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Originally published In Press as doi:10.1074/jbc.M609712200 on January 2, 2007

J. Biol. Chem., Vol. 282, Issue 8, 5468-5477, February 23, 2007
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Inhibition of B56-containing Protein Phosphatase 2As by the Early Response Gene IEX-1 Leads to Control of Akt Activity*

Géraldine Rocher{ddagger}§||, Claire Letourneux{ddagger}§||, Philippe Lenormand**, and Françoise Porteu{ddagger}§||1

From the {ddagger}Institut Cochin, Department of Hematology, Paris F-75014, France, the §INSERM U567, F-75014 Paris, the CNRS, UMR 8104, Paris F-75014, the ||Université Paris Descartes, Faculté de Médecine René Descartes, UMR-S 8104, F-75014 Paris, and the **Institute of Signaling Developmental Biology and Cancer, Centre A. Lacassagne, CNRS UMR 6543, 06189 Nice, France

Received for publication, October 16, 2006 , and in revised form, December 22, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The importance of PP2A in the regulation of Akt/PKB activity has long been recognized but the nature of the holoenzyme involved and the mechanisms controlling dephosphorylation are not yet known. We identified IEX-1, an early gene product with proliferative and survival activities, as a specific inhibitor of B56 regulatory subunit-containing PP2A. IEX-1 inhibits B56-PP2A activity by allowing the phosphorylation of B56 by ERK. This leads to sustained ERK activation. IEX-1 has no effect on PP2A containing other B family subunits. Thus, studying IEX-1 contribution to signaling should help the discovery of new pathways controlled by B56-PP2A. By using overexpression and RNA interference, we show here that IEX-1 increases Akt/PKB activity in response to various growth factors by preventing Akt dephosphorylation on both Thr308 and Ser473 residues. PP2A-B56beta and {gamma} subunits have the opposite effect and reverse IEX-1-mediated Akt activation. The effect of IEX-1 on Akt is ERK-dependent. Indeed: (i) a IEX-1 mutant deficient in ERK binding had no effect on Akt; (ii) ERK dominant-negative mutants reduced IEX-1-mediated increase in pAkt; (iii) a B56beta mutant that cannot be phosphorylated in the ERK·IEX-1 complex showed an enhanced ability to compete with IEX-1. These results identify B56-containing PP2A holoenzymes as Akt phosphatases. They suggest that IEX-1 behaves as a general inhibitor of B56 activity, enabling the control of both ERK and Akt signaling downstream of ERK.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Protein phosphorylation is central to signal transduction by growth factors and the maintenance of cellular homeostasis by controlling many cellular processes such as growth, survival, and differentiation. Deregulation of this activity is a key cause of cancer development. The net activation of a signaling pathway depends on the balance of enzymatic activities between kinases and phosphatases, suggesting that both activation and inactivation signals must be tightly controlled.

Protein phosphatase 2A (PP2A)2 accounts for the majority of serine/threonine phosphatase activity in the cell (reviewed in Refs. 1 and 2)). The predominant form of PP2A is a heterotrimeric holoenzyme, composed of a scaffolding subunit (A), a catalytic subunit (C), and a variable regulatory subunit. The B subunits fall into three major unrelated families, B (PR55), B' (B56/PR61), and B'' (PR72). Each family comprises several isoforms that exhibit significant homology between them. With five different genes, generating at least 8 isoforms named B56 {alpha}, beta, {gamma}, {delta}, and {epsilon}, the B56 family is the most diverse. Many if not most of the components of signaling cascades are PP2A substrates. However, the function of the individual PP2A subunits in these pathways is only beginning to emerge. Interestingly, recent studies indicate that different PP2A holoenzymes regulate positively or negatively signaling by acting at different levels in a cascade. The best example of this type of regulation is the ERK/MAPK signaling pathway. Indeed, Adams et al. (3) showed that PP2As containing B{alpha} and B{delta} are required for activation of the MAPK kinase kinase Raf-1, whereas we demonstrated that the B56 family members shut down the signal by dephosphorylating ERK1 and ERK2 (4). Likewise, the Wnt pathway has been shown to be differently regulated by B56-containing PP2As, which binds to axin and APC (5) and also by PP2As containing the B'' subunit acting at the level of Naked cuticle (6). Unraveling the specific contribution of the various PP2A holoenzymes to signaling pathways, as well as the mechanisms regulating their function, is an important step to understand how kinase cascades are regulated and to find new tools to modulate their activities.

The kinase Akt/PKB, a critical component of a pathway controlling growth and survival, is one of the major targets of PP2A enzymes. Akt exerts its function by phosphorylating several proteins involved in cell cycle regulation and apoptosis, including p21Cip/WAF1, BAD, the Forkhead family of transcription factors and glycogen synthase kinase-3 (GSK3) (reviewed in Ref. 7). Activation of Akt occurs upon recruitment to the plasma membrane through binding of its plekstrin homology domain to phosphatidylinositol 3,4,5-triphosphates, which are produced by growth factor-activated phosphoinositide 3-kinase (PI3K). This allows phosphorylation of Akt at two key regulatory residues: Thr308 in the activation loop and Ser473 in a C-terminal hydrophobic motif. Phosphorylation of Thr308 is catalyzed by PDK1 (8, 9); that of Ser473 is mediated by the mTORC2 complex (10). Turn-off of Akt activity can be mediated by the tumor suppressor phosphatase tensin homologue, which dephosphorylates phosphatidylinositol 3,4,5-triphosphates. Although the importance of PP2A in the regulation of Akt activity has long been recognized (11–15), the nature of the PP2A holoenzyme involved and the mechanisms controlling these dephosphorylation events have not yet been elucidated.

