Carcinogenesis Advance Access originally published online on August 3, 2007
Carcinogenesis 2007 28(12):2443-2450; doi:10.1093/carcin/bgm154
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MAP17 inhibits Myc-induced apoptosis through PI3K/AKT pathway activation
Experimental Therapeutics Programme, Centro Nacional de Investigaciones Oncológicas, Madrid 28029, Spain
* To whom correspondence should be addressed. Tel: +34 91 732 8021; Fax: +34 91 732 8051; Email: acarnero{at}cnio.es
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Abstract |
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MAP17 is a non-glycosylated membrane-associated protein that has been shown to be over-expressed in human carcinomas, suggesting a possible role of this protein in tumorigenesis. However, very little is known about the molecular mechanism mediating the possible tumor promoting properties of MAP17. To analyze the effect of MAP17 on cell survival, we used Rat1 fibroblasts model where Myc over-expression promotes apoptosis in low serum conditions. In the present work, we report that over-expression of MAP17 protects Rat1a fibroblasts from Myc-induced apoptosis through reactive oxygen species (ROS)-mediated activation of the PI3K/AKT signaling pathway. MAP17-mediated survival was associated with absence of Bax translocation to the mitochondria and reduced caspase-3 activation. We show that a fraction of PTEN undergoes oxidation in MAP17-over-expressing cells. Furthermore, activation of AKT by MAP17 as measured by Thr308 phosphorylation was independent of PI3K activity. Importantly, modulation of ROS by antioxidant treatment prevented activation of AKT, restoring the level of apoptosis in serum-starved Rat1/c-Myc fibroblasts. Finally, over-expression of a dominant-negative mutant of AKT in MAP17-expressing clones makes them sensitive to serum depletion. Our data indicate that MAP17 protein activates AKT through ROS and this is determinant to confer resistance to Myc-induced apoptosis in the absence of serum.
Abbreviations: DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; FAC, flow cytometry; GSH, glutathione; NAC, N-acetylcysteine; PBS, phosphate-buffered saline; sROS, reactive oxygen species
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Introduction |
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Although multiple oncogenes that activate signaling pathways directly involved in cell survival or proliferation have been discovered in the last decades, many other genes that provide an advantage to the tumoral cells making them insensitive to physiological signals or altering their normal physiology are still to be found. Functional genetic screens using retroviral delivery of high-complexity cDNA libraries constitute a valuable tool to discover new genes involved in the appropriate phenotypic characteristic of the tumoral cell.
We designed a genome-wide retroviral cDNA screen to search for genes that confer to cancer cells the ability to evade the normal physiological responses directed to maintain tissue homeostasis (1). We identified MAP17, a small non-glycosylated membrane-associated protein of 17 kDa that locates to the plasma membrane and the Golgi apparatus (1,2). The protein has two transmembrane domains and a PDZ-binding domain in the C-terminus (3). MAP17 acts as an atypical anchoring site for PDZK1 and interacts with the NaPi-IIa–PDZK1 protein complex in renal proximal tubular cells (4). The physiological role of MAP17 in proximal tubules is not known but it stimulates specific Na-dependent transport of mannose and glucose in Xenopus oocytes (2) and several human cell lines (1). Ectopic expression of MAP17 in tumor cells prevents tumor necrosis factor-induced G1 arrest by impairing p21waf1 induction. The inhibition of tumor necrosis factor is specific since MAP17 does not alter the response to other cytokines such as IFN
(1). MAP17 is over-expressed in a great variety of human carcinomas (5,6) and, at least in prostatic and ovarian carcinomas, the over-expression of the protein strongly correlates with tumoral progression. Despite the common over-expression of MAP17 among carcinomas of different origins, there are no reports suggesting a possible role of MAP17 in tumorigenesis that could explain this distribution.
In the present work, we show that ectopic over-expression of MAP17 can rescue Rat1 fibroblasts from c-Myc-induced apoptosis. We provide evidence that MAP17 induces survival signaling through reactive oxygen species (ROS)-mediated activation of the PI3K/AKT pathway, and this activation is essential promoting MAP17 survival in the absence of serum.
