INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

Members of the Rho family of small GTPases, including Cdc42, Rac, and Rho, were first identified as proteins that have key roles in regulating the organization of the actin cytoskeleton. They were shown to induce the production of filopodia, lamellipodia, and stress fibers, respectively (17, 30, 37, 38). Subsequently they were found to have other functions, including the regulation of cell proliferation and activation of the JNK and p38 mitogen-activated protein kinase (MAP kinase) pathways (3, 5, 8, 11, 28, 51). When improperly regulated, the Rho GTPases also play key roles in oncogenic transformation and tumor invasiveness (26, 27, 33, 36, 52) which may be directly related to changes in morphology and activation of specific signal transduction pathways.

The characterization of molecular targets for the Rho proteins is important for understanding their functions. The PAK family of serine/threonine kinases interacts directly with GTP-loaded Rac and Cdc42 through a GTPase binding domain (GBD) (1, 2, 4-6, 23, 24, 42). The first members of the family to be identified include the closely related human PAK1 and -2, mouse PAK3, and the rat homologues PAK , , and  (4, 5, 23, 24). One possible function of the PAKs is the regulation of the organization of the actin cytoskeleton. PAK1 has been reported to induce filopodia and membrane ruffles (43) and to localize to polymerized actin (10, 43). These cytoskeletal changes, however, occur independently of PAK1's ability to bind Rho GTPases and are partly independent of PAK1's kinase activity (43). Others have found that PAK  and PAK2 do not induce filopodia or lamellipodia but instead have a role in the dissolution of stress fibers, down-regulation of focal adhesions, and cell retraction (22, 50). Thus, the exact roles for PAKs 1, 2, and 3 in cytoskeletal regulation remain to be fully clarified.

In addition to cytoskeletal organization, the Rho proteins also play key roles in cell proliferation and oncogenic transformation. Constitutively active mutants of the exchange factors for the GTP binding proteins, members of the Dbl family, are potent oncogenes (7). Cdc42, Rac, and Rho were all shown to be necessary for oncogenic transformation by oncogenic Dbl, and each GTPase appears to contribute to different aspects of transformation, including anchorage-independent growth, superoxide production, and loss of contact inhibition, respectively (20). Although PAK1 may have a role in transformation and cell survival induced by oncogenic Ras (44-46) in some cells, a direct role for the PAKs in transformation by the Rho family GTPases has not been demonstrated. In fact, Cdc42C40 and RacC40, effector mutants which cannot bind to PAKs 1, 2, or 3 (1, 13, 18), maintain the ability to induce cytoskeletal changes as well as some of the hallmarks of oncogenic transformation (13, 18). These results indicate that PAKs 1, 2, and 3 may not be essential for the induction of oncogenic transformation in response to these GTPases.

The newest member of the PAK family is PAK4 (1). PAK4 is a new type of PAK protein and it differs significantly from the other PAKs in sequence. PAK4 lacks several key features characteristic of PAKs 1, 2, and 3, including four proline-rich motifs, an autoinhibitory domain, and a putative G binding site (9, 16). PAK4 does contain a modified GBD, however, and it interacts with GTP-loaded Cdc42 and has a weaker interaction with GTP-loaded Rac. Unlike the other PAKs, PAK4 even interacts with the effector loop mutant Cdc42C40 (1). Importantly, PAK4 was found to be a link between Cdc42 and filopodium formation. Expression of Cdc42V12 together with PAK4 leads to a prolonged induction of filopodia (1), and this depends strictly on PAK4's kinase activity and on its binding to Cdc42. PAK4 does not mediate all of Cdc42's functions, however. For example, it is a relatively weak activator of the signaling pathways that lead to activation of the JNK and p38 MAP kinase pathways (1). It is therefore thought to be primarily a mediator of the cytoskeletal changes induced by Cdc42 rather than a regulator of the JNK or p38 pathways.

