The Tumor Promoter 12-O-Tetradecanoylphorbol 13-acetate (TPA) Provokes a Prolonged Morphologic Response and ERK Activation in Tsc2-Null Renal Tumor Cells

Todd M. Kolb* and Myrtle A. Davis{dagger},1

* Program in Toxicology and Department of Pathology, University of Maryland, School of Medicine, Baltimore, Maryland 21201, and {dagger} Toxicology and Drug Disposition, Lilly Research Laboratories, A Division of Eli Lilly and Company, P.O. Box 708, Greenfield, Indiana 46140

Received February 2, 2004; accepted May 17, 2004


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Loss of tumor suppressor function dramatically alters the cellular response to chemicals. The phorbol ester tumor promoter, 12-O-tetradecanoylphorbol 13-acetate (TPA), stimulates cell proliferation through rapid activation of protein kinase C (PKC), followed by gradual degradation of the kinase. TPA also activates the GTPase Rap1 in some cell types. The tumor suppressor protein Tsc2 has a proposed GTPase activating protein (GAP) function for Rap1, providing a common mechanistic target for Tsc2 and TPA. We compared the cellular response of Tsc2-null (ERC-18) and Tsc2-competent (NRK-52E) renal epithelial cells to TPA treatment. Treatment of ERC-18 cells with 100 ng/ml TPA for 24 h resulted in loss of cell-cell contact, retraction of the cell periphery and rounding. These changes were reversed 1 h after treatment in NRK-52E cells and were apparent 24 h after treatment of ERC-18 cells. Expression of Tsc2 in ERC-18 cells abrogated the prolonged morphologic response. TPA treatment rapidly increased phosphorylation of ERK, a reported downstream effector of both PKC and Rap1, in ERC-18 cells, but induced weak Rap1 activation. TPA-induced ERK phosphorylation was prolonged in ERC-18 cells compared to NRK-52E cells and expression of Tsc2 in ERC-18 cells did not inhibit prolonged ERK activation. The selective PKC inhibitor, bisindolylmaleimide VIII, however, inhibited TPA-induced changes in morphology and ERK activation. These results imply that TPA-induced changes in morphology and ERK activation are mediated primarily through PKC and not Rap1 in renal epithelial cells. These data also imply that Tsc2 expression modulates TPA-induced changes in renal epithelial cell morphology via an ERK-independent mechanism.

Key Words: Tsc2; protein kinase C; TPA.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Multiple potential functions of the Tsc2 gene product (tuberin) have been identified through intensive study over the past decade. Tsc2 has recently been identified as a regulator of the insulin signaling pathway that promotes translation of ribosomal proteins and proteins involved in cell cycle progression (Gao and Pan, 2001Go; Goncharova et al., 2002Go; Inoki et al., 2002Go; Kenerson et al., 2002Go; Tee et al., 2002Go). In addition, Tsc2 has been shown to regulate the activity of several GTPases, including Rap1, Rab5, Rho, and Rheb (Astrinidis et al., 2002Go; Wienecke et al., 1995Go; Xiao et al., 1997Go; Zhang et al., 2003Go). These small GTP-binding proteins are upstream regulators of many signaling cascades, and control numerous cellular functions, including proliferation, differentiation, adhesion, migration, and cell shape (for detailed review, see Hall, 2000Go). Tsc2 has been shown to regulate Rap1 activity in vitro, purportedly via the proposed GTPase activating protein (GAP) activity of the C-terminal region (Wienecke et al., 1995Go).

The phorbol ester 12-O-tetradecanoylphorbol-13 acetate (TPA) is the active component of croton oil and a potent tumor promoter. TPA mimics the second messenger diacylglycerol to activate protein kinase C (PKC) and alter cell signaling pathways (reviewed in Gottlicher, 1999Go). TPA has been used extensively to investigate the role of PKC in biologic responses, but recently has been shown to activate Rap1 in neutrophils and fibroblasts (M'Rabet et al., 1998Go; Zwartkruis et al., 1998Go). The activation of Rap1 by TPA has been shown to be PKC-independent in neutrophils, and is presumably dependent on the ability of the phorbol ester to directly activate diacylglycerol-specific Rap1 guanine nucleotide exchange factors (M'Rabet et al., 1998Go). Given the hypothesized role of Tsc2 in Rap1 regulation, we compared the cellular response to TPA between Tsc2-null and Tsc2-expressing cells. We show that TPA rapidly activated PKC{alpha} and the extracellular-signal regulated kinase (ERK) in both cell types, but caused weak Rap1 activation in ERC-18 cells only. TPA induced unique focal accumulations of PKC{alpha} in ERC-18 cells, and ERK activation was prolonged in ERC-18 cells when compared to NRK-52E. Furthermore, Tsc2-null cells had a prolonged morphologic response to the tumor promoter that was PKC-dependent and Tsc2-modulated.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals and reagents. The phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) was purchased from Alexis Biochemicals (Carlsbad, CA) and prepared as a 10 mg/ml stock solution in dimethyl sulfoxide. This stock was further diluted in dimethyl sulfoxide prior to preparation of working dilutions in aqueous media. SYTOX-green and 4',6-diamidino-2-phenylindole, dihydrochloride (DAPI) were purchased from Molecular Probes (Eugene, OR). Bromodeoxyuridine (BrdU) was purchased from Oncogene Research Products (San Diego, CA).