IEX-1 (immediate early response gene X-1), also known as IER3, DIF2, or Gly96, is an ubiquitous early response gene product involved in cell proliferation and survival, which is rapidly induced by various growth factors, cytokines, chemical carcinogens, or viral infections (16). Conflicting data have been reported concerning the role of IEX-1 in apoptosis. Indeed, IEX-1 was found to increase apoptosis in response to UV radiation or upon serum deprivation (17, 18) but it also plays a key role in cellular resistance to various apoptotic triggers and contributes to growth factor-mediated survival activities (19–23). These differential effects might be due to the fact that IEX-1 acquires its pro-survival function upon phosphorylation by ERK (19, 23). In addition to this function, we have demonstrated that IEX-1 behaves as an inhibitor of B56-containing PP2A enzymes (4). IEX-1 binds to both B56 family members and to active ERKs, enhances the phosphorylation of B56 by ERK in this complex and triggers the dissociation of the A/C core from the B56 subunit. Inhibition of PP2A-B56 activity by IEX-1 was found to increase ERK signaling in response to various growth factors (4). In the hematopoietic cell line UT7, the specific induction of the IEX-1 protein by thrombopoietin (TPO), correlates with the unique capacity of this cytokine to sustain the ERK signal and is required for this effect (4, 19, 24). IEX-1 has no effect on other B family of PP2As. Thus, studying IEX-1 contribution to signaling cascades should lead to the discovery of new pathways controlled by B56-containing PP2As. The functions of IEX-1 and its induction by various growth factors prompted us to analyze whether it can affect Akt activation. We show here that IEX-1 and PP2A-B56 regulate Akt activity in an opposite manner by controlling the phosphorylation state of both Thr308 and Ser473. These data add to our understanding of both PP2A and Akt regulatory pathways.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids—Plasmids encoding IEX-1, WT, or deleted of its ERK binding site (IEX-1-{Delta}BD), were described previously (19). IEX-1 was inserted downstream of HA or His tags. pcDNA expression vectors for HA-B55{alpha} and 4xHA-B56beta were generously provided by Dr. D. Virshup (University of Utah). pcDNA encoding HA-tagged B56{gamma} was given by Dr. N. Nojima (Osaka university). B56beta-S368A and B56{gamma}1-S327A were previously characterized (4) and were generated by the QuikChange site-directed mutagenesis kit (Stratagene). The insert was cloned downstream of HA4. Mouse ERK1 cDNA was cloned from reverse transcription of NIH3T3 mRNA. Mouse cDNA for ERK2 was a gift from Dr. M. Weber (University of Virginia Cancer Center). ERK1 and ERK2 dominant negative mutants were generated by mutating their Mg2+ binding pockets (consensus DFG in all kinases). The coding sequences were inserted into pcDNA3 to create plasmids pcDNA3-ERK1-D185A and pcDNA-ERK2-D165A. pcDNA-HA-Elk1 was a gift of R. Hipskind (Montpellier, France). For RNA interference, the target nucleotide sequences and the vectors that drive the expression of small hairpin shRNA for human IEX-1, B56beta, or B56{gamma} were described previously (4, 25, 26). Two shRNA targeting different nucleotides of human B56beta sequence were used: pSM2c-shB56beta-1 and pSM2c-shB56beta-2 (Open Biosystems); pMKO-1-shB56{gamma} was a gift of Dr. W. Hahn (Dana Farber Cancer Institute, Boston). The controls used were either sh0 (nucleotides 398–418 in the IEX-1 sequence), which was found to be ineffective at down-regulating IEX-1 expression, or shGFP (4). The cassette encoding the H1 promoter-shIEX-1 or shRNA-control was introduced in a self-inactivating lentiviral/human immunodeficiency virus vector (TRIP{Delta}U3). This vector also expresses GFP under the EF1{alpha} promoter. Production and titration of infectious particles were done as described (24).

Cell Cultures—Chinese hamster ovary cells expressing the erythropoietin (EPO) receptor (CHO-ER) cells were described previously (19). They were cultured in Dulbecco's minimal essential medium nut Mix F-12 (Ham's) medium containing 7% fetal calf serum (FCS). HEK-293 and HeLa cells were maintained in Dulbecco's minimum essential medium supplemented with 10% FCS. UT7 cells (27) were grown in {alpha}-minimal essential medium supplemented with 10% FCS and 1 unit/ml EPO (Roche Molecular Biochemicals).

Transfections and Viral Infections—CHO-ER and HeLa cells (60–80% confluence) were transiently transfected with 1–2 µg of DNA, using the Lipofectamine Plus reagent (Invitrogen), according to the manufacturer's instructions. Stable clones of HeLa cells expressing the shRNAs targeting B56{gamma} and B56beta were previously described (4). In some experiments, the vectors carrying shRNA were transiently introduced in HeLa cells and the cells were harvested 48 h later. HEK-293 and UT7 cells were incubated with TRIP{Delta}U3-H1-shIEX-1 or control lentiviruses over a period of 36 h. The transduction efficiency, determined by the percentage of GFP positive cells by fluorescence-activated cell sorter analysis, usually reached 98%.

