Introduction

PtdIns(3,4)P₂ and PtdIns(3,4,5)P₃ levels in cells are regulated by PTEN, a phosphatase that dephosphorylates both these phosphoinositides at the 3-position (Maehama and Dixon, 1998; Dahia, 2000). The PTEN gene was originally identified as a tumor suppressor gene located at 10q23.3. This gene is deleted in a wide variety of human cancers such as prostate, breast and endometrial cancers, as well as in glioblastomas and melanomas (Furnari et al., 1997; Li et al., 1997; Risinger et al., 1997; Steck et al., 1997; Cairns et al., 1998). PTEN acts opposite to PI 3-kinase by inducing growth suppression via cell cycle arrest and/or induction of apoptosis, and by inhibiting cell adhesion and migration (Dahia, 2000).

Pten mutations have been found in far advanced metastatic cancers, particularly in high-grade gliomas (44%) and certain types of endometrial cancers (55%) (Mertens et al., 1997; Wang et al., 1997; Hahn et al., 1999). Introduction of PTEN into PC-3 prostate cancer and B16F10 melanoma cells has been shown to suppress the metastasis of these tumors in vivo by a yet uncharacterized mechanism (Celebi et al., 2000; Hwang et al., 2001; Davies et al., 2002).

The PI 3-kinase pathway is a major pathway involved in regulating cellular actin remodeling in response to growth factor stimulation (Rijken et al., 1991; Wennstrom et al., 1994; Nobes et al., 1995; Ojaniemi and Vuori, 1997; Tsakiridis et al., 1999; Dong et al., 2000). An array of different proteins bind to the lipid products of PI 3-kinase and are recruited to the plasma membrane. These proteins typically contain a pleckstrin homology (PH) domain that mediates binding to PtdIns(3,4)P₂ and PtdIns(3,4,5)P₃ (Lemmon and Ferguson, 2000)_. Examples of PH-domain-bearing proteins include guanine nucleotide exchange factors (GEFs) for small G proteins (Musacchio et al., 1993; Franke et al., 1997; Stephens et al., 1998; Balendran et al., 1999; Rameh and Cantley, 1999; Lemmon and Ferguson, 2000; Vanhaesebroeck et al., 2001), Akt (protein kinase B) and 3'-phosphoinositide-dependent kinase-1 (PDK-1).

The actin cytoskeleton is predominantly regulated by members of the Rho family GTPases (Burridge and Wennerberg, 2004). Rho GTPases (Rho, Rac and Cdc42) are activated by GEFs which promote the exchange of GDP for GTP upon binding to PtdIns(3,4,5)P₃ (Han et al., 1998; Scita et al., 2000). Activation of Rho results in formation of stress fibers and focal adhesion, whereas activation of Rac and Cdc42 results in lamellipodia and filopodia formation, respectively (Burridge and Wennerberg, 2004). Rho activates many proteins including Rho-kinases (ROCKs), mDia and protein kinase N (PKN) (Burridge and Wennerberg, 2004). Stress fiber formation is promoted when ROCK phosphorylates and activates LIM-kinase (LIMK), which subsequently phosphorylates and inactivates cofilin, an actin-depolymerizing factor, resulting in net polymerization of actin filaments (Riento and Ridley, 2003).

We recently discovered another PI 3-kinase-dependent pathway that regulates the actin cytoskeleton (Dong et al., 2000). In this pathway, insulin-induced loss of actin stress fibers and accumulation of cortical actin are mediated by PDK-1 phosphorylation and activation of PKN. This actin phenotype, which can be induced by overexpression of either PDK-1 or PKN in 3T3-L1 and Rat1 cells, appears to be similar to the actin phenotype of Pten^-/- MEFs. PDK-1 is a cytoplasmic Ser/Thr kinase that translocates to the plasma membrane upon binding of PtdIns(3,4)P₂ and PtdIns(3,4,5)P₃, where it phosphorylates Akt (Anderson et al., 1998). PKN acts further downstream of PI 3-kinase and is activated by either RhoA binding or phosphorylation by PDK-1 (Amano et al., 1996; Watanabe et al., 1996; Dong et al., 2000).