Cell culture and treatment. Tsc2-null ERC-18 cells were derived from Eker rat renal tumors (Freed et al., 1990Go) and were kindly provided by Dr. Cheryl Walker (MD Anderson Cancer Center; Smithville, TX). Eker renal tumors arise from tubular epithelial cells following loss of functional tuberin expression (Everitt et al., 1992Go). The NRK-52E cell line was used as a control Tsc2-expressing renal tubular epithelial cell line, and was purchased from American Type Culture Collection (Manassas, VA). ERC18-FLAG-Tsc2 and ERC18-FLAG-2B lines were generated by stably expressing a FLAG epitope-tagged Tsc2 construct (ERC18-FLAG-Tsc2) or the empty expression vector (ERC18-FLAG-2B) in the ERC-18 cell line. A detailed discussion of the development and phenotype of these cell lines is presented elsewhere (manuscript in review). Briefly, a 5357 base pair HindIII fragment of the pcDNA3-Tsc2 vector (provided by Dr. Cheryl Walker) containing the rat Tsc2 sequence was subcloned into the pCMV-Tag-4 vector (Stratagene; La Jolla, CA), to create the pCMV-Tag-Tsc2 plasmid. The plasmid encodes a 1738 amino acid tuberin construct with the C-terminal 42 amino acids removed and replaced with a FLAG-epitope tag. The pCMV-Tag-Tsc2 plasmid sequence was verified by multiple restriction endonuclease digests. A similar Tsc2 construct, lacking 55 amino acids from the C-terminus, completely inhibited N-ethyl-N-nitrosurea-induced renal carcinoma formation in transgenic Eker rats (Momose et al., 2002Go). ERC-18 cells were transfected with the linearized pCMV- Tag-Tsc2 plasmid or with the linearized empty pCMV-Tag vector (control) using Effectene transfection reagent (QIAGEN; Valencia, CA) according to the manufacturer's protocol. Individual G418-resistant colonies were screened by immunoblot analysis for expression of FLAG with an anti-FLAG-M2 antibody (Sigma; St. Louis, MO) diluted to a concentration of 1 µg/ml.

ERC-18, ERC18-FLAG-2B (empty plasmid vector) and ERC18-FLAG-Tsc2 (pCMV-Tag-Tsc2) cells were routinely maintained in a 1:1 mixture of Dulbecco's modified Eagle media (DMEM) and Nutrient mixture F12 (Ham) supplemented with 5% fetal bovine serum, 2 mM L-glutamine, 1.6 µM ferrous sulfate, 50 nM sodium selenite, 12 µM vasopressin, 10 nM cholesterol, 200 nM hydrocortisone, 1 nM tri-iodothyronine (T3), 10 pg/ml transferrin, and 25 µg/ml insulin. NRK-52E cells were maintained in DMEM supplemented with 5% fetal bovine serum and 2 mM L-glutamine. All cell lines were grown in a humidified atmosphere of 37°C and 5% CO2/95% room air. Cells were grown to 90% confluence in 60 or 100-mm plastic culture dishes (Western blot, Rap1 activity assay) or 35-mm dishes with glass coverslips (immunocytochemistry), then incubated in low-serum treatment media (DMEM/F12, 1% FBS) for 24 h prior to treatment. Cells were treated with 50 or 100 ng/ml TPA in treatment media for 5 min, 15 min, 1 h, or 24 h, or with an equivalent volume of vehicle (dimethyl sulfoxide) as a control. In PKC inhibitor experiments, cells were co-treated with 1 µM bisindolylmaleimide VIII (Bis VIII), a selective PKC inhibitor with increased specificity for the PKC{alpha} isoform (Wilkinson et al., 1993Go), or with 1 µM bisindolylmaleimide V (Bis V), an inactive structural analogue. At the indicated time points, cells were photographed on a Leitz Diavert inverted microscope.

Immunoblot analysis. Following treatment, cells were lysed on ice with RIPA buffer (50 mM Tris-Cl, pH = 7.4; 150 mM NaCl; 1% NP-40; 0.5% sodium deoxycholate; 0.1% sodium dodecyl sulfate; 1 mM ß-glycerolphosphate; 2.5 mM sodium pyrophosphate) supplemented with protease inhibitor cocktail P8340 and phosphatase inhibitor cocktails P2850 and P5726 (Sigma), sonicated for 10 s, and clarified by centrifugation (17,000 x g, 10 min.). Protein concentrations were measured using the BCA protein assay (Pierce; Rockford, IL), and 20 µg of protein was separated by SDS-PAGE using standard techniques (Laemmli, 1970Go) and transferred to a PVDF membrane (Bio-Rad Laboratories; Hercules, CA) in 25 mM Tris, 192 mM glycine, and 20% methanol overnight at 100 mA. Proteins were detected by immunoblot analysis with antibodies specific for phospho-ERK (1:1000), PKC{alpha} (1:1000), and caspase 3 (1:1000) according to the manufacturer's protocols. All antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Band intensities were quantified from scanned images using Molecular Analyst software (Bio-Rad Laboratories; Hercules, CA), and mean values from multiple independent experiments were compared by randomized block ANOVA with Dunnett's multiple comparisons post-test, unpaired t-test, or two-way ANOVA, as described in the text. Statistically significant differences were judged at a p value <0.05. Immunoblots were stripped and reprobed for actin (1:1000; Santa Cruz) to confirm equal protein loading and transfer. Expression of the 200-kDa FLAG-Tsc2 construct was confirmed in 20 µg of protein from nuclear extracts prepared with the Pierce NE-PER kit according to the manufacturer's protocol.

Rap1 activity assay. GTP-bound (active) Rap1 was measured using a previously described method (Franke et al., 1997Go). Briefly, cells were treated with 100 ng/ml TPA (or vehicle) for 5, 15, or 60 min, then lysed with RIPA buffer on ice and clarified by centrifugation (17,000 x g, 10 min.). Protein concentrations were measured using the BCA assay, and 600 µg of lysate (diluted to 0.6 mg/ml in RIPA buffer) was incubated for 1 h (4°C) with 5 µg of a RalGDS GST-RBD construct (provided by Dr. Johannes Bos; Utrecht University, The Netherlands) pre-coupled to glutathione-agarose beads. The RalGDS GST-RBD construct has a high and specific affinity for GTP-bound Rap1 (Herrmann et al., 1996Go). Beads were washed several times with RIPA buffer, and bound Rap1-GTP was purified by boiling in Laemmli sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 25% glycerol, 2% ß-mercaptoethanol, 0.01% bromophenol blue). Pulldown products were separated by SDS-PAGE and transferred to PVDF membranes as described above, and Rap1 levels were detected by immunoblotting with a Rap1 specific antibody (Santa Cruz; 1:500). Immunoblots were stripped and reprobed with a glutathione-S-transferase (GST) specific antibody (Santa Cruz; 1:2000) to confirm that equivalents amount of the ~ 38 kDa RalGDS GST-RBD construct were used to precipitate Rap1-GTP from each sample. Individual Rap1 band intensities were quantified and compared from four independent experiments using randomized block ANOVA, with statistically significant differences judged at a p value <0.05.

