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Biochemical and Biophysical Research Communications 349 (2006) 789–798

BBRC

Akt phosphorylation is essential for nuclear translocation and retentionin NGF-stimulated PC12 cells

Truong Le Xuan Nguyen a,1, Joung Woo Choi a,1, Sang Bae Lee a, Keqiang Ye b,Soo-Dong Woo c, Kyung-Hoon Lee d, Jee-Yin Ahn a,*

a Department of Molecular Cell Biology, Center for Molecular Medicine, Samsung Biomedical Research Institute, Sungkyunkwan University School

of Medicine, Suwon 440-746, Republic of Koreab Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA 30322, USA

c Department of Plant Medicine, College of Agriculture, Life and Environment Sciences, Chungbuk National University, Cheongju 361-763, Republic of Koread Department of Anatomy, Sungkyunkwan University School of Medicine, Suwon 440-746, Republic of Korea

Received 15 August 2006Available online 28 August 2006

Abstract

Nerve growth factor (NGF) elicits Akt translocation into the nucleus, where it phosphorylates nuclear targets. Here, we describe thatAkt phosphorylation can promote the nuclear translocation of Akt and is necessary for its nuclear retention. Overexpression ofAkt-K179A, T308A, S473A-mutant failed to show either nuclear translocation or nuclear Akt phosphorylation, whereas expressionof wild-type counterpart elicited profound Akt phosphorylation and induced nuclear translocation under NGF stimulation. Employingthe PI3K inhibitor and a variety of mutants PI3K, we showed that nuclear translocation of Akt was mediated by activation of PI3K, andAkt phosphorylation status in the nucleus required PI3K activity. Thus the activity of PI3K might contribute to the nuclear translocationof Akt, and that Akt phosphorylation is essential for its nuclear retention under NGF stimulation conditions.� 2006 Elsevier Inc. All rights reserved.

Keywords: Akt; Nerve growth factor (NGF); Phosphoinositide 3-kinase (PI3K); PC12 cells; Phosphorylation

Akt/PKB performs a crucial function in the intracellularsignaling pathway that is associated with cell survival andproliferation. The activation of Akt is mediated by thegrowth factor receptor, stimulated by Phosphoinositide 3-kinase (PI3K), which is known to be wortmannin-sensitive[1]. During its activation, Akt is recruited to the plasmamembrane, where it binds to the PI3K products,PI(3, 4,5)P3 and PI(3, 4)P2, and exposes a pair of threo-nine-308 and serine-473 residues for phosphorylation by3-phosphoinositide-dependent kinase 1(PDK1), as well asby some as-yet-unidentified kinases [2]. This double phos-phorylation results in a full activation of Akt [3,4].

Once Akt has been activated, it induces the phosphory-lation of a number of nuclear and cytosolic proteins that

0006-291X/$ - see front matter � 2006 Elsevier Inc. All rights reserved.

doi:10.1016/j.bbrc.2006.08.120

* Corresponding author. Fax: +82 31 299 6139.E-mail address: jyahn@med.skku.ac.kr (J.-Y. Ahn).

1 These authors contributed equally to this work.

regulate cell metabolism, growth, and survival. Akt func-tions prior to the release of cytochrome c, via the exertionof regulatory effects on the activities of Bcl-2 family mem-bers and mitochondrial activity, and also operates aftercytochrome c release [4,5]. Akt directly phosphorylatesCREB and inhibits the expression of caspase proteases[6,7]. PI3K and Akt are predominantly cytoplasmicallylocated, but have also been detected within the nucleus[8–10]. This indicates that they either originate in the nucle-us, or are translocated into the nucleus upon stimulation[8,9]. As an example of this, Akt has been shown to trans-locate to the nucleus after 20–30 min of treatment withgrowth factors, and there modulates Forkhead box tran-scription factors, including FKHR, FKHRL1, and AFX,which results in the inhibition of their ability to inducethe expression of death genes [11–13]. Akt has also beenshown to both phosphorylate the p53 tumor suppressorand inhibit its activity [14]. The nuclear targets of Akt

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include nuclear SRK (S6 kinase–related kinase) and Nur77,a transcription factor which has previously been implicatedin T-cell receptor-mediated apoptosis [15].