Western Blotting, Immunoprecipitation, and Kinase Assays— Akt activation was assessed in total lysates by Western blotting with anti-pAkt-Ser473 or pAkt-Thr308 antibodies (Cell Signaling). To follow the kinetics of Akt inactivation, 24 h post-transfection the cells were either treated with the PI3K inhibitor LY294002 or washed 3 times in phosphate-buffered saline and loaded with serum-free medium. The cells were returned to 37 °C and harvested at various times. In some experiments, the cells were deprived of serum overnight, and stimulated with EPO or FCS for various times before lysis. For immunoprecipitation experiments, the cells were lysed in 50 mM Tris, pH 7.5, buffer containing 137 mM NaCl, 0.5% Nonidet P-40, 10% glycerol, 1 mM EDTA, 1 mM orthovanadate, 20 mM beta-glycerophosphate, 20 mM NaF, 1 mM sodium pyrophosphate, and a protease inhibitor mixture (Roche). Gel electrophoresis, immunoblotting, and immunoprecipitation were carried out as previously described (4). In vitro Akt kinase activity was assessed with the non-radioactive Akt kinase kit (Cell Signaling). The other antibodies used in this study were: anti-phospho: pERK, pGSK3, pFoxo3a, pan-Akt substrate, and pElk1 (Cell Signaling); anti-Akt (sc-9312), anti-ERK1 (sc-94), anti-ERK2 (sc-154), and anti-B56beta from Santa Cruz Biotechnology; anti-HA (Roche). Anti-IEX-1 antiserum was as previously described (19). Rabbit anti-B56{gamma} was a gift from Dr. M. Mumby (University of Texas).


Figure 1
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  		FIGURE 1.
IEX-1 potentiates Akt activity. A–C and E, CHO-EpoR cells were transfected with empty vector or IEX-1. The cells were grown in FCS and either directly harvested 24 h post-transfection (A) or starved of serum for 3 h prior to stimulation with EPO, as indicated. D, stable clones of UT7 cells expressing HA-IEX-1 or empty pcDNA (Neo) were stimulated for various times with 1 nM TPO. The activity of endogenous Akt was analyzed by Western blotting of total lysates with anti-pAkt-Ser473 and pAkt-Thr308 (A–D) or in Akt immunoprecipitates by in vitro kinase assay on its substrate GSK3 (E). Quantification are shown normalized to total Akt levels and expressed relative to the levels of phosphorylation at the zero time point of cells not expressing IEX-1.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
IEX-1 Increases Akt Activation—IEX-1 can be induced by serum and various growth factors (4, 16, 19, 24, 28). To determine whether IEX-1 could affect other growth factor-induced signaling pathways in addition to ERK, we measured Akt activity in cells expressing IEX-1. Maximal Akt activation requires phosphorylation at the two activating residues Ser473 and Thr308 (8–10, 29). Thus, CHO cells were transiently transfected with HA-IEX-1 or empty vector and the cells were maintained in serum to activate Akt. Endogenous Akt activation was assessed both by Western blotting with Ser473 and Thr308 phosphospecific Akt antibodies and by immunoprecipitation kinase assays. As shown in Fig. 1A, Akt phosphorylation was greatly enhanced at both Ser473 and Thr308 in cells expressing IEX-1. To determine whether IEX-1 could affect Akt activation in response to growth factors, CHO cells expressing the EPO receptor (CHO-ER, (19)) were transfected with IEX-1 and analyzed after 3 h starvation in a serum-free medium and stimulation with EPO, under conditions leading to increased ERK activation in the presence of IEX-1 (Ref. 19 and Fig. 1, B and C). No phosphorylation of Akt could be detected in non-stimulated starved cells whether they expressed IEX-1 or not. However, IEX-1 expression resulted in a great increase in EPO-induced Akt phosphorylation on both Ser473 and Thr308, at all time points and doses tested (Fig. 1, B and C). Endogenous Akt phosphorylation was also enhanced and prolonged in UT7 cells stably transfected with IEX-1, as compared with control cells (neo), upon stimulation with TPO (Fig. 1D).

To examine IEX-1-dependent Akt activation further, we measured Akt kinase activity, by using an in vitro kinase assay on its substrate GSK3. Fig. 1E shows that the Akt kinase activity that could be immunoprecipitated from CHO cells expressing IEX-1 was increased, as compared with cells transfected with an empty vector. An increased and sustained in vivo GSK3 phosphorylation that paralleled pAkt levels was also detected in IEX-1-overexpressing cells (Fig. 1B). In addition, the phosphorylation levels of several Akt substrates, detected using an anti-pan phospho-Akt substrate antibody, were greatly increased in the presence of IEX-1 (Fig. 1B). Thus, expression of IEX-1 increases Akt activity and Akt downstream signaling.


Figure 2
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  		FIGURE 2.
Endogenous IEX-1 expression is involved in sustained Akt activation induced by various growth factors. A, UT7 cells were starved overnight and stimulated for various times with either EPO (10 units/ml) or TPO (3 nM). IEX-1 expression and phosphorylation of Akt and its downstream substrates were analyzed by Western blotting with the indicated antibodies. B, UT7 cells were infected with lentiviral vectors encoding IEX-1 shRNA (shIEX-1) or control shRNA (shGFP and sh0). The effect of shRNA on endogenous IEX-1 expression was assessed on maximally induced IEX-1 after stimulation with 10 nM TPO in the presence of the proteasome inhibitor N-acetyl-L-leucinyl-L-leucinol-L-norleucinol for 2 h (left panel). Akt phosphorylation was analyzed upon stimulation with TPO alone for various times (right panel). C, HEK-293 cells expressing shGFP or shIEX-1 constructs were starved of serum overnight before being stimulated for various times with 2% serum. pAkt levels were quantified and normalized to total Akt levels and expressed relative to the levels of phosphorylation at the zero time point of control cells.