Immunocytochemistry. Following TPA treatment, cells were fixed with 2% paraformaldehyde at 4°C for 1 h, then permeabilized with 0.1% Triton X-100 for 30 min. Nonspecific antibody binding sites were blocked for 45 min with 5% normal goat serum in phosphate-buffered saline (PBS) supplemented with 0.5% BSA and 0.15% glycine. Cells were incubated with a PKC{alpha}-specific antibody (Santa Cruz; 2 µg/ml) for 1 h at 37°C, followed by Alexa-488 conjugated anti-rabbit IgG antibody (Molecular Probes; 1 µg/ml) for 1 h at room temperature. As a negative control, cells were subjected to the staining protocol with primary antibody replaced with dilution buffer (PBS, 0.5% BSA, 0.15% glycine). Coverslips were mounted on clean glass slides with MOWIOL mounting medium supplemented with 1 µg/ml DAPI, and photographed on a Nikon TE200 Eclipse inverted microscope.

Cell proliferation assay. NRK-52E and ERC-18 cells were plated in black, clear-bottom 96-well tissue culture dishes at a density of 104 cells/well. Cells were grown in complete media for 24 h, then in low-serum DMEM/F12 treatment media for 24 h prior to treatment. Cells were treated in duplicate wells with TPA (10, 50, 100 ng/ml or the equivalent volume of dimethyl sulfoxide) in the presence of 100 µM bromodeoxyuridine (BrdU) for 24 h. BrdU incorporation was measured using the BrdU Proliferation Assay Kit (Oncogene Research Products) according to the manufacturer's instructions. Mean BrdU incorporation was determined from duplicate wells in three independent experiments, and the percentage change in cell proliferation from control in TPA-treated cells was compared within each cell type using randomized block ANOVA. Statistically significant differences were judged at a p value <0.05.

Measurement of cell death (membrane integrity loss). Cells were plated in six-well tissue culture dishes, then grown to 90% confluence and incubated in DMEM/F12 low-serum treatment media for 24 h. Cells were then treated with 10, 50, 100, 200, or 500 ng/ml TPA (or the equivalent volume of vehicle) in the presence of the membrane-impermeant nuclear dye SYTOX green (0.5 µM, Molecular Probes). Fluorescence was measured after 0, 24, and 48 h of treatment using a Cytofluor 2350 (Millipore; Bedford, MA). After the 48 h reading, all cells were permeabilized with 100 µg/ml saponin, and total fluorescence was determined. At each treatment and time point, fluorescence values were normalized to the total cellular fluorescence (saponin).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
TPA Induces a Prolonged Morphologic Change in ERC-18 Cells
We treated Tsc2-null ERC-18 cells and Tsc2-expressing NRK-52E cells with 50 and 100 ng/ml TPA for 24 h and observed the morphological response of each cell line to the phorbol ester. Within 1 h of treatment with TPA, both cell types exhibited a change in cellular morphology. Both cell types showed increased refractility and a retraction of the cell periphery (see "Bis V + TPA" in Fig. 6A). Loss of cell-cell contact and discrete areas of cell attachment to culture dishes were unique to TPA-treated ERC-18 cells. After 24 h of treatment, these morphologic changes persisted only in the ERC-18 line, with NRK-52E cells returning to the morphology of control cells within 2 h. Figure 1A shows a representative image of cellular morphology after 24 h of treatment with TPA or vehicle from 10 independent experiments. As shown in the figure, ERC-18 cells retained a rounded/retracted phenotype with discrete focal contacts at the cell periphery (arrows) after 24 h of TPA treatment. Although many cells had rounded completely, no cell detachment was observed, even after gentle shaking.



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FIG. 6. PKC inhibitor bisindolylmaleimide VIII abrogates TPA-induced changes in cell morphology and decreases ERK activation. Cells were treated with 100 ng/ml TPA or DMSO (cont) in the presence of the highly specific PKC inhibitor bisindolylmaleimide VIII (BisVIII, 1 µM) or an inactive structural analogue (BisV, 1 µM). In (A), cells were photographed after 1 h of treatment. In (B), the effect of BisVIII on TPA-induced ERK phosphorylation was measured. Protein lysates were prepared after 15 min and 1 h of treatment, and ERK phosphorylation was measured using immunoblot analysis from equal amounts of lysate as described above. Shown is the relative increase in ERK phosphorylation compared to the corresponding control (set at 1.0) (**p < 0.01; ***p < 0.001). Error bars indicate SEM from three independent experiments.

 


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FIG. 1. TPA induced prolonged morphologic alteration in Tsc2-null ERC-18 cells. The effects of Tsc2 expression on the morphologic response of renal epithelial cells to TPA is shown in (A). Confluent cultures of Tsc2-null (ERC-18, ERC18-FLAG-2B) and Tsc2-expressing (NRK-52E, ERC18-FLAG-Tsc2) renal epithelial cells were incubated in treatment media (DMEM/F12, 1% FBS) for 24 h prior to treatment with 100 ng/ml TPA (TPA) or an equivalent volume of DMSO (control). Cells were photographed after 24 h of treatment. Cell lines are indicated above photos; 2B = ERC18-FLAG-2B, Tsc2 = ERC18-FLAG-Tsc2. In (B), expression of the 200 kDa FLAG-Tsc2 construct is shown in the ERC18-FLAG-Tsc2 cell line, as indicated by the arrow. Cell lines are indicated above each lane, and the apparent sizes of molecular weight standards (not shown) are indicated to the left.

 
To determine if the prolonged morphologic response to TPA was dependent on Tsc2 expression, Tsc2-expressing ERC-18 cells (ERC18-FLAG-Tsc2) were also treated with 100 ng/ml TPA for 24 h, and their morphology was compared to ERC-18 cells expressing the empty plasmid vector (ERC18-FLAG-2B). Expression of the 200-kDa Tsc2-FLAG construct in the ERC18-FLAG-Tsc2 cell line (but not in the control ERC18-FLAG-2B line) is shown in Figure 1B. In concordance with the results described above, the Tsc2-null ERC18-FLAG-2B line showed retraction of the cell periphery and discrete focal contacts, detachment from neighboring cells, and extensive rounding after 24 h of TPA treatment. This response is consistent with that observed in the Tsc2-null ERC-18 cell line. Conversely, Tsc2-expressing ERC18-FLAG-Tsc2 cells had returned to their control appearance after 24 h of TPA treatment (Fig. 1A).