In order to gain a better understanding of the functions of PAK4, we have generated and analyzed a constitutively active PAK4 mutant, PAK4(S445N). Expression of PAK4(S445N) in fibroblasts resulted in a transient induction of filopodia, a reduction of stress fibers, decreased adhesion to the extracellular matrix, and cell rounding. Interestingly, cells expressing PAK4(S445N) had a transformed morphology. These cells formed colonies in soft agar, similar to what has been described for cells expressing activated Cdc42 (19, 20, 34). Furthermore, dominant-negative PAK4 partially inhibited focus formation in response to oncogenic Dbl in fibroblasts. Taken together, our results suggest that PAK4 is an essential mediator of anchorage-independent growth induced by Cdc42 and by Dbl family exchange factors.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Plasmids. Expression plasmids encoding influenza hemagglutinin (HA)-tagged PAK4wt, HA-tagged PAK4(S474E), and HA-tagged PAK4(K350M) are described in reference 1. HA-PAK4R (1) containing the PAK4 regulatory domain was generated by inserting the HindIII/PstI site from 3HA-PAK4-pBS into the HindIII/BglIII site of the SR vector, together with a pair of BglII/PstI adapter primers. PAK4RGBD (1) is the same as PAK4R but lacks the first 113 amino acids, which include the GBD. To generate activated PAK4, serine 445 on PAK4 was changed to an asparagine using site-directed mutagenesis (Stratagene QuickChange kit), and asparagine 445 was introduced into both SR-PAK4wt and -PAK4(S474E) to generate PAK4(445N) and PAK4(474E/445N), respectively. PAK4(445N) and PAK4(474E/445N) had equivalent levels of kinase activity and induced identical types of morphological changes in transient transfections (including a rounded morphology and loss of stress fibers and focal adhesions). However, PAK4(474E/445N) was expressed at higher levels (its expression level was comparable to that of wild-type PAK4) and was therefore used in all subsequent experiments. This mutant is subsequently referred to as PAK4(S445N). To construct Myc-tagged PAK4(S445N), an EcoRI fragment containing the entire coding sequence of PAK4(S445N) (without the HA tag) was removed from SR and inserted into the EcoRI site of the pCAN-Myc2 vector. To subclone PAK4 into the pIRES2-EGFP vector (Clontech), HA-PAK4wt was removed pBluescript KS II(+) as an XhoI/StuI fragment and inserted into the XhoI/SmaI cut of the pIRES2-EGFP vector. To construct the pIRES2-EGFP vector containing PAK4(S445N), an EcoRI/BamHI fragment of pIRES2-EGFP-PAK4 was replaced by the EcoRI/StuI fragments of SR-PAK4(S445N). To construct pLPC-PAK4-WT and pLPC-PAK4 mutants, HindIII/XhoI fragments from pCAN-Myc2-PAK4 were inserted into the HindIII/XhoI sites of the pLPC vector. The pLPC vector is a retroviral expression vector with a puromycin resistance marker (a gift from R. Prywes). JNK, Rac2L61, Dbl, and Cdc42V12 have been described previously (28).

Antibodies and phalloidin. The anti-HA antibody (MMS-101P) was obtained from Covance, and anti-Myc (sc-40) and anti-Rho antibodies (sc-418) were obtained from Santa Cruz Biotechnology. Mouse monoclonal anti-PAK4 antibody was generated against full-length human PAK4 protein (purified using the Gibco BRL Bac to Bac baculovirus system) in conjunction with PharMingen. Fluorescein isothiocyanate (FITC)-conjugated phalloidin (F-432) was obtained from Molecular Probes, antivinculin antibody (hVin-1) was obtained from Sigma, and rhodamine-conjugated goat anti-mouse immunoglobulin G (IgG) antibody (31663) was obtained from Pierce. Antipaxillin antibody (tetramethyl rhodamine isothiocyanate conjugated) was obtained from Transduction Laboratories. Alkaline phosphatase-conjugated goat anti-mouse IgG (59296) and peroxidase-conjugated goat anti-mouse IgG (55550) for Western blots were obtained from ICN. Antibodies to the regulatory 20-kDa myosin light chain (MLC₂₀) were produced by immunizing rabbits with MLC₂₀ purified from chicken gizzard smooth muscle. The antibodies were affinity purified by applying the immune serum to an MLC₂₀-Sepharose 4B column. Western blot analyses showed that these antibodies react specifically with purified smooth muscle MLC₂₀ and MLC₂₀ found in cell extracts prepared from smooth muscle and nonmuscle cells. Antibody against phosphorylated MLC was a gift from F. Matsumura.

Cell culture and transfection. 293T and Rat1 cells were grown at 37°C in 5% CO₂ and cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum. NIH 3T3 cells and v-Ki-Ras-transformed NIH 3T3 cells (31) were cultured in DMEM containing 10% bovine calf serum. Rat1 and NIH 3T3 cells stably transfected with either pLPC, PAK4, or PAK4 mutants were grown as described above in the presence of 1.5 or 2.0 µg of puromycin per ml, respectively. All media were supplemented with 100 U of penicillin/ml, 100 µg of streptomycin/ml, and 1 mM glutamine. Transient transfection assays were carried out using the Lipofectamine (Gibco-BRL) method according to the manufacturer's protocol. Stable cell lines were generated by retroviral infection. Briefly, 293T cells were transfected with empty pLPC vector, pLPC-Myc-PAK4, pLPC-Myc-PAK4(S474E), or pLPC-Myc-PAK4(S445N), together with helper plasmid  by the calcium phosphate precipitation method. Supernatants containing the released viruses were collected from 293T cells 2 days after transfection and filtered through a 0.45-µm-pore-size filter. The virus was then used to infect either Rat1 cells or NIH 3T3 cells. Cells were selected with puromycin (1.5 µg/ml for Rat1 cells and 2.0 µg/ml for NIH 3T3 cells) and colonies were picked approximately 2 weeks after selection. Expression of PAK4 was determined by Western blotting and immunofluorescence microscopy using a monoclonal antibody against the Myc tag.