A growing body of evidence suggests that the introduc-tion of growth factors may induce Akt nuclear transloca-tion [16]. However, few data are currently available withregard to the mechanisms underlying the regulation ofAkt within the nucleus. Whether or not Akt nuclear trans-location relies on Akt phosphorylation status and thisevent is mediated in a PI3K-dependent manner is currentlyunknown. It also remains to be determined whether or notnuclear Akt remains the downstream target of PI3K. Here,we report that PI3K is a prerequisite for the nuclear trans-location of Akt, and that the phosphorylation of Akt iscritical for its intranuclear permanence and retention with-in PC12 cells.

Materials and methods

Cells and reagents. Cells were maintained in medium A (DMEMsupplemented with 10% fetal bovine serum (FBS), 5% horse serum, 2 mg/ml of glutamine, and 100 units of penicillin–streptomycin) at 37 �C, in ahumidified incubator, in a 5% CO2 atmosphere. All of the PC12 cellsemployed in the experiments were in naı̈ve form and remained undiffer-entiated. Mouse monoclonal anti-HA-horseradish peroxidase, anti-Myc-horseradish peroxidase, and anti-tubulin antibodies were obtained fromSigma. Mouse monoclonal anti-phospho-Akt Ser-473, anti-Akt, anti-phospho-GSK 3a/b(Ser 21/9), anti-poly (ADP-ribose) polymerase1(PARP), and anti-Lamin A/C antibodies were obtained from Cell Sig-naling. Rabbit polyclonal anti-p85 antibody was acquired from SantaCruz, Inc. The GSK-3 fusion protein and Crosstide used in this study wereobtained from Cell Signaling. The FuGen 6 was purchased from Roche.The [c-32P] ATP and enhanced chemiluminescence (ECL) solution wereacquired from Amersham Phamacia Biotech. All chemicals not specificallyreferenced above were obtained from Sigma.

In situ immunofluorescence for GFP-Akt and RFP-Akt translocation

studies. The PC12 cells were plated onto coverslips coated with poly-L-lysine in 24-well plates. Approximately 16 h later, the cells were trans-fected with 1 lg DNA with FuGene 6 (Roche, German) for 4–6 h. After24 h of incubation, the GFP-Akt transfected cells were serum-starved for8 h, and either stimulated with 100 ng/ml of nerve growth factor (NGF),or left untreated, for 0, 10, or 30 min. The RFP-Akt and its mutants weretreated in an identical fashion. In experiments involving kinase inhibitors,the cells were preincubated for 30 min with wortmannin (20 nM),LY294002 (100 lM), or PD98059 (50 lM). After treatment, the cells werefixed for 20 min at room temperature in phosphate-buffered saline con-taining 4% paraformaldehyde, and the cells were washed three times inPBS. The cells were then stained with 4 0-6-diamido-2-phenylindole-2 HCl(DAPI). The GFP/RFP-tagged Akt and DAPI-stained nuclei were thenanalyzed via fluorescence microscopy (Olympus Fluo View).

Subcellular fractionation. The nuclear and cytoplasmic fractions of thePC12 cells were separated using an NE-PER nuclear and extractionreagent kit, in accordance with the manufacturer’s specifications (Pierce).

Akt kinase activity assay. The nuclear fractions were preincubated with5 lg of normal rabbit IgG, and 10 lg of Protein A/G conjugated agarosefor 5 h at 4 �C, then washed three times with cell lysis buffer (50 mM Tris,pH 7.4, 40 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, 1.5 mM Na3VO4,50 mM NaF, 10 mM sodium pyrophosphate, 10 mM sodium b-glycero-phosphate, 1 mM phenylmethylsulfonyl fluoride (PMSF), 5 mg/ml ofaprotinin, 1 mg/ml of leupeptin, and 1 mg/ml of pepstatin (A) The sam-ples were then incubated for an additional 3 h with 10 lg of immobilizedanti-Akt antibody at 4 �C and under constant agitation. The immuno-precipitates were then washed three times with lysis buffer, and once with1· Akt kinase buffer (20 mM Tris, pH 7.5, 10 mM MgCl2, 5 mM b-glyc-

erophosphate, 2 mM dithiothreitol, 0.1 mM Na3VO4). The precipitateswere subsequently incubated in 40 ll of kinase reaction buffer supple-mented with 20 lM ATP, 30 lM Crosstide, and 10 lCi [c-32P] ATP(3000 Ci/mmol) for 30 min at 30 �C. The reaction mixture (25 ll) wasspotted onto P81 paper, and washed three times in 0.75% phosphoric acidsolution, and once in 75% ethanol. After the paper had been dried, theradioactivity was quantified using a liquid scintillation counter. Using0.5 lg GSK3 fusion protein as a substrate, the kinase assays were haltedafter 30 min of incubation via the addition of 3· Laemmli buffer and 3 minof boiling. The proteins were resolved on 10% SDS–PAGE and analyzedwith anti-phospho-GSK 3a/b (Ser 21/9) antibody.