TPA-Induced Morphologic Change Does Not Represent Increased Proliferation or Apoptosis
The rounded morphology observed following TPA treatment is similar to cellular rounding observed as an early phenotypic alteration during proliferation or apoptosis. Indeed, TPA has been shown to promote and inhibit both proliferation and apoptosis in a variety of cell types. Therefore, we examined the effects of TPA on cell proliferation using BrdU labeling as a measure of proliferation. We treated NRK-52E and ERC-18 cells with 10, 50, or 100 ng/ml TPA for 24 h in the presence of BrdU. Remarkably, TPA caused a significant decrease in cell proliferation in NRK-52E cells at all doses (p = 0.003; randomized block ANOVA), with no apparent dose-response (Fig. 2). The tumor promoter had no effect on cell proliferation in the ERC-18 cell line (p = 0.97; randomized block ANOVA). Cleavage of caspase-3 was also examined as a measure of apoptosis, and caspase cleavage product was not observed in either cell type, even after treatment with 100 ng/ml TPA for 48 h. We also measured cell permeability as an indicator of cell death in both cell lines after treatment with 10–500 ng/ml TPA for 24–48 h. No increase in cell death was apparent in either cell type, even after treatment with 500 ng/ml TPA was carried out to 48 h. These data indicate that the TPA did not induce proliferation or apoptosis in either cell type.



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FIG. 2. TPA did not induce cell proliferation in NRK-52E or ERC-18 cells. Incorporation of BrdU into NRK-52E and ERC-18 cells was measured during 24 h of treatment with 10, 50, or 100 ng/ml TPA (or an equivalent volume of DMSO; control). Cells were grown in 96-well plates, and treated with TPA in the presence of 100 µM BrdU for 24 h prior to lysis. Incorporated cellular BrdU was detected in the wells by measuring the fluorogenic product created by peroxidase-conjugated IgG bound to anti-BrdU antibody, and measured with a Wallac 1420 Victor 2 plate reader (PerkinElmer Life Sciences; Boston, MA). Shown are the mean levels of BrdU incorporation (relative to the control values) from three independent experiments (*p < 0.05, **p < 0.01). Error bars indicate SEM.

 
TPA Induces Prolonged Activation of ERK in ERC-18 Cells
The extracellular signal-regulated kinase (ERK) is a downstream effector of both PKC and Rap1, and therefore represents a likely effector of the cellular response to TPA regardless of its direct target. Therefore, we wanted to determine the time-course of ERK phosphorylation and activation in response to the tumor promoter, and to compare any potential differences in this response between Tsc2-null and Tsc2-expressing renal epithelial cells. Representative blots are shown in Figure 3, with mean levels of ERK phosphorylation measured from three independent experiments shown immediately below. Actin immunoblots are included to confirm equivalent protein loading. ERK phosphorylation was markedly increased within 5 min of treatment with 100 ng/ml TPA in both NRK-52E and ERC-18 cells (Fig. 3). ERK phosphorylation rapidly decreased to control levels (15 min–1 h) in NRK-52E cells, but phosphorylation of the kinase remained elevated even after 1 h of TPA treatment in ERC-18 cells. Comparison of phosphorylated ERK band intensities showed that TPA treatment significantly increased ERK phosphorylation in both cell types in the first hour of treatment (NRK-52E, p = 0.01; ERC-18, p < 0.0001; randomized block ANOVA). Statistically significant increases over control ERK phosphorylation were observed after 5, 15, and 60 min of TPA treatment in ERC-18 cells (p < 0.01; Dunnett's multiple comparisons post-test), while only the 5 and 15 min ERK phosphorylation levels were significantly increased versus control in the NRK-52E cell line (p < 0.05; Dunnett's multiple comparisons post-test). ERK phosphorylation was not increased versus control in either cell line after 24 h of TPA treatment (NRK-52E, p = 0.74; ERC-18, p = 0.68; unpaired t-test). To determine whether prolonged ERK phosphorylation induced by TPA was dependent on Tsc2 expression, we measured ERK phosphorylation during the first hour of treatment with 100 ng/ml TPA in ERC18-FLAG-Tsc2 and ERC18-FLAG-2B cell lines. Tsc2 expression did not alter the pattern of TPA-induced ERK phosphorylation, as both ERC18-FLAG-Tsc2 and ERC18-FLAG-2B cell lines showed the prolonged ERK phosphorylation characteristic of the Tsc2-null ERC-18 cell line (Fig. 3).



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FIG. 3. ERC-18 cells show a prolonged activation of ERK in response to TPA, but this effect is not modulated by Tsc2. Cells were grown and treated with 100 ng/ml TPA or DMSO as described above. Protein lysates were prepared with RIPA buffer after 5 min, 15 min, and 1 h of treatment. ERK phosphorylation was measured in equal amounts of lysate with a mouse monoclonal antibody (Santa Cruz Biotechnology). Representative blots are shown, with actin levels directly below. Band intensities (p-ERK) were quantified from three independent experiments and compared to identify statistically significant differences from control at each treatment time point (*p < 0.05; **p < 0.01). Cell lines are indicated above blots; 2B = ERC18-FLAG-2B, Tsc2 = ERC18-FLAG-Tsc2. Error bars indicate SEM.

 
TPA Activates PKC{alpha} in NRK-52E and ERC-18 Cells, but Activates Rap1 Only in ERC-18 Cells
In order to identify the molecular target for the TPA-induced changes in cell morphology and ERK phosphorylation described above, we measured changes in the location of PKC{alpha} and in the levels of GTP-bound Rap1 following treatment with 100 ng/ml TPA. The location of PKC{alpha} was determined using immunocytochemistry in NRK-52E and ERC-18 cells fixed after 5, 15, or 60 min of TPA treatment. In both cell lines, PKC{alpha} was mobilized to the plasma membrane within 5 min of treatment with the phorbol ester (Fig. 4), and remained at that location in both lines in many cells even after 1 h of treatment. In the ERC-18 cell line, the kinase appears to be localized at discrete areas of focal contacts (arrows in Fig. 4).