Protein kinase assays. To assay the kinase activity of PAK4, NIH 3T3 cells were transfected with either empty SR expression vector or expression vectors containing either wild-type PAK4, PAK4(S474E), or PAK4(S445N) fused with the indicated epitope tags. Cells were harvested in M2 buffer (29) 48 h after transfection. PAK4 was immunopurified from approximately 100 µg of cell extracts using antibody generated against the epitope tag. PAK4 protein kinase activity was then assessed as described previously (1). JNK, ERK, and p38 activities were measured as described previously (28). MLC kinase (MLCK) assays were carried out as described previously (41).

Western blots. Western blots were carried out as described previously (1).

Immunofluorescence microscopy. Cells stably expressing wild-type or mutant PAK4 were grown on 22-mm glass coverslips in complete medium and 24 h later were fixed in 4% paraformaldehyde for 10 min at room temperature. For transient transfection experiments, cells were fixed 48 h after transfection. Fixed cells were permeabilized with phosphate-buffered saline (PBS) containing 0.1% Triton X-100 for 20 min. Cells were then incubated with the primary monoclonal anti-HA antibodies or anti-Myc antibodies for 60 min. The coverslips were washed with PBS containing 0.1% Triton X-100 and incubated for 30 min with the secondary rhodamine-conjugated anti-mouse antibody (Molecular Probes). To visualize F-actin, cells were washed again and were incubated with FITC-conjugated phalloidin. For vinculin staining, the antivinculin monoclonal antibody (hVIN-1; Sigma) was used as the primary antibody, and the rhodamine-conjugated anti-mouse antibody was used as the secondary antibody. For paxillin staining, cells were stained with paxillin-tetramethyl rhodamine isothiocyanate for 60 min after fixation and permeabilization. Fluorescence photomicroscopy was carried out with appropriate filters for fluorescence detection.

Soft agar assays. Approximately 10⁴ cells were suspended in 2 ml of 0.3% Bacto agar in DMEM containing 10% fetal bovine serum (Rat1 cells) or 10% bovine calf serum (NIH 3T3 cells), antibiotics, and glutamine and were overlaid on 2 ml of 0.6% Bacto agar in the same medium in 35-mm-diameter dishes. Each cell line was tested in duplicate wells. Colonies were visualized under an inverted light microscope after 2 to 3 weeks.

Focus formation assays. Focus formation assays with NIH 3T3 cells were carried out as described previously (28).

Cell spreading assays. Equal numbers of cells (5 × 10³) were plated onto either 96-well plates or 35-mm-diameter plates in the spreading medium (DMEM, 0.5% bovine serum albumin, 2 mM L-glutamine, penicillin-streptomycin, 20 mM HEPES) and incubated at 37°C. At 10- or 15-min intervals the medium from each well was removed and cells were fixed with fixative solution (4% paraformaldehyde in phosphate buffer) for 30 min at 4°C followed by incubation with dye solution (0.5% [wt/vol] toluidine blue [Sigma] in fixative solution) at 4°C for 1 h. Cells were rinsed with PBS and then visualized under a 10× objective, and the fraction of spread cells was determined at each time point.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

