Akt enhances Mdm2-mediated ubiquitination and degradation of p53

p53 plays a key role in DNA damage-induced apoptosis. Recent studies have reported that the phosphatidylinositol 3-OH-kinase-Akt pathway inhibits p53-mediated transcription and apoptosis, although the underlying mechanisms have yet to be determined. Mdm2, a ubiquitin ligase for p53, plays a central role in regulation of the stability of p53 and serves as a good substrate for Akt. In this study, we find that expression of Akt reduces the protein levels of p53, at least in part by enhancing the degradation of p53. Both Akt expression and serum treatment induced phosphorylation of Mdm2 at Ser186. Akt-mediated phosphorylation of Mdm2 at Ser186 had little effect on the subcellular localization of Mdm2. However, both Akt expression and serum treatment increased Mdm2 ubiquitination of p53. The serum-induced increase in p53 ubiquitination was blocked by LY294002, a phosphatidylinositol 3-OH-kinase inhibitor. Moreover, when Ser186 was replaced by Ala, Mdm2 became resistant to Akt enhancement of p53 ubiquitination and degradation. Collectively, these results suggest that Akt enhances the ubiquitination-promoting function of Mdm2 by phosphorylation of Ser186, which results in reduction of p53 protein. This study may shed light on the mechanisms by which Akt promotes survival, proliferation, and tumorigenesis.

However, this activation of p53 by mitogenic signals must be suppressed during normal cell proliferation, to prevent p53 from inducing cell cycle arrest or apoptosis.
Therefore it appears reasonable to assume that mitogenic signals elicit both p53activating and -inactivating signals.
Recent studies have indeed shown that Ras can inhibit or activate p53, depending on the cellular contexts and the duration of Ras activation (24,25). The Raf-MEK-MAPK pathway has been shown to mediate Ras activation of p53 (26), most likely through induction of p19 ARF , which in turn inactivates Mdm2. The PI3K-Akt pathway has recently been reported to inhibit the transcriptional activity of p53 and reduce the pro-apoptotic functions of p53 (27,28) (Y.O. and Y.G., unpublished data).
Therefore, it is possible that the PI3K-Akt pathway opposes the MAPK pathway in activation of p53. However, it has yet to be determined how Akt suppresses p53.
Here we show that Akt does not affect the mRNA levels of p53, but promotes ubiquitination and degradation of p53 protein. We confirmed very recent studies showing that Mdm2 serves as a good substrate for Akt (29,30). Although they have shown that Akt promotes nuclear translocation of Mdm2, we could not detect any effect of Akt on Mdm2 subcellular localization. Instead, we found that Akt facilitates the functions of Mdm2 to promote p53 ubiquitination by phosphorylation of Ser186.
These findings may explain how mitogenic signal and Ras inhibit p53 during normal cell proliferation, and may also provide a mechanism by which Akt promotes survival. µl of LIPOFECTAMINE Reagent per dish. For 293T cells, transfection was carried out by using FuGENE6 Tranfection Reagent (Roche) in 6 cm dishes (2 x 10 6 cells, 5 µg of total DNA and 12 µl of FuGENE6 Transfection Reagent per dish). For luciferase assay for p53 transcriptional activity, cells were transfected with PG13-Luc together with various constructs and a β-galactosidase-expression plasmid. The β-galactosidase expression was driven by a CMV promoter, and used for a standard to normalize transfection efficiency. Luciferase and β-galactosidase activities were assessed 24 h after transfection.

RT-PCR
Total RNA was isolated from MCF-7 cells using the TRIzol Reagent (Invitrogen) and  All the products were assayed in the linear range of the RT-PCR amplification process.

In vitro kinase assays
Recombinant active Akt and kinase negative Akt were prepared as described previously (32,33). The phosphorylation reaction was also carried out without radiolabeled ATP, and the samples were resolved by SDS-PAGE and subjected to Western blot analysis with antiphospho Ser186 Mdm2 antibody.

Immunostaining
Cells grown on coverslips were fixed for 10 min in PBS containing 3.7% formaldehyde.