Adenoviral infection. Adenovirus-expressing Myc-p110*, dominant-negative p85, and Akt-K179AT308AS473A were purified via CsClbanding at 1011–1012 plaque forming units, introduced into the PC12 cells,and cultured overnight. GFP was monitored under an immunofluorescentmicroscope. Overexpressions of proteins were confirmed via Westernblotting using anti-myc, anti-p85, and anti-Akt antibodies.

Results

Akt phosphorylation is coupled to Akt nuclear translocation

in response to NGF stimulation

The phosphorylation of T308 and S473 on Akt results inthe full activation of its kinase activity. In order to deter-mine whether or not Akt activation could be correlatedwith its nuclear translocation, we conducted a subcellularfractionation assay, in an attempt to assess subcellularAkt levels or phosphorylated Akt levels within our experi-mental PC12cells. Although Western blotting analysisrevealed that the total Akt expression level exhibited nosignificant changes with regard to subcellular Akt distribu-tion, as the following phosphorylation of Akt levelincreased, the total Akt level decreased slightly (Fig. 1A,lane 2). However, an increase was observed in the levelsof phosphorylated Akt in response to 10 min of NGF treat-ment, coupled with a decline in the levels of Akt within thecytosolic fractions, whereas the quantity of phosphorylatedAkt within the nucleus was observed to lag, with a maximalincrease at 30 min, after which a slow decay was registered(Fig. 1A, lane 1). Maximal intranuclear translocation levelswere observed after 30 min of NGF treatment, which wasdetected against anti-Ser-473 phospho-Akt antibody(Fig. 1A, lane 1). The identity and purity of both the cyto-solic and nuclear fractions were, respectively, verified usinganti-Lamin A/C and anti-tubulin antibodies (Fig. 1A, lanes3 and 4).

Akt kinase activity, which was monitored via measure-ments of the phosphorylation of GSK3a, a downstreamtarget of Akt, was correlated with the distribution of phos-phorylated Akt (Fig. 1B). In the cytoplasm, the activity ofAkt kinase was shown to increase after 5–10 min of NGFtreatment, exhibiting a decay period 30 min later. Howev-er, we detected negligible phospho-Akt or phospho-GSK3alevels in the nucleus after 5–10 min of NGF treatment.

Collectively, these observations show that the applica-tion of NGF treatment could induce a rapid accumulationof phosphorylated Akt and a subsequent stimulation ofAkt kinase activity in either the cytoplasm or the nucleus.

Fig. 1. Akt phosphorylation is coupled to its nuclear translocation. Serum-starved PC12 cells were treated with NGF for 0, 5, 10, 30, 60, and 90 min,respectively, and fractionated into cytoplasmic and nuclear fractions. (A) Western blotting analysis of phosphorylated Akt (first panel) or total Akt(second panel) from each nuclear and cytoplasmic fraction. The purity of each of the fractions was verified using anti-tubulin antibodies (third panel andfourth panel) and anti-Lamin A/C. (B) In vitro kinase assays were conducted with the immunoprecipitated endogenous Akt. The GSK-3-fusion proteinwas employed as a substrate, and its phosphorylation status was analyzed with a specific phosphor GSK-3 antibody.

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Nuclear translocation of GFP-Akt by NGF is PI3-kinase-

dependent

Previous studies have indicated that Akt is translocat-ed into the nucleus upon the administration of growthfactor treatment. However, it remains somewhat unclearas to whether or not the intranuclear translocation ofAkt by NGF is regulated in a PI3k-dependent fashion.To determine whether exogenously transfected GFP-Akttranslocation in PC12 cells occurs in response to100 ng/ml NGF treatment, we transfected GFP-Akt intothe PC12 cells and subsequently conducted immunofluo-rescent staining assays. Our immunohistochemical analy-ses demonstrated the substantial nuclear translocation ofGFP-Akt, which, in the unstimulated PC12 cells, wasdetected predominantly within the cytoplasm. Upon theadministration of NGF treatment, we observed anincrease in the immunostaining intensity of the nuclearinterior, which exhibited maximal levels after 30 min oftreatment (Fig. 2A).