The cell migration process – lamellipodia formation, cell body contraction and detachment of adhesion at the rear of the cell – is driven by changes in the actin network (Raftopoulou and Hall, 2004). MEFs and prostate cancer cells deficient in PTEN exhibit loss of actin stress fibers (Liliental et al., 2000) characteristic of malignant transformation of fibroblasts by oncogenic proteins such as Ras (Bar-Sagi and Feramisco, 1986; Barbacid, 1987; Maruta et al., 1999), and accumulation of cortical actin. The signaling pathway responsible for mediating loss of actin stress fibers and cortical actin accumulation in PTEN-deficient cells is unknown, whereas the increased migration of these cells has been attributed to increased Rac and Akt activities (Liliental et al., 2000; Higuchi et al., 2001). In this study, we investigated the potential link between actin rearrangement and increased motility of the Pten^-/- MEFs. We found that the Rho pathway but not the PDK-1 pathway is involved in mediating the cortical actin formation of Pten^-/- MEFs. In contrast, cell migration is regulated by the PDK-1/Akt pathway but not the Rho pathway. Our results indicate that PDK-1 plays a novel and potentially important role in promoting cell migration downstream of PTEN.

Results

Characterization of Pten^-/- cell line

In order to identify the signaling molecules involved in mediating actin reorganization and cell migration in Pten^-/- cells, we examined the expression profiles of components of the PI 3-kinase pathway. Western blot analysis indicated that both Pten^+/+ and Pten^-/- cell lines expressed downstream effectors of PI 3-kinase, such as PDK-1, Akt, PKN and PKC-related kinase 2 (PRK2) (data not shown). In agreement with our previous findings (Liliental et al., 2000), the actin cytoskeleton of the Pten^-/- cells is strikingly different compared to the Pten^+/+ cells (Figure 1a, panels II and I, respectively). Treatment of cells with the PI 3-kinase pharmacological inhibitor, LY294002 (Figure 1a, panel IV), in the presence of serum led to approximately a 20–30% reduction in the number of cells exhibiting cortical F-actin compared to cells treated with vehicle (Figure 1a, panel III). Maximal inhibition of approximately 40% was obtained after 4 h of treatment with LY 294002 in the absence of serum (data not shown). These results indicate that cortical actin formation is partially dependent upon PI 3-kinase activity. To verify that loss of PTEN is responsible for this actin phenotype, PTEN WT, C124S (lipid and protein phosphatase inactive) or G129E (lipid phosphatase inactive) mutants were expressed in Pten^-/- cells. Overexpression of PTEN WT significantly inhibited cortical actin formation and restored stress fibers of Pten^-/- cells (Figure 1b and c), whereas overexpression of either the C124S or the G129E mutant had no effect. These results support the hypothesis that cortical actin is caused by loss of PTEN, and that loss of the lipid phosphatase activity of PTEN is responsible for this actin phenotype.

Figure 1.

Cortical actin formation is caused by the absence of PTEN. (a) Treatment of cells with PI 3-kinase inhibitors partially inhibits cortical actin formation. Filamentous F-actin staining of wild type (panel I) or Pten^-/- cells (panel II). Pten^-/- cells were incubated with either DMSO (panel III) or 50 M LY294002 (panel IV) for 1 h in the presence of serum. (b) Loss of the lipid phosphatase activity of PTEN is responsible for cortical actin formation. Pten^-/- cells transiently transfected with PTEN WT, C124S or G129E were stained with a polyclonal PTEN antibody, followed by Alexa Fluor 350 (left panels), and counterstained for F-actin (right panels). (c) Graphical representation of two independent experiments showing the effect of PTEN overexpression on the frequency of cortical actin formation in Pten^-/- cells. Approximately 200 cells were counted per experiment

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The Rho pathway is involved in regulating cortical actin formation in Pten^-/- MEFs

In a previous study, we showed that the Ser/Thr kinases PDK-1 and PKN are involved in regulating the insulin-induced loss of actin stress fibers and accumulation of cortical actin in 3T3-L1 and Rat1 cells (Dong et al., 2000). Here we examined whether PDK-1 and PKN produced similar effects in Pten^+/+ cells. PDK-1 overexpression led to an approximately 10–15% increase in the number of cells exhibiting actin reorganization, which is predominantly loss of actin stress fibers, whereas approximately 50% of Pten^+/+ cells overexpressing PKN exhibited loss of stress fibers and/or increased cortical F-actin formation (Figure 2). Overexpression of PRK2, a PKN isoform, had only a slight effect on actin structure in a small proportion of cells (Figure 2). The cortical actin induced by PKN overexpression in Pten^+/+ cells is not as thick as those seen in untransfected Pten^-/- cells, which may indicate that other factors, in addition to PKN, are contributing to cortical actin formation in the knockout cells.

Figure 2.