Ubiquitination assays
Cells were transfected with HA-p53 and Flag-Ub together with various constructs. Cells were exposed to β-lactone (5 µM) (CALBIOCHEM) for 2 h before the preparation of cell lysates to inhibit proteasome-mediated degradation of ubiquitinated proteins. Cell lysates were immunoprecipitated with anti-HA antibody.
Immunoprecipitates were resolved by SDS-PAGE and transferred to a PVDF membrane.
For detection of p53, the blot was probed with anti-p53 antibody. For detection of ubiquitinated p53, the blot was probed with anti-Flag antibody or anti-p53 antibody. 12

Akt reduces p53 protein by enhanced degradation
Akt has been shown to suppress p53-dependent apoptosis triggered by hypoxia (28), etoposide, γ-irradiation (data not shown) or ectopic expression of p53 ((28) and data not shown). Previous reports have shown that Akt is capable of inhibiting the transcriptional activity of p53 (28,29). We confirmed that expression of active Akt reduced the transcriptional activity of a p53 reporter plasmid in MCF-7 cells (Fig. 1A).
However, the underlying mechanisms of Akt inhibition of p53 remain unclear.
To dissect the mechanisms by which Akt inhibits the transcriptional activity of p53, we first investigated whether Akt expression has any effect on the protein and mRNA levels of p53. To examine this, we transfected MCF-7 cells with Akt constructs (the transfection efficiency was about 70%). The amounts of endogenous p53 protein were markedly reduced by expression of active Akt (Fig. 1B). In contrast, the mRNA levels of p53 detected by RT-PCR were unchanged by expression of active Akt (Fig. 1C). These results indicate that Akt reduces the levels of p53 protein, but not p53 mRNA in MCF-7 cells.
Since it is well established that the level of p53 protein is regulated largely by stability, we then asked whether the stability of p53 was affected by Akt. Flag-tagged p53 was ectopically expressed in MCF-7 cells along with an Akt plasmid. Titration of the amount of co-transfected Akt plasmid showed that increasing amounts of active Akt correlated with decreased levels of p53 protein (Fig. 1D). The stability of p53 protein 13 was then assessed by the addition of cycloheximide, a translational inhibitor. Two hours of cycloheximide treatment decreased p53 protein by 40% in control cells, whereas the same treatment decreased p53 protein by 80% in active Akt-expressing cells ( Fig. 2A), indicating that degradation rate of p53 protein was greater in active Aktexpressing cells. The amounts of p53 protein were then estimated by the use of a densitometry. As shown in Fig. 2B, p53 decayed faster when active Akt was expressed.
The degradation enhanced by Akt was blocked by treatment with MG132, a proteasome inhibitor (data not shown). When MCF-7 cells were treated with LY294002, a PI3K inhibitor, the stability of endogenous p53 protein increased (Fig. 2C). These results suggest that the PI3K-Akt pathway accelerates p53 degradation.

Akt phosphorylates Mdm2 at Ser186
We asked whether Akt might regulate p53 stability by a direct phosphorylation of p53.
We found that immunoprecipitated active Akt was not able to phosphorylate p53 in vitro (data not shown), suggesting an indirect regulation of p53 by Akt. The major way in which p53 is degraded is by Mdm2-mediated ubiquitination. Mdm2 is phosphorylated at multiple sites in vivo (35). Interestingly, analysis of human Mdm2 sequence revealed two sites (Ser166 and Ser186) that conform to the consensus site phosphorylated by Akt (R x R x x S/T), and recent studies have shown that Mdm2 can be phosphorylated by Akt at these sites in vitro and in IGF-1-treated cells (29,30).
The first site (Ser166) is not conserved across species, however the second site (Ser186) is conserved among species as far as we know, suggesting its possible functional 14 importance. We confirmed that active Akt, but not kinase negative Akt, was capable of inducing Mdm2 phosphorylation in vitro (Fig. 3A). To further examine if Akt phosphorylates Mdm2 at Ser186, we generated a polyclonal antibody that specifically recognizes phosphorylated Ser186 of Mdm2. Upon Western blot analysis, this antibody detected Mdm2 that had been phosphorylated by Akt in vitro (Fig. 3B). The specificity of this antibody was confirmed by its failure to recognize the Mdm2 mutant in which Ser186 was mutated into Ala (S186A Mdm2) (Fig. 3B).
By the use of anti-phospho Ser186 Mdm2 antibody, we found that serum stimulation increased Ser186 phosphorylation (Fig. 3C). The increase in Ser186 phosphorylation was blocked by LY294002, suggesting that PI3K is required for serum induction of Ser186 phosphorylation (Fig. 3C). We also found that active Akt expression was sufficient for inducing Ser186 phosphorylation of Mdm2 in vivo. In addition, expression of kinase negative Akt blocked the serum induction of Ser186 phosphorylation (Fig. 3D). These results strongly support that the PI3K-Akt pathway mediates Mdm2 phosphorylation at Ser186 in vivo.

