is located in the N-terminal transcription activation domain, which has also been shown to be critical for the binding of TATA-binding protein-associated factors (26, 27), whereas the His¹⁷⁵ mutation resides in the central DNA binding domain of p53, which is the site of a majority of mutations seen in tumors (28). We observed that the Gln²²/Ser²³ mutant was able to rescue the migration phenotype of p53^/ cells similar to wild type p53, but the His¹⁷⁵ mutant was inactive (Fig. 3B). These results suggest that specific transcription activity controlled by p53 is responsible for the regulation of cell migration by the p19^Arf-p53 tumor suppressor pathway.

PI 3-kinase has previously been implicated in the regulation of cell migration (29). To investigate whether PI 3-kinase contributes to p19^Arf- and p53-regulated cell motility, the endogenous PI 3-kinase activity and phospho-Akt(Ser⁴⁷³) content, which is indicative of the endogenous PI 3-kinase activation status of Arf ^/, p53^/, and Arf ^//p53^/ MEFs, were examined, and the requirement of PI 3-kinase in p19^Arf- and p53-mediated cell migration was assessed by using specific pharmacological inhibitors wortmannin or LY294002 at defined concentrations. As shown in Fig. 4, A and B, genetic deletion of Arf, p53, or Arf and p53 led to 3-5-fold increases in PI 3-kinase activity and phospho-Akt(Ser⁴⁷³), which were readily reversed by the reintroduction of Arf or p53. The expression levels of p85 and p110, as well as that of Akt, were not altered in these cells (Fig. 4B; data not shown). The enhanced PI 3-kinase activities are apparently required for the migration phenotype observed in these cells, because inhibitors of PI 3-kinase (wortmannin or LY294002) caused complete reversion of the migration phenotype without affecting the basal movement of the cells (Fig. 4C). Under these conditions, wortmannin or LY294002 potently inhibited PI 3-kinase activity (Fig. 4C and data not shown). On the other hand, the expression level of PTEN, a PIP₃ phosphatase that could be transiently regulated by p53 transcriptional activity (30), was not altered in the Arf^/, p53^/, or Arf^//p53^/ cells nor in Arf^/ or p53^/ cells that were forced to express Arf or p53 (Fig. 4D). We conclude that Arf and/or p53 deletion up-regulates PI 3-kinase activity, resulting in increased cell motility.

View larger version (37K): [in this window] [in a new window]   Fig. 4.   PI 3-kinase is required for the p19Arf- and p53-regulated cell migration. A, the PI 3-kinase activities of the knock-out or reconstituted cells were assayed. The cells were starved in a medium containing 0.5% serum for 12 h and harvested for anti-p85 immunoprecipitation. The PI 3-kinase activities in the immunoprecipitated fractions were measured by an in vitro lipid kinase assay using exogenous phosphatidylinositol as substrate. WT, wild type. B, log phase cells were starved in 0.5% serum for 12 h. The relative amounts of total Akt and phospho-Akt (P-Akt (S473)) in the cell lysates containing equal amount of proteins were measured by Western blotting. C, PI 3-kinase inhibitors reduced the PI 3-kinase activity and blocked the migration phenotype of p19Arf- and/or p53-deficient cells. The cells were treated with 50 nM wortmannin, 20 µM LY294002, or Me2SO alone for 10 h before the migration distances and the PI 3-kinase activities were measured. D, the amount of PTEN and -actin in the cell lysates containing equal amount of proteins was determined by Western blotting.