Overexpression of PKN in wild-type fibroblasts results in actin reorganization. (a) Pten^+/+ cells transiently expressing Myc-PDK1, Myc-PKC, FLAG-PKN and FLAG-PRK2 were stained for the respective proteins (left panels) and filamentous actin (right panels). The arrows indicate cells expressing the tagged proteins. (b) A graphical representation of the percentage of cells exhibiting actin change. Actin change refers to loss of actin stress fiber and/or accumulation of cortical actin. Approximately 100 transfected cells were counted per experiment and the bars in the graph represent the means.d. (standard deviation) of two independent experiments

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In order to identify the kinases responsible for regulating cortical actin formation in Pten^-/- cells, we transiently expressed kinase-inactive forms of PDK-1 (Myc-PDK-1^K114G), PKN (FLAG-PKN^K644D) and PRK2 (and FLAG-PRK2^K686E). The negative control used is kinase-dead LIM-kinase 1 (FLAG-LIMK1^D460E). Overexpression of LIMK1, a regulator of actin dynamics, acts downstream of Rho GTPases (Frame and Brunton, 2002), producing a different actin phenotype in Pten^+/+ cells (actin clumping, data not shown) compared to either PDK-1 or PKN. Overexpression of Myc-PDK-1^K114G, FLAG-PRK2^K686E or FLAG-LIMK1^D460E did not affect cortical actin formation of Pten^-/- cells (Figure 3a). In contrast, overexpression of FLAG-PKN^K644D resulted in significant inhibition of cortical actin formation (Figure 3a and b). However, stress fibers were not restored in cells expressing FLAG-PKN^K644D. These findings suggest that PKN plays a role in cortical actin but not stress fiber formation of Pten^-/- cells.

Figure 3.

Overexpression of kinase-inactive PKN blocks the accumulation of cortical F-actin in Pten^-/- cells. (a) Pten^-/- cells transiently expressing Myc-PDK-1^KD, FLAG-PKN^KD, FLAG-PRK2^KD and FLAG-LIMK1^KD were stained for protein expression (left panel) and filamentous actin (right panels). Arrows indicate cells expressing the tagged proteins. (b) Graphical representation of two independent experiments, bars in the graph represent the means.d. (standard deviation). Approximately 100 cells were counted per experiment

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PKN is a 120 kDa protein composed of a regulatory N-terminus with leucine-zipper-like motifs and a C-terminal kinase domain that resembles the kinase domain of the protein kinase C family (Mukai and Ono, 1994; Mukai et al., 1994; Palmer et al., 1995). The PKN N-terminus interacts with numerous proteins, most of which are involved in modulating cytoskeletal stability and dynamics. RhoA is the best-characterized activator of PKN; binding of activated Rho to the PKN N-terminus activates PKN both in vitro and in vivo (Watanabe et al., 1996; Amano et al., 1997; Lu and Settleman, 1999). We examined the roles of members of the Rho GTPase family in regulating cortical actin formation by overexpressing dominant-negative (DN) RhoA (Rho^N19), Rac (Rac^N17) and Cdc42 (Cdc42^N17) in Pten^-/- cells. Only overexpression of Rho^N19 inhibited cortical actin formation (Figure 4a and b), indicating that Rho regulates cortical actin formation caused by PTEN deficiency.

Figure 4.

Overexpression of DN RhoA blocks the accumulation of cortical F-actin in PTEN-deficient cells. (a) PTEN-deficient cells transiently expressing HA-RhoA^N19, Myc-Rac1^N17 and Myc-Cdc42^N17 were stained for protein expression (left panels) and filamentous actin (right panels). Arrows indicate cells expressing the tagged proteins. (b) The percentage of cells exhibiting cortical actin formation in the untransfected control was arbitrarily assigned as 100%, and all other measurements were expressed relative to this value. The bars in the graph represent the means.e. (standard error) of three independent experiments. Approximately 100 cells were counted per experiment

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To further verify that the Rho pathway is involved in regulating cortical actin assembly in Pten^-/- cells, we tested whether the N-terminal Rho-binding domain of PKN could act as a DN in blocking the formation of cortical F-actin in the Pten^-/- cells. Plasmid constructs encoding FLAG-tagged human PKN, kinase-inactive (PKN^K644D), PKN N-terminus (PKN-N, amino acids 2–512), PKN C-terminus (PKN-C, amino acids 561–942) and kinase-dead PKN-C (PKN-C^KD) were transfected into PTEN-deficient cells (schematic representation of the constructs is shown in Figure 5b). Overexpression of PKN^K644D but not wild-type PKN inhibited cortical actin accumulation in Pten^-/- cells (Figure 5a and c). Furthermore, PKN-N but not PKN-C or PKN-C^KD inhibited cortical F-actin formation (Figure 5a and c). Taken together, the data strongly suggest that the Rho pathway is involved in cortical actin formation in Pten^-/- cells.

Figure 5.