Akt does not affect the subcellular localization of Mdm2
We next asked whether Akt phosphorylation of Mdm2 at Ser186 has any impact on Mdm2. We first examined if Akt regulates the stability of Mdm2 protein. Western blot analysis indicated that expression of active Akt did not affect the levels of ectopically-expressed Mdm2 (Fig. 4A). In addition, the levels of wild type and S186A mutant of Mdm2 were almost the same when expressed in MCF-7 cells (Fig. 4B).

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Therefore, Ser186 phosphorylation does not appear to alter the stability of Mdm2 protein.
Since Ser186 is located close to the nuclear localization sequence and nuclear export signal of Mdm2 (residues 178-185 and 191-199, respectively), we asked whether Akt phosphorylation of Mdm2 alters its subcellular localization. In MCF-7 cells, endogenous Mdm2 was localized mainly in the nucleus and slightly in the cytoplasm (Fig. 5A). In subcellular fractionation experiments, more than 90% of Mdm2 was found in the nuclear fraction (Fig. 5B). Serum and LY294002 treatment did not change the amounts of Mdm2 protein in the nuclear and cytoplasmic fractions (Fig. 5B).
The localization of Mdm2 did not change upon serum or LY294002 treatment in immunostaining experiments, either (Fig. 5A). Expression of active Akt did not induce nuclear translocation of Mdm2 as shown in Figs. 5B, 5C and 5D. Furthermore, we found that expression of a dominant negative Akt (3A Akt) did not change the localization of endogenous Mdm2 in MCF-7 cells (Fig. 5C). Importantly, the localization of S186A Mdm2 as well as S166A/S186A Mdm2 (2SA Mdm2) was mainly in the nucleus, indistinguishable from that of wild type Mdm2 when expressed in Saos-2 cells (Fig. 5D). Therefore we conclude that Akt does not induce nuclear translocation of Mdm2 in our system, in apparent contradiction to the previous reports (29,30) (see Discussion).

Serum facilitates Mdm2-mediated p53 ubiquitination in a PI3K-dependent manner
We then tested the possibility that Ser186 phosphorylation regulates the function of Mdm2. Mdm2 is known to promote p53 degradation by facilitating ubiquitination (19).
Since serum treatment increased Ser186 phosphorylation (Fig. 3C), we examined the ability of Mdm2 to promote ubiquitination of p53 in the presence or absence of serum.
To detect ubiquitination of p53, MCF-7 cells were transfected with Flag-tagged ubiquitin and HA-tagged p53, and treated with a proteasome inhibitor for 2 h. p53 was immunoprecipitated and subjected to Western blot analysis with both anti-Flag antibody and anti-p53 antibody, to visualize the ubiquitination of p53. As shown in Figs. 6A and 6B, serum treatment markedly enhanced the ubiquitination-inducing effect of Mdm2. This enhancement of p53 ubiquitination was reduced by LY294002 treatment (Fig. 6B), suggesting that serum enhancement of p53 ubiquitination is PI3K-dependent.

Akt facilitates p53 ubiquitination
We examined whether Akt is sufficient to enhance p53 ubiquitination. MCF-7 cells were transfected with Akt constructs together with p53. Expression of active Akt enhanced the ubiquitination of p53 (Fig. 6C). These results, taken together, suggest that growth factor-stimulation activates Mdm2-mediated p53 ubiquitination by way of the PI3K-Akt pathway.