PAK4(S445N) has increased kinase activity compared to wild-type PAK4 but does not lead to an increased activation of the JNK pathway. Two PAK4 mutants were made in an attempt to generate an activated PAK4. The first mutation was PAK4(S474E) in which one of the predicted autophosphorylation sites was converted to a glutamate (1). The second mutant was PAK4(S445N) in which a serine-to-asparagine mutation was introduced at amino acid 445 in the catalytic loop within kinase subdomain VIb. Mutation of this residue is predicted to stabilize the catalytic loop (47). Expression vectors containing epitope-tagged PAK4(S445N), wild-type PAK4, or PAK4(S474E) were transfected into NIH 3T3 cells. After transient expression, PAK4 was immunopurified from cell lysates using antibodies directed against the epitope tags and used to phosphorylate histone H4 (HH4) in an in vitro kinase assay. PAK4(S474E) exhibited a small increase in its autophosphorylation activity compared with wild-type PAK4. Although serine 474 is a predicted autophosphorylation site and is necessary for PAK4 activity (1), our results suggest that additional sites within PAK4 may be weakly phosphorylated by the PAK4(S474E) mutant. In spite of this, PAK4(S474E) was actually weaker than wild-type PAK4 in HH4 phosphorylation (Fig. 1A). Strikingly, however, PAK4(S445N) showed an approximately 30-fold increase in both autophosphorylation and HH4 phosphorylation compared with wild-type PAK4 (Fig. 1A). Both Cdc42 and Rac are strong activators of the JNK and p38 MAP kinase pathways. Although PAK4 can activate the JNK pathway, its activation ability is weak compared with that by activated Rac or Cdc42 and its primary function is most likely cytoskeletal regulation rather than activation of the JNK pathway (1). To determine whether PAK4(S445N) was a stronger activator of the JNK pathway than wild-type PAK4, NIH 3T3 cells were transfected with Myc-tagged PAK4 expression vectors or empty SR vector together with an expression vector for HA-tagged JNK. After transient expression, HA-JNK was immunopurified using antibodies against the HA tag. Immune complex kinase assays were carried out using glutathione S-transferase-c-Jun as substrate. As shown in Fig. 1B, PAK4(S445N) did not activate the JNK pathway any more than wild-type PAK4 did. Like wild-type PAK4, PAK4(S445N) was not an efficient activator of the p38 or ERK pathways (data not shown). These results suggest that despite its high activity, PAK4(S445N) retains the substrate specificity of wild-type PAK4.

View larger version (31K): [in this window] [in a new window]   FIG. 1.   PAK4(S445N) has increased kinase activity compared with wild-type PAK4 but is not enhanced in its activation of the JNK pathway. (A) NIH 3T3 cells were transiently transfected with either empty vector (Con) or vectors containing HA-tagged wild-type PAK4 (PAK4 WT) or HA-tagged PAK4(S474E) (left panel), or they were transiently transfected with empty vector or vectors containing Myc-tagged wild-type PAK4 or Myc-tagged PAK4(S445N) (right panel). After transient expression, PAK4 was immunopurified from cell lysates with anti-HA antibody (left) or anti-Myc antibody (right). Immunopurified PAK4 was incubated with HH4 or without any substrate in the presence of [-32P]ATP and kinase buffer. Substrate phosphorylation or autophosphorylation was analyzed after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. Aliquots of each lysate were subjected to Western blot analysis with anti-HA (left) or anti-Myc (right) antibody to show that PAK4 was expressed at approximately equivalent levels in each transfection. (B) NIH 3T3 cells were transiently transfected with 1 µg of empty vector, Myc-tagged PAK4(S445N) (0.5 or 1.0 µg), or 1 µg of Myc-tagged wild-type PAK4 together with 1 µg of HA-tagged JNK expression vector. After transient transfection, HA-JNK was immunopurified from cell lysates using anti-HA antibody and then incubated with glutathione S-transferase-c-Jun in the presence of [-32P]ATP and kinase buffer. Substrate phosphorylation was analyzed after SDS-PAGE and autoradiography. As a positive control, cells were transfected with Rac2L61 expression vector and HA-JNK.

Transient expression of PAK4(S445N) causes cell rounding in NIH 3T3 cells. To determine whether PAK4(S445N) has an effect on cell morphology, NIH 3T3 cells were transiently transfected with either empty vector, wild-type PAK4, or PAK4(S445N) in a bicistronic vector containing an internal ribosome entry sequence and enhanced green fluorescent protein (EGFP). After 24 h, PAK4(S445N)-expressing cells had a distinctly rounded appearance and adhered poorly to the surface of the dish (Fig. 2). Trypan blue exclusion experiments confirmed the viability of the PAK4(S445N)-expressing cells (data not shown). Wild-type PAK4-expressing cells were more spread out and looked similar to cells containing the empty vector. Similar results were found in several other cell types that were transfected with PAK4(S445N), including 293T cells and PAE cells (data not shown).

View larger version (53K): [in this window] [in a new window]   FIG. 2.   Transient expression of PAK4(S445N) results in cell rounding. NIH 3T3 cells were transfected with 1 µg of either EGFP expression vector (top), PAK4-IRES2-EGFP (middle), or PAK4(S445N)-IRES2-EGFP (bottom). Twenty-four hours after transfection, cells expressing the EGFP vectors were visualized by fluorescence microscopy under a 40× objective.