The N-terminus of PKN acts as a DN in blocking accumulation of cortical F-actin in PTEN-deficient cells. (a) PTEN-deficient cells transiently expressing FLAG-tagged PKN (panel I), PKN^KD (panel II), PKN-N-terminus (PKN-N, panel III), PKN C-terminus (PKN-C, panel IV) and kinase-dead PKN-C (PKN-C^KD, panel V) were stained for protein expression and filamentous actin. Arrows indicate cells expressing the tagged proteins. (b) Schematic representation of full-length and different truncation mutants of PKN. 'LZ' indicates leucine zipper-like motifs. (c) Graphical representation of two independent experiments, approximately 100 cells were counted per experiment and the bars in the graph represent the means.d. (standard deviation)

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The Rho pathway is activated in Pten^-/- cells

Activation of Rho GTPase is achieved via binding of GTP, which is catalysed by the GEFs (Burridge and Wennerberg, 2004). We therefore investigated whether PTEN deficiency leads to activation of the Rho pathway by first examining whether Rho activation is increased in Pten^-/- cells. As only activated Rho (Rho-GTP) binds to rhotekin (Reid et al., 1996), we performed pulldown assays using rhotekin fused to GST to affinity precipitate endogenous activated Rho from Pten^-/- cells and wild-type cells. As shown in Figure 6a and b, a significant increase (approximately twofold) in the amount of Rho-GTP was detected in Pten^-/- cells compared to wild-type cells. On average, the amount of RhoA-GTP pulled down from the Pten^+/+ and Pten^-/- samples represents approximately 4 and 7% of the total cell lysate, respectively.

Figure 6.

The Rho pathway is activated in Pten^-/- cells. (a) Pten^+/+ and Pten^-/- cell lysates were subjected to GST- or GST-Rhotekin affinity precipitation on glutathione agarose beads. The precipitates were analysed by anti-RhoA Western blotting. (b) The amount of co-precipitated RhoA-GTP, as detected by Western blot with anti-RhoA antibody, was normalized to the amount of RhoA in whole-cell lysates. The graph shows means.d. of four independent experiments. (c) In vitro kinase activity of endogenous PKN was performed as described in Materials and methods. Top panel, MBP phosphorylation by PKN (phosphoimager). Bottom panel, Western blot for endogenous PKN. (d) PKN kinase activity was calculated as described in Materials and methods, results for cells growing under normal growth conditions (left panels) are expressed as means.e.m. (standard error of the mean) of three independent experiments. Data for LY294002-treated cells are expressed as means.d. (standard deviation) of two independent experiments

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In order to further verify that the Rho pathway is activated in Pten^-/- cells, we examined the kinase activity of endogenous PKN, a Rho effector. Endogenous PKN from Pten^+/+ and Pten^-/- cells were immunoprecipitated and subjected to in vitro kinase assays using myelin basic protein (MBP) as substrate. Although the autophosphorylation level of endogenous PKN from Pten^+/+ and Pten^-/- cells was similar (data not shown), there was an approximately twofold increase in kinase activity of PKN from Pten^-/- cells compared to Pten^+/+ cells (Figure 6c and d). Treatment with LY294002 inhibited endogenous PKN activity from Pten^-/- cells (Figure 6c and d), indicating that PKN activation in these cells is dependent on heightened PtdIns(3,4,5)P₃ levels. The observation that endogenous Rho and PKN have increased activities in Pten^-/- cells supports the hypothesis that the Rho/PKN pathway is activated and plays a role in regulating cortical actin formation in these knockout cells.

Cortical actin does not play a role in promoting cell migration

Previous studies using pharmacological inhibitors of PI 3-kinase indicated that the PI 3-kinase pathway plays a role in regulating cell motility of cancer cell lines. For example, wortmannin has been shown to inhibit hepatocyte growth factor-stimulated migration of primary hepatocellular carcinoma (HCC) cells from different patients (Nakanishi et al., 1999). Treatment of the highly invasive PC-3 prostate cancer and MDA-MB-231 breast cancer cell lines with wortmannin and/or LY294002 attenuated basal cell migration (Zheng et al., 2000; Sliva et al., 2002). Consistent with these findings, Pten^-/- MEFs was shown to possess increased motility compared to Pten^+/+ cells (Liliental et al., 2000). Rac1, Cdc42 (Liliental et al., 2000) and their downstream effector, Akt (Higuchi et al., 2001), are implicated in regulating Pten^-/- MEF migration.