 
IEX-1 is an early response gene product that is induced by various growth factors. Thus Akt activity should be more sustained in response to growth factors triggering IEX-1 expression. To test this, we made use of the UT7 cell line. Indeed, whereas both EPO and TPO stimulate Akt activation in this cell line, only TPO can induce IEX-1 protein expression (Ref. 24 and Fig. 2A). As shown in Fig. 2A, Akt phosphorylation on both Ser473 and Thr308 was much higher and more sustained in cells stimulated with optimal doses of TPO, as compared with EPO. This increase in Akt phosphorylation translated into an increased Akt activity, as shown by the higher levels of phosphorylation of the Akt substrates GSK3 and Foxo3a observed upon TPO stimulation. Likewise, Western blotting with a pan-Akt substrate phospho-antibody shows that in TPO-treated, IEX-1 expressing cells, the global Akt-mediated phosphorylation is greatly increased, as compared with cells treated with EPO. Thus, the specific induction of the IEX-1 protein by TPO in UT7 cells correlates with the unique capacity of this cytokine to sustain Akt phosphorylation and Akt downstream signaling.

To further confirm the above results, we examined Akt activation in cells in which TPO-induced IEX-1 expression was impeded by the presence of IEX-1-specific shRNA. Thus, we generated UT7 cell lines expressing shRNA for IEX-1. Expression of endogenous IEX-1 upon TPO stimulation was severely inhibited in the presence of this shRNA (Fig. 2B, left panel). As a control, we used cell lines expressing either a shRNA designed from the IEX-1 nucleotide sequence (sh0, see "Materials and Methods") but that was found unable to down-regulate endogenous IEX-1 levels (Fig. 2B), or a shRNA targeting GFP. As shown in Fig. 2B (right panel), TPO-mediated phosphorylation of both Akt and its downstream substrate GSK3 was greatly reduced in shRNA-IEX-1 expressing cells, as compared with control cells. This effect was particularly striking after 1.5 and 3 h TPO treatments, time points at which IEX-1 is maximally induced by the cytokine. The decreased Akt activation upon down-regulation of IEX-1 expression is not specific to TPO signaling or to the particular cell context of UT7 cells, as shown by the impaired serum-induced Akt phosphorylation at both Ser473 and Thr308 in HEK293 expressing IEX-1-shRNA (Fig. 2C). In these cells, basal expression of IEX-1 was observed but its level increased following serum stimulation. These results show that the induction of IEX-1 by various growth factors plays an important role in their capacity to control Akt activity.

IEX-1 Prevents Akt Dephosphorylation—We next determined by which mechanism IEX-1 increased Akt activity. The marked ability of IEX-1 to sustain Akt signal at long stimulation time points (Fig. 1) suggested that IEX-1 may prevent Akt inactivation rather than activate upstream kinases. To test this possibility, CHO-ER cells, transfected or not with IEX-1, were first subjected to a growth factor pulse with an optimal dose of Epo (10 units/ml) to activate Akt before suppression of upstream activating signals by adding the PI3K inhibitor LY294002. pAkt levels were measured at different time points after inhibition. In agreement with previous reports (30, 31), blocking PI3K activity after growth factor exposure led to a rapid decrease in Akt phosphorylation, as shown by the rapid drop of reactivity with both anti-pAkt-Ser473 and anti-pAkt-Thr308 5 min after addition of the inhibitor. This shows that Akt is rapidly dephosphorylated at its two activating residues by endogenous active phosphatases. By contrast, in cells expressing IEX-1, although the basal Akt activation after optimal EPO stimulation was unchanged, the rate of disappearance of pAkt upon addition of LY294002 was greatly delayed (Fig. 3A). This suggests that IEX-1 reduces the rate of Akt dephosphorylation rather than increases its activation.


Figure 3
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  		FIGURE 3.
IEX-1 inhibits Akt dephosphorylation at both Ser473 and Thr308. CHO-ER cells were transfected with either empty or HA-IEX-1 encoding plasmids. 24 h post-transfection, the cells were either stimulated for 7 min with 10 units/ml EPO prior to addition of 30 µM LY294002 (A) or with 10% FCS for 10 min and then starved for various times in serum-free medium (B). Akt phosphorylation and IEX-1 expression were analyzed by Western blotting of total lysates. Quantification of pAkt levels in control or IEX-1 expressing cells, normalized to total ERK (A) or Akt (B) levels, is expressed relative to the levels of phosphorylation at their zero time point.