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FIG. 4. TPA activates PKC{alpha} in NRK-52E and ERC-18 cells. Cells were grown and treated with 100 ng/ml TPA or DMSO (con) as described above. Immunocytochemical localization of PKC was used as a measure of PKC{alpha} activation after TPA treatment for 5 min, 15 min, and 1 h. Arrows indicate sites of discrete PKC{alpha} localization in ERC-18 cells treated with TPA for 1 h.

 
Since TPA has also been shown to activate Rap1 in fibroblasts and neutrophils, we wanted to determine whether the phorbol ester activated the GTPase in renal epithelial cells. Cells were treated with 100 ng/ml TPA for 5, 15, or 60 min, and endogenous GTP-bound Rap1 was precipitated from cell lysates using a GST-fusion protein containing the Ras-binding domain of RalGDS, as previously described (Franke et al., 1997Go). A representative Rap1 blot and the mean Rap1 band intensities from four independent experiments are shown in Figure 5. Randomized block ANOVA showed no significant change in Rap1-GTP levels at any time point in either cell type (NRK-52E, p = 0.78; ERC-18, p = 0.09). While not quite statistically significant, the mean levels of Rap1-GTP in ERC-18 cells appeared to indicate a weak activation of Rap1 by TPA during the first 15 min of treatment. Equal amounts of the RalGDS GST-RBD construct were used to precipitate Rap1-GTP from each sample, as shown by the representative GST immunoblot in Figure 5.



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FIG. 5. TPA induces weak activation of Rap1 in ERC-18 cells. Cells were grown and treated with 100 ng/ml TPA or DMSO (cont) as described above. Protein lysates were prepared with RIPA buffer after 5 min, 15 min, and 1 h of treatment. Endogenous levels of active, GTP-bound Rap1 were measured from lysates as described in the Methods section. Shown are a representative Rap1 blot and the mean level of Rap1-GTP at each treatment time point from four independent experiments. Error bars indicate SEM. Corresponding levels of RalGDS GST-RBD are shown to demonstrate equivalent use of the construct in each sample (GST).

 
Inhibition of PKC Activation Abrogates TPA-Induced Changes in Cell Morphology and Decreases ERK Phosphorylation
Since TPA activated PKC{alpha} in both cell types, but activated Rap1 only in ERC-18 cells, it was unclear which TPA target mediated the morphologic change and increased ERK phosphorylation in each cell type. In order to clarify the role of PKC in the response of renal epithelial cells to TPA, NRK-52E and ERC-18 cells were treated with 100 ng/ml TPA in the presence of the selective PKC inhibitor bisindolylmaleimide VIII (BisVIII, 1 µM). Bisindolylmaleimide compounds inhibit PKC isoforms with similar potency but improved selectivity when compared to PKC inhibitors like staurosporine (Toullec et al., 1991Go). Bisindolylmaleimide compounds also have increased selectivity for the PKC{alpha} isoform, and BisVIII has an IC50 of 53 nM for PKC{alpha} (Wilkinson et al., 1993Go). As a control, cells were treated with 100 ng/ml TPA in the presence of an inactive, structural analogue of BisVIII, bisindolylmaleimide V (Bis V, 1 µM). Morphology was observed over the first hour of treatment, and cell lysates were prepared after 15 min and 1 h of treatment to compare ERK phosphorylation. As shown in Figure 6A, BisVIII completely inhibited the morphologic change observed in both cell types after 1 h of TPA treatment. Conversely, TPA treatment in the presence of BisV resulted in the characteristic TPA-induced changes in cell morphology (increased refractility, retraction of the cell periphery, loss of cell-cell contact, rounding) described above.

BisVIII cotreatment was able to dramatically reduce TPA-induced ERK phosphorylation in both cell types. Figure 6B shows the relative increase in ERK phosphorylation (compared to control) induced by TPA treatment in the presence of BisVIII and BisV in both cell lines. BisVIII cotreatment significantly reduced TPA-induced ERK activation in NRK-52E cells and ERC-18 cells (p < 0.01; two-way ANOVA and p < 0.001; two-way ANOVA, respectively).

TPA-Induced PKC{alpha} Degradation Is More Extensive in NRK-52E Cells than in ERC-18 Cells
As PKC{alpha} appears to be an important regulator of both the morphologic change and increased ERK phosphorylation induced by TPA, we wanted to examine potential differences in the ability of the phorbol ester to down-regulate the kinase between NRK-52E and ERC-18 cells. Cells were treated with 50 or 100 ng/ml TPA for 24 h, and PKC{alpha} expression was measured in whole cell lysates. A dose-dependent decrease in PKC{alpha} expression was observed in both cell types after 24 h of TPA treatment (not shown). Comparison of PKC{alpha} levels after 24 h of treatment with 100 ng/ml TPA showed that this decrease was much more pronounced in NRK-52E cells (Fig. 7A). Indeed, comparison of the relative decrease in PKC{alpha} expression (from control) after 24 h treatment with 100 ng/ml TPA showed a statistically significant difference between NRK-52E and ERC-18 cells (p = 0.02; unpaired t-test), with only 6% of control PKC{alpha} remaining in NRK-52E cells and 32% remaining inERC-18 cells after TPA treatment (Fig. 7B).



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FIG. 7. TPA treatment results in a more dramatic down-regulation of PKC{alpha} in NRK-52E cells than in ERC-18 cells. Protein lysates were prepared from cells treated with 100 ng/ml TPA or DMSO (con) for 24 h. Expression of PKC{alpha} was measured in equal amounts of lysate using immunoblot analysis as described above. A representative blot is shown in (A), with actin levels shown immediately below. In (B), the relative decrease in PKC{alpha} expression after treatment with 100 ng/ml TPA for 24 h is compared between the cell types (*p < 0.05). Error bars indicate SEM from five independent experiments.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Morphologic changes observed in vitro may provide clues to the cellular response to toxicant exposure in vivo. Cell culture models also provide the advantage of allowing study of specific molecular defects as they relate to the toxic response. In the present study, we used Tsc2-null renal tumor cells and Tsc2-expressing renal epithelial cells to gain insight about the effects of Tsc2 expression on the response of renal epithelial cells to the phorbol ester tumor promoter TPA. TPA is the active component of the naturally occurring tumor promoter croton oil, and functions by directly targeting cell signaling pathways. We showed that Tsc2 expression modulates the morphologic response of renal epithelial cells to TPA, but may not directly influence TPA-induced ERK activation.