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Materials and methods |
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Cell culture conditions
Rat1 fibroblasts and LinXE ecotropic retrovirus producer cells (7) were grown in Dulbecco's modified Eagle's medium (DMEM) with glutamax supplemented with 10% fetal bovine serum (FBS), penicillin, streptomycin and fungizone. Retroviral constructs were introduced into packaging cells by standard calcium phosphate transfection. Retroviral-mediated gene transfer was performed as previously described in Carnero et al. (7). Briefly, packaging cells were transfected by calcium phosphate. After 48 h, the virus-containing medium was filtered and added to 8 µg/ml polybrene (Sigma) and fresh medium. Target cells were incubated overnight with the appropriate viral supernatant. The infected cell population was purified using the appropriate culture selection. MAP17 full cDNA was cloned in pBabepuro and clones were generated by retroviral-mediated gene transfer. Numerous clones were selected and tested. All behave similarly. In this work, three representative clones are shown.
Treatments
Both N-acetylcysteine (NAC) and reduced glutathione (GSH) were purchased from Sigma and cells were incubated with 10 mM of either NAC or GSH as indicated.
Cell death measurement
Triplicate samples of 104 cells per well were seeded in 96-well plates. After 24 h, fresh medium with the appropriate treatment (with or without serum) was added (day 0). Every 24 h, cells were fixed and stained with crystal violet. After extensive washing, crystal violet was resolubilized in 15% acetic acid and quantified at 595 nm as a relative measure of cell number. Values are expressed as the percentage of cell growth. Untreated control cells represent 100% of cell growth.
Reverse transcriptase–polymerase chain reaction
Total RNA was purified using the TRI-REAGENT (Molecular Research Center, Cincinnati, OH). Reverse transcription was performed with 5 µg of RNA using MMLV reverse transcriptase (Promega) and oligodT primer according to the manufacturer's recommendations. The following primers were used to amplify specific cDNA regions: MAP17: forward 5'-CAGCCATGTCGGCCCTCA-3' and reverse 5'-TTATTTCACAGAAATTAGGGCC-3'; β-actin: forward 5'-AGGCCAACCGCGAGAAGATGAC-3' and reverse 5'-GAAGTCCAGGGCGACGTAGCA-3'. cDNA was subjected to polymerase chain reaction and products were analyzed by 1% agarose gel electrophoresis.
Western blot analysis
Cells were washed twice with ice-cold phosphate-buffered saline (PBS) and lysed by sonication in lysis buffer (50 mM Tris–HCl, pH 7.5, 1% Nonidet P-40, 2 mM Na3VO4, 150 mM NaCl, 20 mM Na4P2O7 and complete protease inhibitor cocktail (Roche Molecular Biochemicals). The protein content of the lysates was determined by the modified method of Bradford. Membranes were incubated overnight at 4°C with the primary antibody, washed and incubated with anti-mouse (1:10000) or anti-rabbit (1:5000) horseradish peroxidase-conjugated secondary antibody. Bands were visualized by using the enhanced chemiluminescence Western Blotting detection system (Amersham Pharmacia Biotech) exposed to Kodak-X-Omat LS film (Kodak). The following primary antibodies were used: phospho-AKT(Ser473) (#9271), phospho-AKT(Thr308) (#9275), total AKT (#9272), PTEN (#9552), eIF4G (#2441) and PDK1 (#3062) antibodies were supplied by Cell Signaling Technology; c-Myc (Santa Cruz 9E10), FKRHL1-P (Upstate Cell Signaling #06-952). Monoclonal antibody anti-β-tubulin (Sigma 9026 1:10000), horseradish peroxidase-labeled rabbit anti-mouse (Promega diluted 1:5000) and goat anti-rabbit (Calbiochem 401315, diluted 1:4000) are the secondary antibodies.