 
To confirm these results, CHO cells were stimulated with FCS and Akt phosphorylation was measured after various times following arrest of Akt activation induced by FCS starvation. As above with the addition of LY294002, removal of the stimulus resulted in a rapid decrease in Akt phosphorylation (Fig. 3B). IEX-1 expression greatly delayed this response, as shown by the presence of pAkt up to 1 h following stimulus removal. Quantification experiments showed that the half-life for the pAkt-Ser473 signal increased from 12 ± 4 min in control cells to 59 ± 15 min (mean ± S.E., n = 7) in IEX-1-expressing cells. That of pAkt-Thr308 varied from 16 ± 3 to 60 ± 11 min (n = 4) in control and IEX-1-transfected cells, respectively. Thus, altogether, these results indicate that IEX-1 increases Akt activity by reducing its rate of dephosphorylation on both Ser473 and Thr308.


Figure 4
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  		FIGURE 4.
B56 subunits of PP2A reverse IEX-1 effect on Akt activation. CHO-ER cells were transfected with 0.5 µg of IEX-1 together with 0.5 µgof either empty vector (V) or vector encoding various PP2A B-regulatory subunits: 4xHA-B56beta or HA-B{alpha} (A); HA-B56{gamma}1(B); and FLAG-Bbeta (C). 24 h following transfection, the cells were stimulated with 10 units/ml EPO for 5 min. Akt phosphorylation at Ser473 and Thr308 was determined after various times (A) or 30 min (B and C) of growth factor starvation. pAkt levels were determined as described in the legend to Fig. 3.

 
IEX-1 Inhibits Akt Dephosphorylation by PP2As Containing the B56 Regulatory B Subunits—PP2A is the main phosphatase known to act on both Ser473 and Thr308 (11–15). We previously found that IEX-1 increases ERK activation by preventing its dephosphorylation specifically by PP2A holoenzymes containing the B56 family of B regulatory subunits (4). The kinetics of Akt phosphorylation and dephosphorylation upon growth factor removal paralleled that of ERK (Figs. 1, B and C, and 3B). These similar regulations of Akt and ERK activities by IEX-1 prompted us to examine whether the same mechanism operates on the two pathways.

To test the possibility that IEX-1-mediated inhibition of B56-containing PP2As is responsible for its ability to increase Akt activity, IEX-1 was transfected in CHO cells in the presence or absence of various B56 or B regulatory subunits and the decay of Akt phosphorylation was followed after serum starvation. As shown in Fig. 4A, expression of B56beta strongly reduced the capacity of IEX-1 to prolong Akt phosphorylation on both Ser473 and Thr308. A similar effect was observed with B56{gamma}1 (Fig. 4B). In contrast, neither B family member B{alpha} (Fig. 4A) nor Bbeta (Fig. 4C) could compete with IEX-1 to induce extension of Akt phosphorylation. This suggests that the ability of IEX-1 to delay Akt dephosphorylation on both Thr308 and Ser473 is related to its capacity to inhibit specifically B56- but not B-containing PP2A phosphatases.


Figure 5
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  		FIGURE 5.
B56-containing PP2As dephosphorylate Akt. A and B, the B56- but not the B-family of PP2A dephosphorylates Akt. CHO cells were transfected with 1 µg of 4xHA-B56beta (A) or HA-B{alpha} (B), stimulated with EPO for 5 min and then washed. Akt phosphorylation was determined at the indicated times after starvation. C and D, down-regulation of B56 subunits leads to increased Akt phosphorylation. C, specificity of the various shRNA. CHO cells (left panel) or HeLa cells (right panel) were transfected with vector expressing shRNA for B56beta (shB56beta-1 and/or shB56beta-2) or B56{gamma}, alone or together with either HA-B56beta or HA-B56{gamma}. The expression of the B regulatory subunits was assessed by Western blotting with anti-HA or anti-pan-B56{gamma} or anti-B56beta antibodies, as indicated. D, increased Akt phosphorylation in shB56-expressing cells. Left panel, HeLa cells were transfected with vectors expressing B56beta, B56{gamma}, or control (GFP) shRNAs and Akt phosphorylation was analyzed after 48 h of culture in the presence of serum. Right panel, HeLa cells expressing the indicated shRNA were starved overnight, stimulated with serum for 5 min, and starved again. Akt phosphorylation was determined at various times after serum removal. Quantification of pAkt is expressed relative to the levels of phosphorylation at their zero time point after normalization to Akt levels.

 
To confirm these data, we next wished to examine whether Akt phosphorylation can indeed be regulated by the family of B56-containing PP2As. To study the effect of B subunits on Akt dephosphorylation specifically without interference with a possible effect on its upstream activation, CHO cells transfected with either B or B56 subunits were pulse-stimulated in the presence of FCS or EPO as above and the phosphorylation of Akt was measured at various time intervals following growth factor starvation. Fig. 5A shows that expression of the B56beta subunit accelerated the disappearance of both Akt-Ser473 and -Thr308 phosphorylated forms, after activation stopped. Given that the transfection efficiency in CHO cells reaches about 60%, these data reveal that Akt phosphorylation was almost abolished in cells expressing the B56 subunits. In contrast, as above with IEX-1, Akt phosphorylation was not reduced in the presence of B{alpha} (Fig. 5B).