Previous studies have shown that cytosolic PKC{alpha} rapidly translocates to the plasma membrane upon TPA-induced activation (Nakashima, 2002Go), and that PKC can activate the MAPK signaling pathway via Raf-1 dependent (Kolch et al., 1993Go) and independent (Chao et al., 1994Go) mechanisms following phorbol ester activation. In addition, TPA has been shown to activate Rap1 in a PKC-independent manner in neutrophils and fibroblasts (M'Rabet et al., 1998Go; Zwartkruis et al., 1998Go). In the present study, TPA-induced changes in cell morphology and ERK activation in both cell types appear to be at least partially PKC-dependent. TPA caused translocation of PKC{alpha} to the plasma membrane within 5 min in both cell types, temporally consistent with a causal role for the kinase in morphologic change and ERK activation. More importantly, changes in cell morphology and ERK activation were abrogated by cotreatment with the selective PKC inhibitor bisindolylmaleimide VIII. These findings clearly indicate that TPA functions through PKC to induce morphologic changes and ERK activation in NRK-52E and ERC-18 cells. However, while TPA-induced morphologic change appears to be primarily dependent on PKC activation, the prolonged ERK activation observed in TPA-treated ERC-18 cells may be mediated by the effects of the tumor promoter on Rap1. Since Rap1 activity increased in response to TPA in ERC-18 cells, and since Rap1 has been shown to mediate sustained ERK activation (York et al., 1998Go), it is possible that basal Rap1 activity is partially responsible for the prolonged ERK activation observed in ERC-18 cells. However, since reexpression of Tsc2 did not abrogate the prolonged ERK activation in ERC-18 cells, it is unclear whether Tsc2 is required for this effect. It may be possible that levels of Tsc2-FLAG expression were not high enough to affect Rap1 activity in the current study. Alternatively, the differential activation of ERK in NRK-52E and ERC-18 based cell lines may be due to cell-type specific differences not involving Tsc2. Regardless, Rap1 activation is not likely to be responsible for the prolonged morphologic response of ERC-18 cells treated with TPA since bisindolylmaleimide VIII completely abrogated TPA-induced morphologic change in both cell types. In addition, expression of Tsc2 in ERC-18 cells abrogated only the prolonged morphologic response of these cells to TPA, implying that the prolonged changes in ERC-18 cell morphology following TPA treatment are not the result of prolonged ERK activation.

Previous studies have indicated that TPA-induced changes in cell morphology may arise from the phosphorylation of key focal adhesion (FA) substrates by PKC. A previous study of TPA-induced changes in renal epithelial cell morphology showed that the tumor promoter caused a disruption of focal contacts, redistribution of the FA protein vinculin, and reorganization of the actin cytoskeleton (Rahilly and Fleming, 1992Go). PKC{alpha} binds the FA proteins vinculin and talin (Hyatt et al., 1994Go) and was co-localized with talin at focal contacts in rat embryo fibroblasts (Jaken et al., 1989Go). In the current study, PKC{alpha} was localized to distinct focal contacts after 1 h of TPA treatment in ERC-18 cells. Focal localization of PKC{alpha} was not observed in NRK-52E cells after TPA treatment, and may be an important mediator of the prolonged morphologic response of ERC-18 cells to TPA. Expression of Tsc2 in ERC-18 cells abrogated the prolonged morphologic response, emphasizing the importance of Tsc2 in the regulation of this response.

While TPA rapidly activates PKC{alpha}, prolonged TPA treatment results in down-regulation of the kinase (Blumberg et al., 2000Go). We observed a more pronounced decrease in PKC{alpha} in NRK-52E cells compared to ERC-18 cells following prolonged TPA treatment. Since the morphologic response required PKC activation, it may be possible that the prolonged morphologic response of ERC-18 cells to TPA is due to the persistent expression of the kinase in these cells even after 24 h of treatment. In renal epithelial cells, PKC{alpha} is normally degraded following prolonged activation via ubiquitination and proteasomal degradation (Lee et al., 1996Go). Tsc2 loss may alter the ability of renal epithelial cells to degrade PKC{alpha} via normal proteasomal mechanisms. Alternatively, expression of PKC{alpha} may be increased in Tsc2-null cells. Indeed, we have consistently observed elevated PKC{alpha} expression in ERC-18 cells compared to NRK-52E cells, although it is unclear whether this increase is Tsc2-dependent (unpublished observation). Additional studies are required to directly test these possibilities.

Activation of PKC{alpha} has been implicated in the regulation of proliferation, apoptosis, differentiation, cell migration, and adhesion (reviewed in Nakashima, 2002Go). The morphologic changes observed in the current study, combined with evidence from previous studies, suggest that PKC{alpha}-dependent adhesion and/or migration may be altered in Tsc2-null cells. Changes in cell morphology observed in the current study were not the result of increased cell proliferation or apoptosis. In a previous report, nearly identical changes in cell morphology (loss of intercellular adhesion, retraction of cell periphery, increased refractility, rounding) were induced in primary cultures of renal epithelial cells treated with 100 ng/ml TPA and caused cell detachment after 2 h (Rahilly and Fleming, 1992Go). In the current study, TPA did not induce detachment in either cell type, even after agitation. This difference may be due to phenotypic differences between the primary cultures used in the previous study and the cell lines used in our study. Alternatively, differences in the culture vessels used (laminin- or fibronectin-coated glass vs. tissue culture-treated plastic) may account for the differences in detachment between studies.