To investigate whether cortical actin is essential for cell migration, we examined the effect of Rac1^N17, RhoA^N19 PKN^K664D or PDK-1^K114G overexpression in Pten^-/- cells. Overexpression of PKN^K664D or RhoA^N19 did not significantly reverse the increased motility of Pten^-/- cells (Figure 7a, left panel, columns 3 and 5 vs column 2). Intriguingly, kinase-dead PDK-1 inhibits migration of Pten^-/- cells to the same extent as Rac^N17, indicating that PDK-1 may play a role in regulating cell migration downstream of PTEN. To further verify these results, we introduced kinase-dead PDK-1 (PDK-1^K114G) into Pten^-/- cells by adenoviral infection. Cells infected with adenovirus encoding both GFP and PDK-1^K114G inhibited Pten^-/- cell migration compared to those infected with adenovirus encoding GFP alone (Figure 7a, right panel). Taken together, the data suggest that disruption of cortical actin has no effect on migration of Pten^-/- cells, and that these two are separate events. This hypothesis is further supported by the observation that DN Rac (Rac^N17) and Akt (Akt^{K197A, T308A, S473A}), both previously reported to inhibit the migration of Pten^-/- cells (Liliental et al., 2000; Higuchi et al., 2001), did not inhibit cortical actin formation in Pten^-/- cells (Figure 4; data not shown).

Figure 7.

Cortical actin is not essential for migration of Pten^-/- cells. (a) Left panel, migration assay of Pten^+/+ or Pten^-/- cells transiently expressing GFP alone or GFP along with Rho^N19, Rac^N17, PKN^K664D or PDK-1^K114G. All the migration values were expressed relative to the wild-type migration value. Error bars are standard error of the mean (s.e.) from three independent experiments. Right panel, migration of cells infected with either the control (GFP) or PDK-1^K114G adenoviruses, results representative of two independent experiments. (b) In vitro kinase activity of endogenous PDK-1 towards PKN-C^KD was detected using the phosphoimager (top panel). Bottom panel, immunoprecipitated endogenous PDK-1 loading was detected by Western blotting. (c) Graphical representation of PDK-1 kinase activity, result is expressed as means.e.m. (standard error of the mean) of three independent experiments. (d) Top panel, confocal microscope images showing localization of Myc-PDK-1 in Pten^+/+ or Pten^-/- cells. Bottom panel, graph showing membrane localization frequency of Myc-PDK-1 in Pten^+/+ and Pten^-/- cells, results are meanstandard deviation of two independent experiments. (e) Top panel, graph showing Akt activation (pT308A phosphorylation) in Pten^-/- cells infected with either GFP (control) or PDK-1^K114G adenovirus, bars represent means.e.m. of three independent experiments. Bottom panel, graph showing PKN activation in Pten^-/- cells infected with either GFP (control) or PDK-1^K114G adenovirus, bars represent means.e.m. of four independent experiments

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PDK-1 activation in Pten^-/- cells

The observation that kinase-dead PDK-1 inhibits migration of Pten^-/- cells suggests that this kinase may be activated in these cells. Regulation of PDK-1 function could occur via two distinct mechanisms that may not be mutually exclusive: increase in intrinsic PDK-1 kinase activity or increase in translocation of PDK-1 to the plasma membrane. We first investigated whether PDK-1 is activated in Pten^-/- cells by immunoprecipitating endogenous PDK-1 from Pten^+/+ and Pten^-/- cell lysates and performing in vitro kinase assays using kinase-dead PKN C-terminus (PKN-C^KD) as substrate. No obvious difference was observed in kinase activities of PDK-1 from Pten^+/+ and Pten^-/- cells (Figure 7b and c). However, PDK-1 is a predominantly cytoplasmic protein which translocates to the plasma membrane upon binding the lipid products of PI 3-kinase (Vanhaesebroeck and Alessi, 2000). We therefore examined whether the basal localization of Myc-tagged PDK-1 was increased in Pten^-/- compared to Pten^+/+ cells. As shown in Figure 7d, wild-type PDK-1 is only found at the membrane of 6% of the Pten^+/+ cell population under basal conditions, whereas approximately 34% of the Pten^-/- cells population showed PDK-1 membrane localization, which represents an approx. 6–7-fold in plasma membrane localization. Studies performed with PDK-1^K114G yielded similar results. The increased membrane localization of PDK-1 may explain why Akt phosphorylation is increased in PTEN-deficient MEFs ((Stambolic et al., 1998) and our unpublished observations). The increased PDK-1/Akt signaling in Pten^-/- cells may contribute to the increased motility of these cells.