Conversely, we used RNA interference to down-regulate the expression of B56 members. shRNA targeting specifically either B56beta or B56{gamma} were chosen according to described sequences previously validated by us and/or others (4, 25, 26). Their ability to affect in a specific manner the expression of their target, either B56beta or {gamma}, without affecting other B56 members and to down-regulate the corresponding endogenous B56 species is illustrated in Fig. 5C. Expression of either shB56beta or shB56{gamma} in HeLa cells resulted in a strong increase in the levels of Akt phosphorylation observed 48 h after transfection in cells grown in serum, as compared with control cells transfected with shGFP (Fig. 5D, left panel). To test the effect of down-regulating B56 expression directly on Akt dephosphorylation, HeLa cells stably expressing shB56{gamma} or shB56beta (4) were starved overnight, stimulated with FCS for 5 min to activate Akt, and then starved again for various times. As shown in Fig. 5D (right panel), maximal FCS-induced Akt phosphorylation was rather similar in control and shRNA-expressing cells. However, knock-down of either B56beta or B56{gamma} reduced the rate of disappearance of pAkt after serum starvation. Similar results where observed in HeLa cells stably expressing another shB56{gamma} RNA target sequence (Ref. 25, and data not shown). These results show that down-regulation of either endogenous B56{gamma} or B56beta proteins is sufficient to slow down significantly Akt dephosphorylation. They support the involvement of B56-containing PP2As in the regulation of Akt activity.


Figure 6
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  		FIGURE 6.
The inhibitory effect of IEX-1 on PP2A-mediated dephosphorylation of Akt is ERK dependent. A, IEX-1 does not interact with Akt. CHO cells were transfected with His-IEX-1 and the presence of Akt and ERK was analyzed in anti-His immunoprecipitates. B, the ability of IEX-1 to increase Akt activity requires the ERK binding motif on IEX-1. CHO-ER cells were transfected with pcDNA or vectors encoding HA-IEX-1, WT, or {Delta}BD. After EPO stimulation (5 min at 37 °C), the cells were deprived of growth factors and analyzed for Akt phosphorylation at various times. C, the ability of IEX-1 to increase Akt activity requires ERK kinase activity. CHO cells were transfected with a mixture of ERK1 and ERK2 (0.25 µg each), either WT or kinase-dead (ERK-AFG), along with empty pcDNA, and 0.5 µg of HA-Elk1 (left panel) or IEX-1 (right panel). The phosphorylation of Elk1 or endogenous Akt was assessed 24 h after transfection in cells grown in serum. D, the ability of IEX-1 to increase Akt activity involves ERK-induced B56 phosphorylation. CHO cells were transfected with 0.5 µg of IEX-1 together with 0.5 µg of the indicated B56beta forms and assayed for Akt phosphorylation as above. Quantifications are shown after normalization to the total level of Akt.

 
Inhibition of PP2A-induced Akt Dephosphorylation by IEX-1 Is Dependent on ERK—To explore the mechanism by which IEX-1 prevents the activity of B56-containing PP2As on pAkt, we first examined whether IEX-1 could interact with Akt. Thus, IEX-1 was immunoprecipitated from cells co-transfected with His-tagged IEX-1 and HA-Akt. In agreement with our previous data (19), anti-His antibodies could co-precipitate His-IEX-1 and endogenous active pERK from CHO lysates. In contrast, no HA-Akt, or its phosphorylated species, could be detected in these precipitates (Fig. 6A). Likewise, IEX-1 was not found in anti-Akt precipitates (data not shown). These results suggest that IEX-1 most probably does not act by competing with PP2A for Akt binding.

We previously demonstrated that IEX-1 inhibits B56-containing PP2A activity by a mechanism involving ERK-mediated phosphorylation of the B56 subunits (4). Therefore, if the inhibition of Akt dephosphorylation involves the same mechanism, one would expect this effect to be dependent on ERK activity. To test this possibility we first examined the effect of an IEX-1 protein mutated in its ERK binding site ({Delta}BD-IEX-1). This mutant has completely lost the ability to bind pERK and to inhibit B56-PP2A activity (4, 19). As shown in Fig. 6B, by contrast with the WT protein, {Delta}BD-IEX-1 could not enhance Akt activation or extend the duration of its phosphorylation upon starvation. This shows that IEX-1-mediated Akt activation is dependent on its capacity to interact with ERK.

To confirm these results, we expressed IEX-1 together with ERK1 and ERK2 kinase-dead proteins or their wild-type counter-parts as a control. ERK1/2-AFG forms were created by mutation of the Mg2+ binding site. This mutation destroys the kinase activity of ERKs but preserves their phosphorylation by MEK within the activation loop, so that they behave as dominant negative mutants on the activity of endogenous ERKs. Indeed, as shown in Fig. 6C (left panel), expression of ERK1-AFG and ERK2-AFG led to a decreased phosphorylation of the ERK substrate Elk1, as shown both by Western blotting with anti-phospho-specific Elk1 antibodies and the disappearance of the upper Elk1-shifted band. Expression of dominant negative ERK proteins had no effect on the basal Akt phosphorylation obtained in the absence of IEX-1. This shows that Akt activation is independent of ERK activity. However, it reduced the capacity of IEX-1 to enhance Akt phosphorylation at both Thr308 and Ser473 residues (Fig. 6C, right panel). In four independent experiments, the levels of pAkt-Ser473 reached in IEX-1 expressing cells were decreased by 53.5 ± 7.5%, upon transfection with ERK-AFG, as compared with ERK-WT. These results indicate that the capacity of IEX-1 to protect Akt phosphorylation requires ERK kinase activity.