Fibroblasts stably expressing PAK4(S445N) show distinctive morphological and cytoskeletal changes. To further characterize the morphological changes induced by constitutively active PAK4, stable cell lines were generated in which Rat1 cells and NIH 3T3 cells overexpressed either empty vector (pLPC), Myc-tagged wild-type PAK4, PAK4(S474E), or PAK4(S445N). Expression of PAK4 in the stable cell lines was analyzed by Western blotting (Fig. 3A) and immunofluorescence microscopy. Immunofluorescence analysis indicated that each cell line was homogenous for expression of PAK4 (data not shown). The morphologies of the pLPC- and PAK4(S445N)-expressing Rat1 cells are shown in Fig. 3B and C. Figure 3B shows the morphologies of the cells 10 min and 1 h after plating onto fibronectin-coated surfaces. At 10 min, PAK4(S445N)-expressing cells were rounded but had numerous long filopodia. These filopodia were quite motile as assessed by video microscopy, and a representative cell that was photographed at 20-s intervals approximately 10 min after plating is shown in panels i, ii, and iii. At 1 h, some of the cells had a more flattened morphology, but filopodia could still be visualized at the edges of the cells. See panel iv for an example of a cell that had begun to spread onto the surface. Filopodia could be visualized up to 3 h after plating. The filopodia were somewhat transient, however, because by 24 h filopodia could not be visualized in most of the Rat1 cells (see below). In contrast to the PAK4(S445N) cells, very few filopodia could be detected on the control pLPC cells either 10 min or 1 h after plating (panels v and vi). These results indicate that like wild-type PAK4 (1), PAK4(S445N) can induce filopodia. Unlike wild-type PAK4, however, PAK4(S445N) can induce filopodia even without cotransfection of activated Cdc42.

View larger version (382K): [in this window] [in a new window]   FIG. 3.   Morphological changes in fibroblasts stably expressing PAK4(S445N). (A) Representative Western blot analysis of Myc-tagged PAK4 expression in the different stable cell lines. Cell lysates (25 µg) from Rat1 cells or NIH 3T3 cells stably expressing the indicated plasmids were used in each blot. Blots were probed with anti-Myc antibody (top and middle panels) or anti-PAK4 antibody (bottom panel). (B) Rat1 cells expressing PAK4(S445N) produce filopodia after plating onto fibronectin. Cells expressing empty vector (pLPC) or PAK4(S445N) were plated onto fibronectin-coated coverslips and visualized by Zeiss Axiovert phase-contrast microscopy 10 min and 1 h after plating under a 100× objective. Cell morphology was visualized by time lapse photography and individual frames are shown in the figure. Panels i, ii, and iii show a PAK4(S445N) cell that was photographed at 10-s intervals 10 min after plating. Panel iv shows an example of a PAK4(S445N) cell that had already begun to spread onto the fibronectin-coated surface 1 h after plating. Panels v and vi show control cells 10 min and 1 h, respectively, after plating. (C) Morphological changes in Rat1 cells overexpressing PAK(S445N). Top panels, cells containing either PAK4(S445N) or empty vector (pLPC) were visualized by phase-contrast microscopy under a 10× objective 24 h after plating; middle panels, cells were fixed 24 h after plating onto fibronectin-coated coverslips, and polymerized actin was visualized under a 60× oil lens after staining with FITC-conjugated phalloidin; bottom panels, cells were fixed 24 h after plating, and focal adhesions were visualized by immunofluorescence microscopy under a 60× oil lens after staining with anti-vinculin antibody and rhodamine-conjugated secondary antibody. (D) Morphological changes in NIH 3T3 cells expressing PAK4(S445N). NIH 3T3 cells were analyzed as described for panel C.

PAK4 does not inhibit MLC phosphorylation. The dissolution of stress fibers is reminiscent of previous results seen with activated PAK1. Activated PAK1 was shown to cause the dissolution of stress fibers by phosphorylating MLCK and inhibiting MLCK activity, thereby decreasing MLC phosphorylation on serine 19 (41). PAK1 thus inhibits stress fiber formation, since MLC phosphorylation is required for the actin-myosin interactions that are present in actin stress fibers. In contrast to PAK1, however, activated PAK4 did not phosphorylate MLCK (Fig.4A), although in parallel experiments MLCK was phosphorylated by activated PAK1 (data not shown). To examine MLC phosphorylation in PAK4(S445N)-expressing cells, equal volumes of lysates from pLPC- and PAK4(S445N)-expressing NIH 3T3 cells were blotted and probed with anti-phospho-MLC antibody and anti-MLC antibody (Fig. 4B). Although a lower amount of MLC was detected in the lysates from the PAK4(S445N)-expressing cells, there was not a corresponding decrease in MLC phosphorylation (Fig. 4B). Thus, PAK4 appears to differ from PAK1 in its substrate specificity and in the mechanism by which it induces cytoskeletal reorganization.