Discussion

In this study, we demonstrated that the RhoA pathway is involved in regulating cortical actin accumulation in Pten^-/- MEFs by blocking cortical actin accumulation in Pten^-/- MEFs via overexpression of DN RhoA and PKN. Interestingly, neither DN RhoA nor KD PKN overexpression restored actin stress fibers of Pten^-/- MEFs (Figures 3 and 4) in contrast to overexpression of DN PTEN (Figure 1). A possible explanation for the failure of KD PKN to restore the stress fibers of Pten^-/- cells is that PKN is regulating a subset of Rho-induced responses, which is unrelated to stress fiber formation. As the major pathway responsible for stress fiber formation in cells is the Rho/Rho-kinase (ROCK) pathway (Riento and Ridley, 2003), the observation that DN Rho and KD LIMK1 (downstream ROCK effector) could not restore actin stress fibers of Pten^-/- cells indicates that the loss of stress fibers is regulated by a more complex mechanism.

Our results show that kinase-dead PDK-1 inhibits migration of Pten^-/- cells to the same extent as Rac^N17 (Figure 7a). These findings are in agreement with the observation that DN Akt can inhibit cell migration of Pten^-/- MEFs (Higuchi et al., 2001), and suggest that PDK-1 may act upstream of Akt in promoting cell motility of these fibroblasts. However, overexpression of PDK-1^K114G has no effect on cortical actin, whereas overexpression of kinase-dead PKN inhibited this actin phenotype. As PDK-1 has been reported to activate both Akt and PKN (Alessi et al., 1997; Dong et al., 2000), these observations suggest that the PI 3-kinase/PTEN pathway bifurcates into two distinct pathways in the Pten^-/- cells, the PDK-1/Akt pathway regulates cell motility, whereas the Rho/PKN pathway regulates cortical actin accumulation. In order to investigate this hypothesis, we investigated the effect of adenoviral-mediated PDK-1^K114G overexpression on endogenous Akt and PKN activities. PDK-1^K114G overexpression in Pten^-/- cells inhibited Akt activity, but had no effect on PKN activity (Figure 7e, top vs bottom panels). This observation supports the proposed model that the PI 3-kinase/PTEN pathway bifurcates upstream of PDK-1 into two separate pathways.

The mechanism by which PDK-1^K114G inhibits cell motility induced by PTEN deficiency is unclear. As PDK-1^K114G localization is also increased at plasma membrane of Pten^-/- cells (data not shown), it is possible that PDK-1^K114G acts by sequestering endogenous Akt at the plasma membrane. Thus, it is possible that increased Akt phosphorylation observed in this cell line is due to increased PDK-1 membrane/cortical localization and that PDK-1 may promote increased motility via phosphorylation of Akt at the plasma membrane. We attempted to address this hypothesis by overexpressing PDK-1^PH, a PDK-1 mutant incapable of membrane translocation, in Pten^-/- cells. Surprisingly, overexpression of PDK-1^PH inhibited the migration of Pten^-/- MEFs to the same extent as PDK-1^K114G (data not shown). However, this observation is consistent with a previous publication showing that overexpression of a translocation-deficient PDK-1 mutant prevented Akt from moving to the cell periphery upon insulin stimulation (Filippa et al., 2000). Thus, PDK-1^PH may function as a DN by inhibiting Akt membrane translocation.

Interestingly, we found that overexpression of constitutively active PDK-1 (A280V) alone is not sufficient to promote cell migration in Pten^+/+ cells (data not shown). This finding suggests that PDK-1 may be acting in concert with other signaling proteins downstream of PI 3-kinase to regulate cell migration. The mechanism by which PDK-1 affects cell migration definitely warrants further investigation.

In conclusion, our results suggest that cortical actin polymerization and cell migration in PTEN-deficient cells are regulated by different mechanisms. The former is regulated by the Rho pathway, whereas the latter appears to be regulated by PDK-1 and Akt. Taken together, our results suggest that these two signaling pathways may bifurcate downstream of PI 3-kinase to regulate cortical actin formation and cell migration.

The involvement of PI 3-kinase, PTEN and Akt in many aspects of oncogenesis has been extensively studied (Vara et al., 2004), whereas the role of PDK-1 in promoting oncogenesis has been mostly neglected. Recently, Zeng et al. (2002) showed that forced expression of PDK-1 induced anchorage-independent growth in vitro, and isografts of PDK-1 transforming cell lines into syngeneic mice induced the formation of poorly differentiated mammary carcinomas. Here, we demonstrate that PDK-1 may be involved in promoting cell migration downstream of PTEN inactivation. These results suggest that PDK-1 can promote both tumor formation and progression, and imply that PDK-1 may be an attractive target for the development of anti-cancer drugs.