Ser186 of Mdm2 is essential for the Akt enhancement of its functions
To further examine if Akt facilitates the ability of Mdm2 to induce p53 ubiquitination, we examined the synergy between Akt and Mdm2, and the possible requirement of Ser186 for this synergy. This experiment was performed under low serum conditions 17 (0.1% serum) to reduce the possible contribution of endogenous Akt activity.
Expression of either active Akt alone or wild type Mdm2 alone induced p53 ubiquitination to some extent, but expression of both active Akt and wild type Mdm2 synergistically increased p53 ubiquitination (Fig. 7). If this synergy is due to direct activation of Mdm2 by Akt, S186A mutation should hamper it. The S186A mutation of Mdm2 almost completely abrogated the ubiquitination promoting activity of Mdm2 enhanced by active Akt (Fig. 7), suggesting that Akt activates Mdm2 by direct phosphorylation. The loss of p53 ubiquitination by S186A mutation was not due to reduction of Mdm2 protein, since the protein levels were almost the same between WT and S186A Mdm2 (Fig. 4B).
We then examined the effects of S186A mutation of Mdm2 on the levels of p53 protein, by expression of Mdm2 and p53 in the absence of proteasome inhibitors.
Expression of active Akt as well as wild type Mdm2 reduced the levels of p53 protein, presumably due to the enhanced degradation of p53 (Fig. 8A, also see Fig. 2).
However, the S186A mutation of Mdm2 abrogated its activity to reduce p53 protein ( Fig. 8A). Consistent with this, the inhibitory effect of Mdm2 on the transcriptional activity of p53 was also reduced when Ser186 was mutated to Ala (Fig. 8B). These results strongly suggest that Akt facilitates the functions of Mdm2 to induce ubiquitination and degradation of p53 in a Ser186-dependent manner.

Discussion
In this study, we investigated the mechanism by which Akt antagonizes p53.
Expression of active Akt reduced the levels of p53 protein, but not p53 mRNA. The reduction of p53 protein by Akt appeared to be due at least in part to the reduced stability of p53 protein, because active Akt was capable of reducing the levels of ectopically-induced p53, and because active Akt increased the degradation rate of p53.
This finding is consistent with the report by Mayo and Donner (29), but is apparently degradation through regulation of other targets, such as p19 ARF , p300 and the proteasome. However, the contribution of these other possible targets may be small, as the S186A mutant Mdm2 appeared to behave as a dominant negative preventing the phosphorylation of Mdm2, and its expression reversed the ability of Akt to promote p53 degradation (see Fig. 8A).
Mdm2 has been shown to shuttle between the nucleus and the cytoplasm by utilizing nuclear export signals (NESs) and nuclear localization sequences (NLSs) (36,37), and the nuclear localization of Mdm2 is prerequisite for the degradation of p53 (37).  (38).
Alternatively, Ser186 phosphorylation may affect the ubiquitin ligase activity of Mdm2, 20 or the affinity of Mdm2 toward other proteins including p19 ARF , which has been reported to sequester Mdm2 from p53 (30,(39)(40)(41). The exact roles of Ser186 phosphorylation of Mdm2 await future investigation.
In our experiments, the S186A mutation abolished Mdm2 induction of p53 ubiquitination, but only partially impaired the Mdm2 reduction of the transcriptional activity of p53. This result suggests that Mdm2 suppresses the transcriptional activity of p53 not just by induction of ubiquitination, but also by other mechanisms, as previously described (42)(43)(44), and that Ser186 phosphorylation regulates the former, but not the latter, functions of Mdm2.
It has been shown that mitogenic signals including Ras activation regulate    antibody.
Essentially the same results were obtained in three independent experiments.

Fig. 3 Akt phosphorylates Mdm2 at Ser186
A. Recombinant Mdm2 protein (1 µg each) was phosphorylated with active (CA) or kinase negative (KN) Akt prepared as described (32,33)    with plasmids encoding Mdm2 (either wild type, S186A or S166A/S186A (2SA)) and Akt (either CA or 3A) for 24 h. Sa0s-2 cells were used because the level of endgenous Mdm2 is negligible. The cells were fixed and stained with anti-Mdm2 antibody (green) and with Hoechst33258 (blue). Essentially the same results were obtained in at least three independent experiments in A-D. indicated. One day after transfection, the cells were serum-starved for 12 h, exposed to β-lactone (5 µM) for 2 h and lysed. p53 was immunoprecipitated with anti-HA antibody, and subjected to Western blot analysis with anti-p53 antibody.