View larger version (21K): [in this window] [in a new window]   FIG. 4.   PAK4 does not phosphorylate MLCK or regulate MLC phosphorylation. (A) Wild-type PAK4, PAK4(S474E), or PAK4(S445N) were immunopurified from NIH 3T3 stable cell lines using anti-Myc antibody. Empty pLPC vector cells were used as a control. Immunoprecipitates were incubated with purified MLCK (41) and an in vitro kinase assay was carried out as described previously (41). Phosphorylated MLCK and PAK4 autophosphorylation were visualized after SDS-PAGE and autoradiography. (B) PAK4(S445N) does not lead to a decrease in MLC phosphorylation. Equal volumes of lysates from NIH 3T3 cells expressing pLPC or PAK4(S445N) were analyzed by Western blotting. Filters were probed with anti-phospho-MLC antibody, which recognizes phospho-serine 19 (top panel), or anti-MLC antibody (bottom panel).

Fibroblasts expressing PAK4(S445N) are deficient in their ability to spread on extracellular matrix-coated surfaces. Because of the low level of vinculin staining in the PAK4(S445N)-expressing cells, we were interested in determining whether PAK4-expressing cells had a defect in cell spreading. To analyze cell spreading, the Rat1 stable cell lines were plated onto either fibronectin- or collagen-coated wells. At 10- to 15-min intervals cells were fixed and stained as described in Materials and Methods. The number of spread cells was counted at each time point. The results indicate that Rat1 cells containing PAK4(S445N) spread significantly more slowly on both surfaces than did Rat1 cells containing empty vector (Fig. 5).

View larger version (60K): [in this window] [in a new window]   FIG. 5.   Inhibition of spreading in cells expressing PAK4(S445N). Equal numbers of stable Rat1 cells expressing empty vector (pLPC) (diamonds) or PAK4(S445N) (squares) were plated onto dishes that were coated with fibronectin or collagen. At the indicated time intervals, cells were fixed and stained as described in Materials and Methods and visualized by phase-contrast microscopy. The number of spread cells was counted at each time point and expressed as a percentage of the total cell number.

PAK4-overexpressing cells exhibit anchorage-independent growth in soft agar. The rounded morphologies of PAK4(S445N)-overexpressing cells and the loss of stress fibers and focal adhesions resembled fibroblasts that have been transformed with oncogenes such as the Ras oncogene. One of the hallmarks of oncogenic transformation is the loss of anchorage-dependent growth as demonstrated by the ability to form colonies on soft agar. Because the PAK4(S445N)-expressing cells had a decrease in cell adhesion without any noticeable change in cell viability, we were interested in determining whether PAK4(S445N) cells exhibited a lack of anchorage-dependent growth. We analyzed two independent Rat1 cell lines and two NIH 3T3 cell lines overexpressing PAK4(S445N), wild-type PAK4, or empty pLPC vector. For comparison, we analyzed NIH 3T3 cells that were stably transfected with oncogenic v-Ki-Ras (31). Equal numbers of each cell type were plated in soft agar as described in Materials and Methods. We found that all of the Rat1 and NIH 3T3 cell lines containing PAK4(S445N) produced colonies on soft agar similar to Ras-transformed NIH 3T3 cells (Fig. 6A and B). In each case [both PAK4(S445N)- and v-Ki-Ras-transformed cells] approximately 18% of the total plated cells produced foci. Neither Rat1 cells nor NIH 3T3 cells expressing wild-type PAK4 or pLPC formed any colonies in soft agar (Fig. 6A and B). Likewise, PAK4(S474E)-expressing cells formed very few colonies in soft agar (data not shown). These differences reflect the anchorage-independent growth characteristic of PAK4(S445N)-expressing cells rather than an increase in their growth rates, as PAK4(S445N)-expressing cells did not grow at an enhanced growth rate compared with the pLPC-containing cells under normal adherent culture conditions (data not shown).

View larger version (129K): [in this window] [in a new window]   FIG. 6.   PAK4(S445N) induces anchorage-independent growth, and PAK4 is required for oncogenic Dbl-induced focus formation. (A and B) PAK4(S445N) induces anchorage-independent growth. Soft agar growth assays were carried out as described in Materials and Methods. PAK4(S445N)-expressing cells (NIH 3T3 cells and Rat1 cells) produced colonies on soft agar similar to those of Ras-transformed cells. Pictures of the plates containing each indicated cell line were taken 2 weeks after plating. The larger colonies are visible by eye, as shown in panel A (no magnification). Representative foci of each indicated cell line were visualized by phase-contrast microscopy under a 10× objective and are shown in panel B. (C) PAK4 is required for focus formation by oncogenic Db1. NIH 3T3 cells were transfected with oncogenic Dbl or Ras (200 ng) together with either empty vector (CMV) or the indicated concentrations of PAK4(K350M), PAK4R, PAK4RGBD, or Cdc42N17. Cells were grown for 2 weeks following transfection and foci were counted. The number of foci is indicated as the percent of foci induced by Db1 or Ras plus empty vector. Db1 and Ras plus empty vector each produced approximately 500 foci per µg of transfected Db1 or Ras DNA. The results are the averages of two independent experiments.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