ROS fluorescent detection
To visualize intracellular ROS levels, RatMyc cells grown on cover slips were washed twice with warm PBS and then incubated at 37°C with 8 µM of CM-H2DCFDA (Molecular Probes) in warm PBS supplemented with 2.5 mM glucose for 15 min. Then, PBS was replaced with 10% FBS-supplemented DMEM, and cells were incubated 10 min in the same conditions. Cells were washed with warm PBS and fixed with 4% paraformaldehyde (Sigma) at room temperature for 5 min. The fixed cells were washed three times with PBS and the cover slips mounted in mowiol. Intracellular ROS were visualized using a Confocal Ultra-spectral microscope Leica TCS-SP2-AOBS-UV.
Caspase assay
Cells were seeded, grown to 70% confluence and incubated in the presence of 10% (controls) or 0.1% serum for 24 h. Plates were washed twice with Tris-buffered saline and then harvested by adding cold lysis buffer containing 50 mM Tris–HCl, 150 mM NaCl, 1% Nonidet P-40, 100 mM dithiothreitol and protease inhibitor cocktail (Roche Molecular Biochemicals). We used Promega's CaspACE assay system (fluorimetric). The assays were performed following the manufacturer's instructions.
Immunohistochemistry of MAP17
Cells were trypsinized and cytospinned onto glass cover slips. The following day cells were fixed with acetone for 10 min and then incubated with MAP17 monoclonal antibody for 30 min. Cells were washed three times with PBS and incubated for additional 30 min with a secondary goat anti-mouse antibody (DAKO Cytomation) diluted 1:50 in FBS. After washing, slides were mounted with Aquatex (Merck). Photos were taken in an Olympus Provis Microscope AX70.
Monoclonal anti-MAP17 antibody was generated by the Monoclonal Antibody Unit of the Centro Nacional de Investigaciones Oncológicas. We used a glutathione-S-transferase–MAP17 fusion protein purified from Escherichia coli as immunogen. Different antibodies were tested and validated by antigenic competition.
Immunostaining and confocal analysis
Cells were seeded at a density of 104 cells/cm2 onto glass cover slips and cultured for 24 h. Then we changed the cells to fresh media with the appropriate serum concentrations. After 24 h, cells were incubated with 75 nM Mitotracker RED CMXRos for 45 min at 37°C and washed with PBS. Cover slips were fixed in 2% paraformaldehyde for 15 min at room temperature and washed 5 times with PBS. Samples were incubated in blocking solution (PBS containing 3% bovine serum albumin) at room temperature for 15 min, followed by incubation overnight at 4°C with anti-Bax antibody. After washing with PBS, cells were incubated with species-specific Alexa 633-conjugated secondary antibody diluted 1:200 in blocking buffer for 1 h at room temperature in the dark. The nuclei were stained with Hoechst 33258 for 10 min at room temperature in the dark prior to mounting with mowiol (Calbiochem). Images were collected by confocal laser microscopy (model TCS-SP2-AOBS, Leica, Germany). The 543 and 633 nm lines of the Helio/Neon laser were used for the excitation of Mitotracker Red CMXRos and Alexa 633, respectively.
Annexin staining
Exponentially growing cells (106) were incubated in different serum conditions: DMEM + 10% FBS, DMEM + 0.5% FBS, DMEM + 0.01% FBS and 24 h later the detached cells in the supernatant were obtained and mixed with trypsinized cells. They were centrifuged for 5 min at 1100 r.p.m. and the pellets washed with PBS and resuspended in binding buffer 1x (BD Pharmingen; 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2). Then cells were incubated with 5 µl annexin V (BD Pharmingen) and 10 µl propidium iodide (Sigma) during 15 min in the dark. Flow cytometry (FAC) analysis was made with a Beckton and Dickinson FACScalibur cytometer and data were analyzed with Cell Quest Pro software.
Clonogenic survival assay
Cell survival following transfection with empty pcDNA3 or pcDNA3 containing AKT K179M was determined by clonogenic assay. Transfection of RatMyc control cells and RatMyc cells expressing MAP17 was achieved by a standard CaPO4 precipitation method using 2 µg plasmid DNA and 18 µg sheared salmon sperm DNA carrier. Selection of geneticin-resistant colonies was obtained by adding 0.7–1 mg/ml G418 (Sigma) to the medium. After selection for 10 days, cells were rinsed in PBS and fixed in 0.5% glutaraldehyde (Sigma). Fixed fibroblasts were stained for 30 min in 0.1% crystal violet solution.