Rho family members Rac1, RhoA, and Cdc42 are downstream signaling components of PI 3-kinase and have been implicated in the regulation of cell migration (31). To determine whether these Rho proteins might be involved in p19^Arf- and p53-regulated migration, we examined the activation status of these Rho proteins in Arf ^/ and p53^/ cells by specific effector domain pull-down assays. As shown in Fig. 5A, in the absence of serum stimulation, the endogenous levels of GTP-bound active Rac1 and RhoA in p19Arf ^/ or p53^/ cells increased by 2- and 6-fold, respectively, compared with those in wild type cells, whereas Cdc42-GTP increased by less than 20%. The Rac1, RhoA, or Cdc42 expression levels were not affected by p19Arf or p53 deletion. When p19Arf or p53 was reintroduced into the knock-out cells, the active Rho protein contents were decreased to the level of wild type cells or lower (Fig. 5A), indicating that p19^Arf and p53 suppress the Rho protein activities. Furthermore, among the dominant negative mutants of Rac1 (Rac1N17), RhoA (RhoAN19), and Cdc42 (Cdc42N17), only Rac1N17 was able to completely reverse the migration phenotype in both Arf^/ and p53^/ cells, reducing their migration rates to that of the wild type MEFs, similar to the effect of reintroducing the Arf or p53 gene (Fig. 5B). The three Rho GTPase mutants were expressed equally well in the cells (Fig. 5B). These results indicate that Rac1 is a specific mediator of p19^Arf- and p53-regulated cell migration.

View larger version (33K): [in this window] [in a new window]   Fig. 5.   Rac1 is an essential mediator of p19Arf- and p53-regulated cell migration. A, endogenous Rac1, RhoA, or Cdc42 activities in the knock-out cells were assayed by using log phase cells that were serum-starved for 12 h prior to lysis and subject to GST-PAK1, GST-Rhotekin, or GST-WASP pull-down analysis. The amount of Rac1-GTP, RhoA-GTP, or Cdc42-GTP was detected by Western blotting of the respective co-precipitates with anti-Rac1, anti-RhoA, or anti-Cdc42 antibody and was normalized to that of the total amount of Rac1, RhoA, or Cdc42 in respect to that in wild type MEFs (WT). The results shown in the lower panels are means ± S.D. from three experiments. B, dominant negative Rac1, but not RhoA or Cdc42, inhibited the migration phenotype of the knock-out cells. GFP or GFP together with Rac1DN-, RhoADN-, or Cdc42DN-expressing primary MEFs was generated by retroviral induction. The amount of the three Rho GTPase mutants (N-terminal hemagglutinin (HA)-tagged) expressed in p53/ cells was probed by anti-hemagglutinin Western blot.

The p53 and Rb genes and their regulatory molecules are central to the growth malfunction of most tumor cells (1). The p19^Arf-p53 pathway, specifically, constitutes a checkpoint in cell cycle progression that is responsive to the proliferative signals normally required for cell proliferation (3). Mostly through its transcriptional activity, p53 can inhibit cell cycle progression or induce apoptosis in response to stress or DNA damage. Moreover, p53 is known to be a critical factor in maintaining genomic stability and in eliciting DNA repair reactions (22, 32). Some earlier evidence also pointed to a regulatory role of p53 in transcriptionally activating a number of genes that could be involved in modulating cell movement (8-12). Two recent studies have begun to address the possibility that p53 might contribute to the regulation of cell movement and polarity (14, 15). By genetic deletion of the tumor suppressors, we show that p19^Arf and p53, but not p27^Kip1 or pRb, have a profound negative effect on cell motility and migration. p19^Arf likely depends on p53 for migration regulation, and specific transcriptional activity, or specific target genes controlled by p53, may serve as the link between the nuclear events and the cytoskeleton, focal adhesion contacts, or extracellular matrix components to promote the migration phenotype. We are currently examining the candidate gene products whose expression profiles are altered by the p19^Arf and p53 deficiencies in an effort to identify the molecule(s) responsible for the observed cellular effects.