PAK4 was originally identified as a molecular target for the Rho GTPase Cdc42, and it plays a key role in Cdc42's ability to induce filopodia (1). Here we have generated a constitutively active mutant of PAK4 [PAK4(S445N)] in order to better characterize the function of this serine/threonine kinase. Fibroblasts expressing PAK4(S445N) transiently induced filopodia when they were plated onto fibronectin, which is consistent with PAK4's function as a target for Cdc42. Activated PAK4 also had a number of other functions which were not revealed previously by studying wild-type PAK4. Most importantly, expression of PAK4(S445N) caused a transformed morphology in fibroblasts. Fibroblasts stably expressing PAK4(S445N) lost their anchorage-dependent growth requirement and formed colonies in soft agar. Similar results were shown previously for cells expressing activated Cdc42 (19, 20, 34). Consistent with this, dominant-negative PAK4 mutants inhibited focus formation by oncogenic Dbl, an exchange factor for the Rho GTPases. We propose that PAK4(S445N)'s ability to transform cells is due at least in part to its ability to induce morphological changes, including cell rounding, loss of stress fibers and focal adhesions, and decreased spreading.

PAK4(S445N) is the first full-length activated PAK4 to be generated. PAK4 as well as the PAK4 Drosophila melanogaster homologue mushroom body tiny (mbt) (25) and an uncharacterized Caenorhabditis elegans homologue, C45B11.1, all encode for proteins with a serine at residue 445. Most serine/threonine protein kinases, including other members of the PAK family, however, have an asparagine residue at the corresponding site. The asparagine at this position serves to stabilize the catalytic loop by hydrogen bonding to a conserved aspartate (corresponding to D440 in PAK4) within the loop (47). The serine-to-asparagine conversion in PAK4 is therefore thought to function by strengthening the catalytic loop. Although PAK4(S445N) has strong kinase activity, its substrate specificity appears to be unaltered. Like wild-type PAK4, for example, PAK4(S445N) does not phosphorylate MLCK. Furthermore, PAK4(S445N) does not activate the ERK or p38 pathways and does not activate the JNK pathway any further than wild-type PAK4 does. This is consistent with our previous prediction that PAK4 is primarily a mediator of the cytoskeletal changes induced by Cdc42 rather than a key player in the Cdc42-to-JNK pathway (1).

The morphological changes resulting from PAK4(S445N) expression do not appear to be nonspecific effects due to the high activity of this kinase. Rather, they seem to be an amplification of wild-type PAK4's activity. Overexpression of even wild-type PAK4 in some cells, such as HeLa cells, results in a slightly rounded shape and lowered adhesion (N. Gnesutta and A. Minden, unpublished results). Likewise, another activated PAK4 mutant, PAK4, in which the PAK4 regulatory domain is deleted (1), induced some of the morphological changes induced by PAK4(S445N), such as a decrease in stress fibers and focal adhesions, but not filopodium formation (data not shown). Furthermore, some of the functions of PAK4(S445N) are consistent with PAK4's role as an effector for Cdc42. For example, the transient induction of filopodia and the ability to confer anchorage-independent growth are functions which are shared by PAK4 and activated Cdc42 (17, 19, 20, 30, 34). Importantly, the activities of PAK4(S445N) appear to be specific to PAK4. For example, unlike activated PAK4, constitutively active PAK1(T423E) does not induce anchorage-independent growth (data not shown) (45). PAK4(S445N) also differs from activated PAK1 in substrate specificity and the mechanism by which it induces cytoskeletal changes. In particular, we have found that unlike PAK1, the dissolution of stress fibers by PAK4 does not appear to be mediated by MLCK phosphorylation and the subsequent regulation of MLC phosphorylation.

While many of the functions of PAK4(S445N) are characteristic of Cdc42 functions, some of its functions, such as cell rounding and the dissolution of stress fibers, may be distinct from Cdc42 functions. The identification of new PAK4 activators other than Cdc42 will be important in order to better understand whether it also serves as a target for other types of signaling and cytoskeletal regulatory proteins. The mechanisms by which PAK4 induces its various morphological effects are not yet clearly understood. For example, as discussed above, unlike for PAK1, the dissolution of stress fibers by PAK4 is not due to a decrease in MLC phosphorylation. It is interesting to note, however, that PAK4 does lead to a consistent decrease in MLC expression which could potentially contribute to the dissolution of stress fibers. Another possibility is that PAK4 could inhibit Rho activity, which normally functions to stimulate stress fiber formation. We have in fact found that Rho activity is decreased (but not abolished) in PAK4(S445N)-expressing cells (data not shown) and it will be interesting to determine whether this is related to PAK4's role in stress fiber dissolution. It should be noted, however, that activated Rho triggers stress fiber formation by a mechanism that involves multiple steps which include increased phosphorylation of MLC (15) and activation of LIMK1 (21, 32), yet we do not see a decrease in MLC phosphorylation (Fig. 4) or an inhibition of LIMK1 activity (data not shown) in PAK4(S445N)-expressing cells. Ultimately it will be important to identify new substrates for PAK4 in order to fully understand the mechanism by which it induces specific morphological changes.