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MAP17 inhibits Myc-induced apoptosis in low serum conditions
To investigate the contribution of MAP17 to survival signaling, we took advantage of the experimental system described by Evan et al. (8). The human c-Myc gene constitutively driven by an LTR promoter was transduced into Rat-1 fibroblasts and cells stably expressing c-Myc (RatMyc) were selected (9). RatMyc fibroblasts were then infected with a retrovirus carrying the full MAP17 cDNA. We selected several clones and characterized the MAP17 mRNA expression by reverse transcriptase–polymerase chain reaction. All the clones analyzed expressed considerable amounts of MAP17 mRNA in contrast with the RatMyc cells carrying an empty vector (RatMyc-P) that do not express endogenous MAP17. To determine the protein level, we generated mouse monoclonal antibodies using recombinant MAP17 produced in E.coli as immunogen (6). We determined the ectopic over-expression of MAP17 protein in cells by immunohistochemistry and immunofluorescence (Figure 1B and C). All the clones showed a clear staining for MAP17 protein. We confirmed these data by FAC and immunofluorescence (data not shown). As control, we confirmed that all the selected clones maintained similar levels of c-Myc protein expression (Figure 1A).
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Consistent with previous reports, Rat-1 cells that constitutively express c-Myc underwent apoptosis upon serum withdrawal. Control RatMyc cells were cultured at different serum concentrations and the degree of cell death was assessed 48 h later by microscopic examination. Figure 1D shows that the number of parental c-Myc-expressing cells with apoptotic phenotype increased significantly when the serum concentration was reduced. To examine the effect of ectopic expression of MAP17, representative RatMycMAP17 clones were treated as described above. As shown in Figure 1D, all the MAP17-expressing clones showed reduced apoptotic capacity. The level of cell death in MAP17-expressing cells was dramatically reduced even in the complete absence of serum. We quantified this effect in a time-response curve in the absence of serum. Parental cells and MAP17-expressing clones were cultured in low serum conditions during 4 days. While RatMyc parental cells presented a fast response to serum withdrawal and 70% died within the first 24 h, MAP17-expressing clones were resistant to the absence of serum and did not die (Figure 1E). The behavior of MAP17-expressing clones was similar to Rat1 cells (data not shown). This death response was also followed by annexin V/propidium iodide staining (Figure 2A) since they do not die under serum depletion. Serum withdrawal induced an increase of 30% in annexin V staining in parental RatMyc cells, whereas no significant increase in annexin V-positive cells was observed in MAP17-expressing clones.
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To further understand the mechanism by which MAP17 mediates cell survival in serum-deprived RatMyc cells, we examined whether MAP17 was able to reduce caspase activity. We prepared cell lysates from control RatMyc parental cells and from representative MAP17-expressing clones before and after serum deprivation. By using the fluorogenic substrate, Ac-DEVD-MCA, we measured the caspase-3-like protease activity. Ac-DEVD-MCA processing caspase-3-like activity was significantly induced after 12 h of serum starvation in parental cells. The expression of MAP17 reduced caspase-3-like activity between 40 and 60% in serum-starving conditions (Figure 2B).
It has been shown that the apoptotic process initiated by serum withdrawal in Myc-expressing cells rely on the ability of Bax to translocate to mitochondria (10). Therefore, we monitored the intracellular localization of Bax protein. Cells were subjected to serum withdrawal for 24 h and then stained for mitochondria and Bax with fluorescent antibodies. Serum-starved RatMyc parental cells showed an increase in Bax immunofluorescence localized in the mitochondria (Figure 2C). In contrast, the MAP17-expressing clones analyzed showed only a mild increase in Bax and no translocation to the mitochondria (Figure 2C). Almost 50% of serum-depleted parental cells showed Bax traslocation after 24 h, while that phenotype was observed in less than 10% of cells in MAP17 clones (Figure 2D).