Materials and methods

Cell lines, cDNAs and antibodies

Isogenic Pten^+/+ and Pten^-/- MEF cell lines and retroviral vectors encoding PTEN WT, C124S and G129E were described previously (Liliental et al., 2000). cDNAs encoding FLAG-PKN truncations/mutants (Takahashi et al., 1998; Dong et al., 2000) and PDK-1 mutants were described previously (Dong et al., 1999; Wick et al., 2000). The cDNA encoding FLAG-LIMK1^D460E was a kind donation from Dr Ora Bernard (Bernard et al., 1994). Monoclonal anti-FLAG, anti-Myc and anti-HA antibodies were purchased from Sigma, Santa Cruz Biotechnologies and BABCO, respectively. The anti-PKN monoclonal and anti-PTEN polyclonal antibodies were purchased from Transduction Laboratories and Cell Signaling Technology, respectively. The anti-goat PDK-1 antibody was generated by immunizing goats (Ferrell Farms, Inc., OK) with purified recombinant mouse PDK-1 carboxyl terminus (amino-acid residues 285–559) fused to glutathione-S-transferase (GST).

Generation of FLAG-tagged PKN N-terminus (PKN-N)

cDNA encoding the N-terminus of human PKN was generated by PCR by using the PCR primers: 5'-cccggatatccagcgacgccgtgcaga-3' and 5'-cgcacccacgtgtcgacatcgatgttc-3' (added restriction sites underlined). Following EcoRV and SalI restriction digest, the insert was ligated into pFLAG-CMV™-2 (Sigma), in-frame with a sequence encoding the FLAG tag at the N-terminus.

In vitro kinase assays

Subconfluent cells growing in 100 mm plates were pre-incubated with either vehicle (DMSO) or 50 M LY294002 for 1 h in growth medium at 37°C in experiments using PI 3-kinase inhibitors. Pten^+/+ and/or Pten^-/- cells were lysed in either Buffer A (Wick et al., 2000) for PDK-1, or modified Buffer A (Dong et al., 2000) for PKN immunoprecipitations. Lysates were incubated with either goat anti-PDK-1 or mouse anti-PKN antibody pre-absorbed onto protein-G-sepharose at 4°C for 6 h with mixing. Bound proteins were washed three times with ice-cold Buffer B (50 mM HEPES, pH 7.6, 150 mM NaCl, 0.1% Triton X-100 for PDK-1 or NP-40 for PKN) and twice with Buffer C (50 mM Tris, pH 7.5, 5 mM MgCl₂, 1 mM NaF, 0.1 mM Na₄O₇P₂, 0.1 mM Na₃VO₄, 1 g/ml leupeptin, 1 g/ml aprotinin and 1 mM PMSF). PKN in vitro kinase assay was carried out in a 25 l of Buffer C containing 1 Ci of [-³²P]ATP and 0.4 g/l MBP (Sigma) at 30°C for 30 min. To determine PKN activity, one-third of the reaction volume containing MBP was removed from the beads and loaded onto 15% SDS gel. The gels were stained with Coomassie blue and phosphorylation levels were quantified by phosphoimager analysis (ImageQuant 5.0). The protein level of PKN was quantified by eluting bound proteins, SDS–PAGE, and Western blotting with an anti-PKN antibody. The kinase activity was calculated by dividing the MBP phosphoimager values (total counts) by PKN protein levels (NIH Image 1.58) and the relative PKN activity was obtained by comparing the values obtained for Pten^-/- cells against that from Pten^+/+ cells (100%). PDK-1 in vitro kinase assay was carried out for 15 min at 30°C in Buffer C containing 1 Ci of [-³²P]ATP in the presence of kinase-dead PKN C-terminus (PKN-C^KD, amino acids 561–942) immobilized on anti-FLAG/protein-G-sepharose beads. PDK-1 kinase activity was determined by separating the radiolabeled proteins by SDS–PAGE and Western blotting. The nitrocellulose membrane was exposed on the phosphoimager prior to Western blotting with either rabbit anti-PDK-1 or mouse anti-FLAG antibodies. PDK-1 kinase activity was obtained by dividing PKN-C^KD phosphorylation values (total counts) by PDK-1 loading levels. Endogenous PDK-1 kinase activity from Pten^+/+ cells was arbitrarily assigned as 100% and the PDK-1 kinase activity from Pten^-/- cells was expressed relative to this value.

Effect of PDK-1^K114G on PKN kinase activity

Pten^-/- cells were infected with either control (GFP) or GFP and PDK-1^K114G cesium chloride-purified adenovirus and harvested 24 h post-infection. Infection efficiency was typically approximately 90% for the Pten^-/- cells and 50% for the Pten^+/+ cells (as determined by GFP fluorescence) with the same amount of virus, indicating that Pten^+/+ cells are less susceptible to adenoviral infection than Pten^-/- cells. The PKN in vitro kinase activity was performed as described above and the relative PKN kinase activity was obtained by comparing the MBP phosphorylation/PKN loading ratio of GFP-infected cells against PDK-1^K114G-infected cells.