The ability of PAK4(S445N) to induce a transformed phenotype in fibroblasts is especially intriguing because the regulation of cell proliferation, progression through the cell cycle, and oncogenic transformation are important functions of the Rho proteins (14, 18, 34-36). Most of the Dbl family exchange factors are potent oncogenes, and all three GTPases have important contributions to oncogenic transformation by Dbl (20). A number of target proteins for the Rho GTPases have been identified which may be necessary for oncogenic transformation, such as the Rho effector protein ROCK (39, 40, 48) and the Cdc42 target protein -COP (49). Neither of these, however, is sufficient to transform cells on its own. Likewise, activated PAK1 does not induce transformation on its own (44, 45), even though it has been shown to be necessary for transformation by oncogenic Ras in some cells (44, 45). Furthermore, effector loop mutants for Cdc42 and Rac which do not bind to PAKs 1, 2, or 3 can still induce many of the hallmarks of oncogenic transformation (13, 18), suggesting that these PAK family members are not required for transformation by the Rho family GTPases.

In contrast to other PAKs, we have found that activated PAK4 confers anchorage-independent growth on fibroblasts and leads to focus formation in soft agar assays. This work is the first study to show a direct role for a PAK protein in transformation. Importantly, constitutively active and cycling mutants of Cdc42 also induce anchorage-independent growth in fibroblasts (19, 20, 34). The induction of anchorage-independent growth has in fact been shown to be a major contribution of Cdc42 to transformation by oncogenic Dbl (20). Not only does activated PAK4 trigger anchorage-independent growth, but dominant-negative PAK4 also inhibits transformation by oncogenic Dbl, a potent activator of all three Rho family GTPases. This inhibition is not due merely to sequestering Cdc42, because a mutant that lacks the GBD also inhibits transformation. Dominant-negative PAK4 was less efficient at inhibiting Dbl-induced foci than was dominant-negative Cdc42 (Cdc42N17). However, it should be noted that the PAK4 mutants and Cdc42N17 cannot be directly compared with each other because we do not know whether they have the same capacities to act as dominant-negative mutants, i.e., to inhibit their endogenous counterparts. Furthermore, it should be noted that Cdc42N17 can bind directly to Dbl and therefore may be a particularly effective inhibitor of Dbl activity.

The mechanism by which PAK4 can regulate oncogenic transformation is not yet known. Our results suggest that activated PAK4 can induce anchorage-independent growth in the absence of any strong activation of several known signal transduction pathways, such as the JNK, p38, and ERK pathways, that lead to changes in gene expression patterns. Rather, we propose that changes in cell morphology and adhesion play a major role in PAK4's ability to transform cells. We do not know, however, whether all of the different cytoskeletal changes induced by PAK4(S445N) contribute to the oncogenic process. Further investigation will be necessary in order to determine which of the morphological changes induced by PAK4 are directly related to its role in transformation. Furthermore, as discussed above, although PAK4 was originally identified as a target for Cdc42, we cannot rule out the possibility that it could regulate transformation by a mechanism that is either partly or entirely independent of Cdc42. For example, recently we have found that PAK4 can protect cells against apoptosis and inhibit caspase activation in response to a variety of different stimuli, including serum withdrawal, UV irradiation, and tumor necrosis factor alpha stimulation (12). Since inhibition of apoptosis is an important part of oncogenic transformation, such a protective role is also likely to contribute to PAK4's role in oncogenic transformation.

We do not rule out the possibility that PAK4 may also mediate oncogenic transformation or morphological changes in response to other signaling enzymes besides Cdc42. We have found, however, that dominant-negative PAK4 is an inefficient inhibitor of Ras transformation. This is initially surprising because previous work using dominant-negative Cdc42N17 has indicated that Ras requires Cdc42 to produce foci (34). It should be noted, however, that the requirement for Cdc42 by Ras and Dbl cannot be directly compared, because Cdc42N17 is a much more effective inhibitor of Dbl foci than of Ras foci (Fig. 6). In future work it will be interesting to determine whether other oncogenes require signals through PAK4 to induce transformation and whether PAK4 can mediate responses that are independent of Cdc42.