Taken together, these data indicate that MAP17 drastically reduces serum deprivation-induced apoptosis in RatMyc cells.
MAP17 activates PI3K pathway
Activation of PI3K pathway has been shown to inhibit Myc-induced apoptosis in the absence of serum. Since AKT is one of the major downstream effectors of the PI3K survival signaling pathway, we analyzed whether MAP17 apoptotic protection in low serum conditions was due to the activation of AKT. We monitored AKT activation analyzing its phosphorylation at Thr308 and Ser473 with specific antibodies. Figure 3 shows that ectopic expression of MAP17 did not alter AKT phosphorylation at Ser473 in the presence of serum. While in parental cells AKT activation, measured as Ser473 phosphorylation, was reduced more than 50% after serum deprivation, the presence of MAP17 in the different clones rendered AKT phosphorylation insensitive to serum starvation. Interestingly, MAP17 expression led to an increase in Thr308 phosphorylation both in the presence or absence of serum similar to what happened with Ser473; while in parental cells Thr308 phosphorylation was reduced in the absence of serum, it was maintained in all the MAP17-expressing clones in conditions of serum deprivation.
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To investigate the impact of maintained AKT activation upon serum deprivation on downstream signaling events, we analyzed the phosphorylation of eIF4G and FRKHL1, two well-known AKT substrates. In both cases, serum withdrawal triggered dephosphorylation in parental cells but not in MAP17-expressing clones (Figure 3). To rule out alterations in PDK1, PTEN or Myc expression that might account for these results, we confirmed by Western blot that neither of these proteins suffered alterations that could explain our data since ectopic expression of MAP17 did not affect the protein level of PDK1, PTEN or c-Myc.
Induction of ROS by MAP17 is responsible for PI3K pathway activation
Growing evidence suggests that ROS act as second messengers in intracellular signaling cascades that induce and maintain the oncogenic phenotype of tumoral cells. ROS have been described to induce proliferation, survival and cellular migration (11–13). Since ROS have been reported as potent activators of the PI3K/AKT pathway, we measured ROS generation in response to MAP17 expression in RatMyc cells.
The level of ROS in living cells was assessed using the fluorescent probe DCF. MAP17-expressing clones showed a clear increase of ROS respect to their parental counterparts (Figure 4A). We confirmed this result analyzing the whole population by FACs. We measured superoxide generation by DCF and analyzed by FACs the level of DCF fluorescence in a total of 104 cells (Figure 4B) and observed more than a 40% increase in the total population respect controls. The observed increase in cellular ROS was inhibited by treatment with GSH (Data not shown).
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To study the potential role of MAP17-induced ROS in cell survival, we first investigated whether AKT phosphorylation in MAP17-expressing clones was dependent on the production of ROS. Parental RatMyc cells and the MAP17-expressing clones were cultured in the presence of the antioxidants GSH or NAC. The activity of the PI3K/AKT pathway was assessed by Western blot using a specific antibody against AKT phosphorylation on Ser473. We observed that in MAP17-expressing cells the treatment with antioxidants reduced AKT phosphorylation in the absence of serum, confirming that the protection observed in such conditions in MAP17-expressing clones was due to the induction of ROS (Figure 4C). However, no reduction in AKT phosphorylation was observed in 10% serum, indicating that in such conditions ROS are not necessary to maintain AKT activation.
To investigate the link between the production of ROS induced by MAP17 and the protection from apoptosis, we measured the survival of MAP17-expressing clones, serum starved, in the presence of antioxidants. Addition of 10 mM NAC to the serum-free medium clearly reduced the survival of MAP17-expressing clones, whereas NAC does not affect growth in 10% growth (Figure 5). These results suggest that the generation of ROS acting as a mediator of MAP17 induced survival through PI3K/AKT signaling.