PKC{alpha} may also regulate cell adhesion and motility by binding and phosphorylating members of the actin-binding ezrin-radixin-moesin (ERM) family of proteins (Ng et al., 2001Go). Hamartin, the product of the Tsc1 tumor suppressor gene, binds both tuberin and ERM proteins and appears to be required for cell-matrix adhesion (Lamb et al., 2000Go). Over-expression of tuberin in Madine-Darby canine kidney (MDCK) epithelial cells or re-expression of tuberin in Tsc2-null cells has recently been shown to promote cell adhesion, inhibit migration, and activate the Rho GTPase (Astrinidis et al., 2002Go). Interestingly, TPA-induced changes in MDCK cell focal adhesion structure and actin organization were shown to be dependent on the activities of Rho and Rab5 GTPases in one study (Imamura et al., 1998Go). There is also evidence supporting the possibility that tuberin functions as a GAP for Rab5 (Xiao et al., 1997Go). The findings of the current study clearly show that the prolonged morphologic response of renal epithelial cells to TPA is Tsc2-dependent. Taken together, these findings may implicate PKC{alpha} as an additional mediator in the Tsc2-dependent regulation of cell adhesion and migration (Fig. 8). Changes in the migratory response of renal epithelial cells to PKC{alpha} activation may be particularly important in vivo, as PKC activation appears to have an invasion-promoting role during RCC. The in vitro invasiveness of human renal cell carcinoma lines was reduced by PKC inhibitors, and membrane translocation of the PKC{alpha} isoform was correlated with increased invasiveness of the RCC lines used (Engers et al., 2000Go).



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FIG. 8. PKC{alpha} may be an additional mediator of Tsc2-dependent regulation of cell adhesion and migration. Activation of PKC{alpha} can promote proliferation, survival, and cell migration via multiple signaling pathways, as described in the text. We propose that Tsc2 may function to increase PKC{alpha} expression or reduce PKC{alpha} degradation. Solid arrows indicate direct regulation; dashed arrows represent potential regulation.

 
We have shown that Tsc2-null renal tumor cells have a prolonged morphologic response (loss of cell-cell contact, retraction of the cell periphery with discrete sites of substrate contact, increased refractility, rounding) to the phorbol ester tumor promoter TPA when compared to a Tsc2-expressing rat renal tubular epithelial cell line. Expression of Tsc2 abrogated the prolonged morphologic response, but did not inhibit the prolonged ERK activation, of Tsc2-null renal tumor cells. TPA altered cell morphology and increased ERK activity via activation of PKC. These findings may have important implications for the progression of renal tumorigenesis.


    ACKNOWLEDGMENTS
 
This work was supported by National Institute of Environmental Health Sciences (NIEHS) grant F30 ES05925 to T.M. Kolb and ES08157 to M.A. Davis.


    NOTES
 

1 To whom correspondence should be addressed. Fax: (317) 277-6770. E-mail: davisma{at}lilly.com.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Astrinidis, A., Cash, T. P., Hunter, D. S., Walker, C. L., Chernoff, J., and Henske, E. P. (2002). Tuberin, the tuberous sclerosis complex 2 tumor suppressor gene product, regulates Rho activation, cell adhesion and migration. Oncogene 21, 8470–8476.[CrossRef][ISI][Medline]

Blumberg, P., Acs, P., Bhattacharyya, D., and Lorenzo, P. (2000). Inhibitors of protein kinase C and related receptors for the lipophilic second messenger sn-1,2-diacylglycerol. In Signaling Networks and Cell Cycle Control: The Molecular Basis of Cancer and Other Diseases (J. S. Gutkind, Ed.), pp. 347–364.Humana Press, Totowa, NJ.

Chao, T. S., Foster, D. A., Rapp, U. R., and Rosner, M. R. (1994). Differential Raf requirement for activation of mitogen-activated protein kinase by growth factors, phorbol esters, and calcium. J. Biol. Chem. 269, 7337–7341.[Abstract/Free Full Text]

Engers, R., Mrzyk, S., Springer, E., Fabbro, D., Weissgerber, G., Gernharz, C. D., and Gabbert, H. E. (2000). Protein kinase C in human renal cell carcinomas: Role in invasion and differential isoenzyme expression. Br. J. Cancer 82, 1063–1069.[CrossRef][Medline]

Everitt, J. I., Goldsworthy, T. L., Wolf, D. C., and Walker, C. L. (1992). Hereditary renal cell carcinoma in the Eker rat: A rodent familial cancer syndrome. J. Urol. 148, 1932–1936.[ISI][Medline]

Franke, B., Akkerman, J. W., and Bos, J. L. (1997). Rapid Ca2+-mediated activation of Rap1 in human platelets. Embo J. 16, 252–259.[Abstract/Free Full Text]

Freed, J. J., Howard, S., Lerro, A., Dodson, L., Scsok, D., and Knudson, A. G. (1990). Hereditary renal tumors in the rat: Cell lines from adenocarcinomas induced by the Eker mutation. Proc. Am. Assoc. Cancer Res. 31, 317.

Gao, X., and Pan, D. (2001). TSC1 and TSC2 tumor suppressors antagonize insulin signaling in cell growth. Genes Dev. 15, 1383–1392.[Abstract/Free Full Text]

Goncharova, E. A., Goncharov, D. A., Eszterhas, A., Hunter, D. S., Glassberg, M. K., Yeung, R. S., Walker, C. L., Noonan, D., Kwiatkowski, D. J., Chou, M. M., Panettieri, R. A., Jr., and Krymskaya, V. P. (2002). Tuberin regulates p70 S6 kinase activation and ribosomal protein S6 phosphorylation. A role for the TSC2 tumor suppressor gene in pulmonary lymphangioleiomyomatosis (LAM). J. Biol. Chem. 277, 30958–30967.[Abstract/Free Full Text]

Gottlicher, M. (1999). Receptor toxicology. In Toxicology (H. Marquardt, S. Schafer, R. McClellan, and R. Welsch, Eds.), pp. 231–243. Academic Press, San Diego, CA.

Hall, A. (2000). GTPases. In Frontiers in Molecular Biology (B. Hames and D. Glover, Eds.), pp. 67–175. Oxford University Press, New York.