In the B56·IEX-1·pERK complex, B56 subunits are phosphorylated by ERK at a Ser/Pro consensus ERK phosphorylation site that is conserved in all B56 members (Ser368 in B56beta). Mutation of the serine to alanine at this site leads to a B56 species whose activity is resistant toward inhibition by IEX-1 (4). As shown in Fig. 6D, at a dose where the WT-B56beta protein could only reverse partially IEX-1-mediated increase in Akt phosphorylation, the B56beta-S368A completely abolished this effect. Altogether these results indicate that IEX-1-mediated ERK-dependent inhibition of B56-containing PP2As is responsible for its ability to control both ERK and Akt activation.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Given the importance of the Akt signaling pathway on many cellular functions and its frequent deregulation in cancer, considerable efforts have been made in the past few years to identify new regulators of its activity. Nonetheless, in contrast to the flood of information regarding the activation mechanism of Akt downstream of PI3K, we have a more limited understanding about how Akt activity is turned off. In this work we identify two new actors that influence the duration of Akt activity by regulating directly its dephosphorylation in an opposite fashion: (i) the PP2As containing B56-regulatory subunits that rapidly and specifically dephosphorylate Akt; and (ii) the early gene IEX-1, which prolongs its activity by preventing this step. The importance of this regulatory pathway to modulate the amplitude and duration of Akt signaling is illustrated by the reduced or increased phosphorylation of Akt and of its downstream substrates occurring upon down-regulation of IEX-1 or B56 subunits expression, respectively.

Several proteins that regulate positively Akt activity have been described. JIP1 (30) and Ft1 (32) tether Akt to PDK1 and increase its phosphorylation, whereas TCL1, by binding to the Akt plekstrin homology domain, augments directly Akt kinase activity and transport without affecting its phosphorylation (33). In contrast to these mechanisms, we found that IEX-1 acts by preventing Akt dephosphorylation on its activating residues. IEX-1 could prolong Akt phosphorylation on both Thr308 and Ser473 after a short pulse stimulation by growth factors; it reduced by 4–5-fold the rate of decay of Akt-phosphorylated forms induced upon inhibition of upstream kinases by serum starvation or addition of LY294002. This effect of IEX-1 on Akt is similar to that found on ERK (4, 19) suggesting that it may be related to its capacity to inhibit the activity of the B56 family of PP2As. Indeed, expression or down-regulation of B56beta or {gamma} subunits regulated the decay of Akt phosphorylation in a fashion opposite to that of IEX-1. Both subunits could also reverse the IEX-1 effect on Akt. Just as IEX-1, Hsp90 prevents Akt dephosphorylation by PP2A (13). However, whereas Hsp90 binds directly to Akt and prevents its access to the phosphatase, we could not detect Akt in anti-IEX-1 immunoprecipitates, indicating that the effect of IEX-1 on Akt is indirect and relies only on its ability to inhibit the phosphatase activity of B56-containing PP2As. These results indicate that the action of IEX-1 on PP2A is not limited to steric interference that would occur only within the B56·IEX-1·pERK complex. They show that IEX-1 is a more general inhibitor of PP2A-B56 activity than expected. The indirect effect of IEX-1 protecting both ERK and Akt from phosphatase action is reminiscent of that recently described for caveolin-1, a protein that inhibits both PP1 and PP2A through scaffolding interactions (31). Whether these different Akt regulatory mechanisms coexist in a given cellular context remains to be determined.

One of the important results of this study is the identification of PP2As containing B56-regulatory subunits as the holoenzymes controlling Akt dephosphorylation. Indeed, in different cell types, overexpression of B56beta and {gamma}-family members accelerated the dephosphorylation of pAkt, whereas their down-regulation by shRNA increased Akt phosphorylation in serum-cultured cells and delayed Akt dephosphorylation upon starvation. By contrast, expression of the B-family members B{alpha}, Bbeta (Fig. 4B), or B{gamma} (data not shown) had no effect. The ability of IEX-1, which was found to bind to and inhibit specifically B56- but not B-containing PP2As, to increase the half-life of Akt phosphorylation further argues in favor of a role of B56 but not of B holoenzymes in Akt inactivation. Previous studies have shown that PDK1, the upstream Akt Thr308 kinase, may also be a substrate of PP2A (15, 31). We have not tested directly the effect of B56-containing PP2As on PDK1 activity. However, the ability of B56 subunits to increase the rate of disappearance of Akt phosphorylation after addition of the PI3K inhibitor strongly suggests that B56-PP2A holoenzymes affect Akt activity by increasing Akt dephosphorylation directly rather than by preventing its phosphorylation by PDK1. This is also supported by the ability of B56 subunits to regulate Ser473 phosphorylation.

Although many papers have reported the dephosphorylation of Akt by PP2A, very few of them have documented the involvement of specific regulatory subunits in this action. Data in support of a role of B56-containing PP2As in Akt regulation came from studies on the transforming ability of SV40 small t, which was shown to be both dependent on Akt activation (34) and partially mimicked by down-regulation of B56{gamma} (26, 35). Our results confirm the role of B56{gamma} in Akt dephosphorylation. However, by contrast with Chen et al. (35) we found that dephosphorylation of Akt does not seem to be linked solely to the B56{gamma} member. Indeed, expression or down-regulation of either B56{gamma} or B56beta subunits is sufficient to modulate pAkt levels. Although we have not been able to test all B56 subunits, these results suggest that at least B56{gamma} and B56beta enzymes have redundant effects on Akt activity. The preferential participation of one B56 family member to Akt regulation may depend on the cell context and on the level of expression of a given subunit and/or its subcellular localization.