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Since ROS can oxidize PTEN and inactivate its phosphatase activity, we analyzed PTEN oxidation in MAP17-expressing Rat1Myc cells (Figure 6A). Total protein was extracted from exponentially growing cells and PTEN analyzed by polyacrylamide gel electrophoresis–Western blot in non-oxidant conditions (see Materials and methods). Oxidized PTEN can be distinguished by its different mobility (Figure 6A). MAP17 increased 6–10 folds the levels of oxidized PTEN in serum-starved cells (Figure 6A and B). Also, an increased level of oxidized PTEN was found in cells growing in the presence of serum (data not shown). This result suggests that ROS might oxidize and inactivate PTEN maintaining, therefore, the PI3K pathway activated in the absence of serum. Additionally, ROS has been shown to oxidize and inactivate several phosphatases containing cysteine residues in its active centre. In order to investigate additional mechanisms that might support MAP17-mediated survival, we tested the effect of PI3K inhibition on AKT activity in MAP17-expressing cells. Surprisingly, in contrast to phosphorylation at Ser473, which was significantly reduced in the presence of the PI3K inhibitor LY294002, the phosphorylation at Thr308 seemed to be independent of the upstream PI3K activity (Figure 6C). This result suggests the existence of a PTEN-independent target of ROS impacting specifically on AKT phosphorylation at Thr308.
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In order to further analyze the implication of AKT activation in MAP17-mediated protection from c-Myc-induced apoptosis, we performed clonogenic survival assays in the presence of an Akt derivate that carries a K179M point mutation resulting in a loss of kinase activity (14). We monitored the clonogenic survival of RatMyc control cells and RatMyc cells expressing MAP17 after transfection with AKT K179M or with the pcDNA3 vector alone by a crystal violet colorimetric test. Figure 6D shows that the expression of the MAP17 abolished the reduction of clonogenic survival in the absence of serum confirming data shown above. However, co-expression of the dominant-negative form of Akt blocked the survival of MAP17-expressing RatMyc cells in the absence of serum, suggesting an essential role of Akt activation in the suppression of c-Myc-induced apoptosis upon the ectopic expression of MAP17.
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The present study provides insights into the ROS-mediated activation of an anti-apoptotic signaling pathway by MAP17. We evaluated the effects of ectopic expression of MAP17 in Rat1 fibroblasts that stably co-express c-Myc.
Sensitization to apoptotic stimuli is an intrinsic c-Myc activity that is suppressed by the survival factors present in FBS under normal cell culture conditions. Thus, c-Myc induces cell death upon attenuation of the counteracting survival signaling through serum deprivation (8). We have shown previously that the degree of cell death in RatMyc cells expressing myristoylated forms of p110
, p110β, p110
or p110 was dramatically reduced even in the complete absence of serum (9). Hence, introducing MAP17 in this well-established apoptosis model appears to be a suitable strategy to test its efficiency promoting survival signaling. Interestingly, the degree of protection from c-Myc-induced apoptosis conferred by ectopic expression of MAP17 or activated forms of PI3K were very similar. In order to test the hypothesis that PI3K- and MAP17-triggered protection is mediated by a common mechanism, we assessed the impact of ectopic MAP17 expression on the activity of AKT, the greater downstream effector of PI3K signaling. Activation of AKT involves sequential phosphorylation at two key residues located at the catalytic site (T loop) and the C-terminal hydrophobic motif. PDK1 phosphorylates AKT at Thr308 of its T loop, which is essential for AKT catalytic activity. Recent studies strongly suggest that rapamycin-insensitive TOR complex 2 is responsible for Ser473 phosphorylation at the hydrophobic motif. Interestingly, in the presence of serum, ectopic expression of MAP17 triggers a dramatic increase of Thr308 phosphorylation, whereas Ser473 remains unaffected. Furthermore, inhibition of PI3K signaling by treatment with LY294002 only decreases phosphorylation at Ser473 in MAP17 over-expressing cells. In contrast, MAP17 maintains Thr308 phosphorylation even in the absence of upstream PI3K signaling input, suggesting an uncoupling of the two phosphorylation events. In the case of other members of the AGC kinase family, phosphorylation reactions of the activation loop site and hydrophobic site are tightly interdependent. However, multiple lines of evidence indicate that the phosphorylation state of the corresponding sites on AKT does not always correlate. It has been suggested that uncoupling of dephosphorylation accounts for independent kinetics of Thr308 and Ser473 phosphorylation (15).