The PI 3-kinase-Rac1 signaling module has been known to mediate growth factor-stimulated cell motility (31). Activation of PI 3-kinase (similar to deletion of the PTEN tumor suppressor, which is a PIP₃-specific phosphatase that counter-reacts with many PI 3-kinase effects) can cause activation of both Rac1 and Akt and contributes to enhanced cell motility and invasion (20, 29). The cell morphological and actin structural changes we have observed in Arf^/ and p53^/ MEFs, i.e. a round appearance and intense cortical actin staining, are reminiscent of what was observed in the case of PTEN^/ cells (20), suggesting that elevation of the PIP₃ level in the cells may be significant to increased cell movement. Indeed, Rac1 and Akt appear to be important contributors to the migration phenotype in PTEN deficiency (20, 29). A few previous reports support the assumption that Rac may act independently of Akt in signaling to mammalian cell migration (33, 34), but a recent study employing constitutively active and dominant negative Akt in fibroblasts shows that Akt is an essential mediator of Rac-regulated cell motility stimulated through PI 3-kinase activation (29). In the context of our finding that PI 3-kinase and Rac1 of this module are required for p19^Arf- and p53-regulated migration, it seems logical that a hierarchical link among them may be at work to transduce signals resulting from the genetic defects of these tumor suppressors to cell activities related to migration, although the detailed relationship among them remains to be determined. The components that serve to further relay signals from Rac1 and/or Akt to cell motility might include PAK, phosphoinositide 5-kinase, and WAVE-like actin polymerization molecules (31, 35).

Gadea et al. (15) have recently shown that p53 function is necessary for the suppression of Cdc42-induced filopodia formation and may transcriptionally control the initiating components of filopodia. Although conceptually our observation draws a similar conclusion regarding the negative effect of the Cdc42-mediated cell response imposed by p53, the mechanistic details of our cell motility results may differ from the interpretation of Gadea et al. (15), drawn mostly from the Cdc42 and/or p53 overexpression experiments. In the present work, although Rac1 and RhoA activities are shown to be up-regulated by deletion of Arf or p53, Rac1, but not RhoA nor Cdc42, appears to provide the key functional linkage between the p19^Arf-p53 pathway and cell motility. However it remains likely that the contributions of Cdc42-mediated cell polarity and RhoA-mediated cell adhesion are an integral part of the p19^Arf- and p53-regulated cytoskeleton organization and related functional output.

A recent gene array study has identified heparin-binding epidermal growth factor-like growth factor (HB-EGF) as a p53-responsive gene product that could be up-regulated by DNA damage and p53 induction (36). HB-EGF may protect cells from H₂O₂-induced apoptosis through MAPK (mitogen-activated protein kinase) and PI 3-kinase activation, presenting a possible autocrine regulatory loop linking p53 induction with PI 3-kinase and Akt activation. Whether this p53-mediated regulatory pathway is functional in the p53/ background remains unknown. In light of our finding that p53 deletion would cause an up-regulation rather than a down-regulation of PI 3-kinase activity, it is likely that the above proposed p53 to HB-EGF to PI 3-kinase pathway might be suppressed by another more dominant component unleashed by p53 deletion, which preferably stimulates the PI 3-kinase-Rac1-Akt signaling module when the p19^Arf-p53 pathway is impaired. This possibility is currently under investigation.

In summary, we have demonstrated that the p19^Arf-p53 tumor suppressor pathway negatively regulates cell motility, revealing an interesting connection between the established tumor suppressor pathway and the PI 3-kinase-Rac1 signaling module that is known to modulate cell migration. The results provide insights into a previously under-appreciated cellular function of the tumor suppressor pathway and may have important implications in developing strategies that target Rho proteins in anti-cancer therapy.

	ACKNOWLEDGEMENTS

We thank members of Zheng laboratory for help with retroviral production and cDNA constructs and Dr. Martine Roussel for the gift of MEFs and Arf cDNA.

	FOOTNOTES

^* This work was supported by grants from the National Institutes of Health (GM53943) and the Department of Defense Breast Cancer Program (BC990290) (both 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. Tel.: 513-636-0595; Fax: 513-636-3768; E-mail: yi.zheng@chmcc.org.

Published, JBC Papers in Press, February 10, 2003, DOI 10.1074/jbc.M300341200

	ABBREVIATIONS

The abbreviations used are: MEF, mouse embryonic fibroblast; GST, glutathione S-transferase; PAK, p21-activated kinase; PI 3-kinase, phosphoinositide 3-kinase; GFP, green fluorescent protein; PIP₃, inositol 1,4,5-trisphosphate; PTEN, phosphatase and tensin homolog deleted on chromosome 10; WASP, Wiskott-Aldrich syndrome protein.

	REFERENCES

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