Other studies have suggested that PP2As containing the B-family subunits could be involved in Akt regulation. B{alpha} was found in the caveolin-1 complex with PP2A-C and PP1. However, caveolin-1 alters both PDK1 and Akt activities and the role of the B{alpha} subunit in this action was not evaluated (31). Beaulieu et al. (36) showed that beta-arrestin2 promotes dephosphorylation of Akt by forming a complex with Akt, PP2A-C, and B{alpha}. Thus, whereas we found that B56-containing PP2A can dephosphorylate Akt activated by various growth factors, the action of B{alpha}-containing PP2A might be restricted to dopaminergic neurotransmission or other pathways coupled to beta-arrestin2.

Recently, Newton and colleagues (37) identified PHLPP, a phosphatase from the PP1C family, as a new Akt phosphatase acting mostly on Ser473. Based on a greater sensitivity of Thr308 than of Ser473 phosphorylation to the PP2A inhibitor okadaic acid, they suggested that PHLPP would be the key phosphatase for Ser473, whereas PP2A would regulate specifically the inactivation of Akt at Thr308. By contrast, our results show that both Ser473 and Thr308 are sensitive to expression of B56 subunits and are similarly regulated by IEX-1, indicating that both residues can be substrates for B56-containing PP2As. This is in agreement with many other reports showing, both in vitro and in vivo, that PP2A effectively dephosphorylates the two activating residues of Akt (12–14). Phosphorylation of Thr308 by PDK1 is necessary to activate Akt and this activity is augmented about 5–10-fold through Ser473 phosphorylation by the mTORC2 complex (10, 29). This suggests that PP2A may function as the primary Akt phosphatase. Dephosphorylation of both residues by PP2A-B56 holoenzymes and inhibition of this effect by IEX-1 may provide a rapid and complete turn-on and off mechanism to regulate Akt activity. As for activation, the extent of dephosphorylation and therefore inactivation of Akt by PP2A and PHLPP may be regulated separately, creating a modulatory network of regulation that may lead to a fine tuning of Akt signal.

We previously demonstrated that IEX-1-mediated inhibition of B56-containing PP2As results from its ability to allow B56 phosphorylation by active ERK in a ternary B56·IEX-1·pERK complex. By using ERK kinase-inactive dominant negative mutants, IEX-1 proteins unable to bind ERK, as well as the B56 species mutated in their major ERK phosphorylation site (4), we found that IEX-1 capacity to regulate Akt phosphorylation requires ERK activity. This suggests that Akt regulation by IEX-1 lies downstream of ERK activation. ERK and Akt have been shown to cooperate for activation of various common substrates (38–40). Several cross-talk mechanisms between these two signaling pathways have also been described. Notably, depending on the differentiation state, Akt-mediated phosphorylation of Raf-1 can lead to inhibition of ERK activation (41). Conversely, Akt can positively regulate ERK signaling through inhibition of GSK3 activity (42). Likewise, PI3K-independent but ERK-dependent routes to Akt activation have been reported (43) and Raf-induced nuclear accumulation of p21Cip1 was shown to depend on its ability to inhibit Akt (44). However, this is to our knowledge the first evidence for a regulation directly at the level of Akt dephosphorylation by the ERK pathway. Interestingly, IEX-1 expression is high in many tumor cells, as compared with normal tissues (16). In addition, our work (24), as well as data from cDNA arrays, showed that IEX-1 expression is controlled by ERK and is increased in cells expressing constitutively active Ras, Raf, or MEK (45–47). Inhibition of PP2A activity can synergize with phosphatase tensin homologue inactivation to activate Akt in tumor cells (48). This suggests that an increased expression of IEX-1 in response to ERK activation could lead to chronic inhibition of B56-containing PP2A activity in tumor cells and favor both ERK and Akt activation. B56-containing PP2As regulate also several onco-proteins such as c-Myc (49), Bcl2 (50), and beta-catenin (5). Whether IEX-1 contributes to tumor formation and inhibits other B56-controlled signaling pathways downstream of ERK activation will need to be determined.


   FOOTNOTES
 
* This work was supported by the Institut National de la Santé et de la Recherche Médicale, the Centre National de la Recherche Scientifique, and grants from the Ligue Contre le Cancer Comité de Paris, Association pour la Recherche contre le Cancer, Association for International Cancer Research, and Fondation de France. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Hôpital Cochin, Bat. G. Roussy, 27 Rue du Fg St. Jacques, 75014 Paris, France. Tel.: 33-1-40-51-65-15; Fax: 33-1-40-51-65-10; E-mail: Porteu{at}cochin.inserm.fr.

2 The abbreviations used are: PP2A, protein phosphatase 2A; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; GSK3, glycogen synthase kinase-3; IEX-1, immediate early response gene X-1; PI3K, phosphoinositide 3-kinase; TPO, thrombopoietin; WT, wild type; HA, hemagglutinin; shRNA, short hairpin RNA; GFP, green fluorescent protein; EPO, erythropoietin; CHO, Chinese hamster ovary; ER, endoplasmic reticulum; FCS, fetal calf serum; PDK1, phosphoinositide-dependent kinase-1. Back


   ACKNOWLEDGMENTS
 
We are grateful to Drs. D. Virshup, E. Ogris, N. Nojima, W. Hahn, E. Cohen, M. Weber, R. Hipsking, and M. Mumby for the gifts of plasmids and antibodies.



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