Most importantly, the expression of MAP17 completely abolished the reduction of AKT phosphorylation upon serum deprivation, suggesting that the activation of AKT signaling is involved in MAP17-mediated protection of Rat/Myc fibroblasts. In accordance with this notion, AKT exerts its anti-apoptotic activity by preventing the release of cytochrome c into the cytosol, a process triggered by the over-expression of c-Myc in Rat1 fibroblats (16,17). It is thought that mitochondrial cytochrome c release increases caspase-3 proteases activity. We demonstrated that MAP17 decreases significantly the c-Myc-induced caspase-3-like activity in Rat1 fibroblasts under low serum conditions. Additionally and in keeping with the concept of MAP17-induced PI3K/AKT signaling, we report that MAP17 is able to interfere with Bax translocation to the mitochondria.
Furthermore, we shed light on the mechanism by which MAP17 might render RatMyc fibroblast less sensitive to apoptosis. We report that the increased levels of ROS induced by MAP17 might modify the activity of the PI3K pathway through the inhibition of its switch-off signals. The MAP17-mediated increase of ROS induces oxidization of PTEN and might decrease its lipid phosphatase activity as it has been reported. Inhibition of PTEN is known to increase the level of AKT phosphorylation at Thr308 and Ser473. However, we cannot exclude additional mechanisms leading to enhanced AKT activity in our model. Accordingly, we observed only partial oxidization of PTEN upon ectopic expression of MAP17. These data taken together with our observation of independent behavior of Thr308 and Ser473 phosphorylation mentioned above suggest the implication of decreased specific phosphatase acivities. Dephosphorylation of AKT has been associated with PP2A phosphatase activity. A recent study reports the identification of a specific Ser473 phosphatase (15). We hypothesize that MAP17 mediates the inhibition of a Thr308-specific phosphatase activity. Further experiments are required to identify the putative phosphatase that specifically dephosphorylates Thr308 and shed light on the mechanism by which MAP17 might blocks its activity.
On the other hand, ROS can directly modify signaling proteins through nitrosylation, carbonylation, glutathionylation and formation of disulphide bonds (18). The crystal structure of inactive AKT2 reveals a possible redox-sensitive intramolecular disulfide bond in its activating loop (19) between Lys297 and Cys301 (20). We have found that MAP17-induced ROS maintain AKT phosphorylation at Thr308 and Ser473 in the absence of serum. The maintenance of AKT phosphorylation at Thr308 under our experimental conditions is independent of PI3K activity, suggesting that maintaining AKT Thr308 phosphorylation occurs directly at the AKT level, either through the direct modification of AKT that impairs the Thr308 phosphatase activity or by altering the phosphatase itself. It is possible that the regulation of AKT by ROS occurs through changes in the redox state of critical cysteine residues, most likely at positions 297 and 301, resulting in conformational changes which could then affect Thr308 phosphorylation and therefore AKT activity. This redox-dependent regulation of AKT has been shown for AKT2 by Murata et al. (20). However, whereas in cardiac H9c2 cells treated with lethal doses of H2O2, AKT suffers initial activation followed by degradation, in Rat1A fibroblasts increased levels of ROS maintained by constitutive expression of MAP17 enhance AKT activity.
In summary, MAP17 triggers oxidation of PTEN and AKT activation independent of serum. Antioxidant treatments decrease AKT phosphorylation and restore the level of apoptosis upon serum depletion, indicating the essential role of MAP17-mediated ROS production. Our data support a model in which MAP17 represents a new type of oncogenic protein that acts through intracellular ROS as second messengers. The present work provides evidence for MAP17-mediated alterations of cellular processes that might drive tumorigenesis.
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Funding |
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Spanish Ministry of Health (FIS-02/0126); Fundación Mutua Madrileña; the Spanish Ministry of Education and Science (SAF2005-00944).
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Acknowledgments |
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The authors acknowledge the other members of the Assay Development Group at Centro Nacional de Investigaciones Oncológicas for helpful discussions and critical reading of the manuscript.
Conflicts of Interest Statement: None declared.
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