Herrmann, C., Horn, G., Spaargaren, M., and Wittinghofer, A. (1996). Differential interaction of the ras family GTP-binding proteins H-Ras, Rap1A, and R-Ras with the putative effector molecules Raf kinase and Ral-guanine nucleotide exchange factor. J. Biol. Chem. 271, 6794–6800.[Abstract/Free Full Text]

Hyatt, S. L., Liao, L., Chapline, C., and Jaken, S. (1994). Identification and characterization of alpha-protein kinase C binding proteins in normal and transformed REF52 cells. Biochemistry 33, 1223–1228.[ISI][Medline]

Imamura, H., Takaishi, K., Nakano, K., Kodama, A., Oishi, H., Shiozaki, H., Monden, M., Sasaki, T., and Takai, Y. (1998). Rho and Rab small G proteins coordinately reorganize stress fibers and focal adhesions in MDCK cells. Mol. Biol. Cell 9, 2561–2575.[Abstract/Free Full Text]

Inoki, K., Li, Y., Zhu, T., Wu, J., and Guan, K. L. (2002). TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling. Nat. Cell Biol. 4, 648–657.[CrossRef][ISI][Medline]

Jaken, S., Leach, K., and Klauck, T. (1989). Association of type 3 protein kinase C with focal contacts in rat embryo fibroblasts. J Cell. Biol. 109, 697–704.[Abstract]

Kenerson, H. L., Aicher, L. D., True, L. D., and Yeung, R. S. (2002). Activated mammalian target of rapamycin pathway in the pathogenesis of tuberous sclerosis complex renal tumors. Cancer Res. 62, 5645–5650.[Abstract/Free Full Text]

Kolch, W., Heidecker, G., Kochs, G., Hummel, R., Vahidi, H., Mischak, H., Finkenzeller, G., Marme, D., and Rapp, U. R. (1993). Protein kinase C alpha activates RAF-1 by direct phosphorylation. Nature 364, 249–252.[CrossRef][ISI][Medline]

Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.[ISI][Medline]

Lamb, R. F., Roy, C., Diefenbach, T. J., Vinters, H. V., Johnson, M. W., Jay, D. G., and Hall, A. (2000). The TSC1 tumour suppressor hamartin regulates cell adhesion through ERM proteins and the GTPase Rho. Nat. Cell Biol. 2, 281–287.[CrossRef][ISI][Medline]

Lee, H. W., Smith, L., Pettit, G. R., Vinitsky, A., and Smith, J. B. (1996). Ubiquitination of protein kinase C-alpha and degradation by the proteasome. J. Biol. Chem. 271, 20973–20976.[Abstract/Free Full Text]

Momose, S., Kobayashi, T., Mitani, H., Hirabayashi, M., Ito, K., Ueda, M., Nabeshima, Y., and Hino, O. (2002). Identification of the coding sequences responsible for Tsc2-mediated tumor suppression using a transgenic rat system. Hum. Mol. Genet. 11, 2997–3006.[Abstract/Free Full Text]

M'Rabet, L., Coffer, P., Zwartkruis, F., Franke, B., Segal, A. W., Koenderman, L., and Bos, J. L. (1998). Activation of the small GTPase rap1 in human neutrophils. Blood 92, 2133–2140.[Abstract/Free Full Text]

Nakashima, S. (2002). Protein kinase calpha (PKCalpha): Regulation and biological function. J. Biochem. (Tokyo) 132, 669–675.[Abstract]

Ng, T., Parsons, M., Hughes, W. E., Monypenny, J., Zicha, D., Gautreau, A., Arpin, M., Gschmeissner, S., Verveer, P. J., Bastiaens, P. I., and Parker, P. J. (2001). Ezrin is a downstream effector of trafficking PKC-integrin complexes involved in the control of cell motility. Embo J. 20, 2723–2741.[Abstract/Free Full Text]

Rahilly, M. A., and Fleming, S. (1992). A tumour promoter induces alterations in vinculin and actin distribution in human renal epithelium. J. Pathol. 166, 283–288.[ISI][Medline]

Tee, A. R., Fingar, D. C., Manning, B. D., Kwiatkowski, D. J., Cantley, L. C., and Blenis, J. (2002). Tuberous sclerosis complex-1 and -2 gene products function together to inhibit mammalian target of rapamycin (mTOR)-mediated downstream signaling. Proc. Natl. Acad. Sci. U.S.A. 99, 13571–13576.[Abstract/Free Full Text]

Toullec, D., Pianetti, P., Coste, H., Bellevergue, P., Grand-Perret, T., Ajakane, M., Baudet, V., Boissin, P., Boursier, E., Loriolle, F., Duhamel, L., Charon, D., and Kirilovsky, J. (1991). The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C. J. Biol. Chem. 266, 15771–15781.[Abstract/Free Full Text]

Wienecke, R., Konig, A., and DeClue, J. E. (1995). Identification of tuberin, the tuberous sclerosis-2 product. Tuberin possesses specific Rap1GAP activity. J. Biol. Chem. 270, 16409–16414.[Abstract/Free Full Text]

Wilkinson, S. E., Parker, P. J., and Nixon, J. S. (1993). Isoenzyme specificity of bisindolylmaleimides, selective inhibitors of protein kinase C. Biochem. J. 294, 335–337.[ISI][Medline]

Xiao, G. H., Shoarinejad, F., Jin, F., Golemis, E. A., and Yeung, R. S. (1997). The tuberous sclerosis 2 gene product, tuberin, functions as a Rab5 GTPase activating protein (GAP) in modulating endocytosis. J. Biol. Chem. 272, 6097–6100.[Abstract/Free Full Text]

York, R. D., Yao, H., Dillon, T., Ellig, C. L., Eckert, S. P., McCleskey, E. W., and Stork, P. J. (1998). Rap1 mediates sustained MAP kinase activation induced by nerve growth factor. Nature 392, 622–626.[CrossRef][ISI][Medline]

Zhang, Y., Gao, X., Saucedo, L. J., Ru, B., Edgar, B. A., and Pan, D. (2003). Rheb is a direct target of the tuberous sclerosis tumour suppressor proteins. Nat. Cell Biol. 5, 578–581.[CrossRef][ISI][Medline]

Zwartkruis, F. J., Wolthuis, R. M., Nabben, N. M., Franke, B., and Bos, J. L. (1998). Extracellular signal-regulated activation of Rap1 fails to interfere in Ras effector signalling. Embo J. 17, 5905–5912.[Abstract/Free Full Text]





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