Effect of PDK-1^K114G on Akt activation

Pten^+/+ or Pten^-/- cells growing in six-well plates were infected with either control (GFP) adenovirus or kinase-dead PDK-1 adenovirus. At approximately 24 h post-infection, the cells were directly lysed in SDS buffer and the protein separated on SDS gel. Western blot was performed to determine phosphorylation of Akt at Thr308, the PDK-1 phosphorylation site required for Akt activation (Alessi et al., 1997). Akt phosphorylation and loading levels were quantified using Scion Image, and Akt phosphorylation was normalized against protein-loading levels. Akt phosphorylation of GFP-infected cells was arbitrarily assigned as 100% and Akt phosphorylation from PDK-1^K114G-infected cells was expressed relative to this value.

Endogenous RhoA activity assay

The GST-Rhotekin probe containing the RhoA-binding domain was expressed and purified from Escherichia coli as described previously (Li et al., 1999). Cells were washed once with ice-cold phosphate-buffered saline (PBS) buffer prior to lysis in Buffer D (50 mM Tris–HCl (pH 7.4), 100 mM NaCl, 10 mM MgCl₂, 1% Triton X-100, 0.1% SDS, 10 g/ml each of leupeptin and aprotinin, and 1 mM PMSF). Following centrifugation at 13 000 g at 4°C for 10 min, equal amounts of cell lysates were incubated with the GST-Rhotekin probe (10 g/sample) immediately for an hour at 4°C under constant agitation. The precipitated beads were washed three times with the lysis buffer, and the bound RhoA was detected by anti-RhoA (Santa Cruz Biotechnology) Western blotting. Quantification of the Western blots was carried out using an AlphaImager system (Alpha Innotech). To compare the level of the active RhoA-GTP in GST-Rhotekin pulldowns, the amount of co-precipitated RhoA was normalized to the total amount of RhoA in different samples.

Cell staining and actin studies

At 18–24 h post-transfection, Pten^+/+ and/or Pten^-/- cells growing on glass coverslips were fixed with 4% paraformaldehyde/PBS for 20 min, followed by permeabilization with 0.2% Triton X-100/PBS for 3 min at room temperature. Cells were stained with monoclonal anti-FLAG, anti-Myc or anti-HA antibody, followed by incubation with either anti-mouse Alexa Fluor 488 or 350 (Molecular Probes), and counterstained for polymerized/filamentous actin (F-actin) using rhodamine phalloidin (Molecular Probes). Coverslips were mounted on glass slides using the ProLong Antifade Mounting reagent (Molecular Probes).

Cell migration assays

Pten^+/+ or Pten^-/- cells growing in 100 mm plates were transfected with cDNAs encoding Rho^N19, Rac^N17, PKN^K664D, PDK-1^K114G or PDK-1^PH with pEGFP-C1 to a 10 to 1 ratio, respectively, using Lipofectamine (GibcoBRL). After 24 h, the cells were detached by EDTA treatment and approximately 10 000–20 000 GFP-positive cells were added to the top chamber of a 24-well Transwell unit (8 m pore size, Corning). Fibronectin (10 g/ml) diluted in DMEM/0.1% BSA was added to the bottom chamber. A fraction of the detached cells was lysed for Western blotting with an anti-GFP antibody. The rest of the cells were plated on poly-D-lysine-coated glass coverslips and stained for expression of the DN proteins in order to determine co-transfection efficiency. After 4 h incubation at 37°C, the cells in the top chamber of the Transwell unit were removed by swabbing with a cotton tip and the cells adhering to the bottom were fixed with 4% paraformaldehyde. The number of GFP-positive cells from two to three independent fields were counted per well and each experiment was performed in triplicates. The average number of GFP cells was multiplied by the co-transfection efficiency and divided by GFP loading levels (NIH Image 1.58) in order to minimize errors resulting from GFP loading and different co-transfection efficiencies. All the migration values were expressed relative to the Pten^+/+ migration value, which is arbitrarily set as 1. A similar protocol was employed for the adenoviral-infected MEFs, with the exception that the mean number of migrated GFP-positive cells was not calibrated by the co-transfection efficiency.

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Acknowledgements

We thank Derong Hu and Fresnida Ramos for excellent technical assistance. Many thanks are due to Dr Vicki Frohlich (Assistant Director, Digital Optical Imaging Facility) for assistance and advice on confocal microscopy techniques. We are grateful to Drs H Mukai and Y Ono for providing the PKN and PRK2 constructs and to Dr Ora Bernard for providing cDNA encoding LIMK1. This work was supported by a grant from the National Institute of Health Grant DK56166 awarded to Feng Liu and Lily Q Dong.