The nuclear localization of GFP-Akt increased uponNGF administration, whereas the quantity of cytoplasmicGFP-Akt was shown to decrease markedly. We thenattempted to characterize the mechanism responsible forthe effects of NGF on Akt translocation, using kinaseinhibitors. Pretreatment with wortmannin (20 nM), as wellas LY294002 (20 lM) (data not shown), both of which arePI3K inhibitors (Fig. 2B) prevented the intranuclear migra-tion of GFP-Akt after NGF stimulation, whereas theadministration of PD98059 (50 lM), an MEK1 inhibitor,failed to prevent the migration of GFP-Akt (Fig. 2C).

The degree to which nuclear location occurred was evaluat-ed via DAPI staining. In a result consistent with previousfindings [17], Akt nuclear translocation appears to be med-iated by PI3K.

Preferential Akt phosphorylation is required for its nuclear

translocation and retention

In order to determine whether Akt phosphorylation isrequired for its nuclear retention and nuclear translocationin neuronal cells, we transfected PC12 cells either withwild-type HA-Akt, or with one of three HA-Aktmutants—HA-Akt-K179A HA-Akt- T308D, S473D orHA-Akt-K179A, T308D, S473D. The transfected cellswere then stimulated with NGF for 30 min, or left untreat-ed. Immunoblotting analysis showed that the robust nucle-ar translocation and phosphorylation of constitutivelyactivated HA-Akt -T308D, S473D occurred regardless ofgrowth factor stimulation. However, the cells that had beentransfected with the wild-type Akt exhibited demonstrablenuclear translocation and phosphorylation only in theNGF-stimulated cells, thereby indicating that the extremephosphorylation of Akt is largely responsible for its nucleartranslocation.

Interestingly, no phosphorylated Akt was observed inthe nucleus in the cells transfected with the Akt kinase-in-active mutant (K179A), regardless of NGF stimulation sta-tus, although a modest amount of Akt nucleartranslocation was observed in the absence of NGF stimula-tion, and elevated phosphorylation and translocation ofAkt were observed in response to NGF treatment in the

Fig. 2. NGF-induced nuclear translocation of GFP-Akt is prohibited by PI3K inhibitor treatment, but not by MEK1 inhibitor treatment. (A) GFP-tagged nuclear translocation of Akt after NGF treatment. After transfection with 1 lg GFP-Akt, the transfected cells were stimulated with or without100 ng/ml NGF for 0, 10, and 30 min, respectively. (B) PI3K inhibitor pretreatment inhibited GFP-Akt translocation. GFP-Akt transfected cells werepreincubated with 20 nM wortmannin for 30 min at RT and then treated with 100 ng NGF as indicated. (C) MEK1 inhibitor pretreatment had no effect onGFP-Akt nuclear translocation. The GFP-Akt transfected cells were preincubated with 50 lM PD98059 for 30 min at RT prior to exposure to 100 ngNGF.

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cells transfected with the kinase-inactive but constitutivelyphosphorylated HA-Akt mutant that (K179A, T308D,S473D) (Fig. 3A, top and middle panels). No Akt kinaseactivity was detected in the cells overexpressing HA-Akt-K179A or HA-Akt-K179A, T308D, S473D (data notshown). These findings appear to suggest that the activityof Akt kinase may exert some influence on Akt nucleartranslocation and its nuclear retention, but may not consti-tute the critical cause for Akt translocation or nuclearretention. Furthermore, the phosphorylation of HA-Akt-K179A, T308D, S473D resulted in a slight phosphoryla-tion and translocation, even in the cells that were nottreated with NGF. Also, in the cytoplasm of the treatedcells, Akt phosphorylation was shown to occur to a higherdegree even than was observed in that of the wild-typetransfected cells, and the patterns and levels of nucleartranslocation were similar to those seen in the wild-type-transfected cells, which strongly suggests that Akt

Fig. 3. Phosphorylation status of Akt correlated with its nuclear location. (T308D, S473D, and HA-Akt-K179A, T308D, S473D were serum-starved for 1micro gram of both nuclear and cytoplasmic fractions analyzed with anti-phoexpression levels of HA-Akt were detected by immunoblotting against anti-HAinfected with wild-type or dominant-negative Akt-K179A-, T308A-, S473A-exp10, or 30 min, the infected cells were fractionated and analyzed with anti-pInfection efficiency was verified via Western blotting against anti-Akt antibod

phosphorylation is sufficient for both its nuclear transloca-tion and nuclear retention. Equal amounts of transfectedAkt were confirmed (Fig. 3A, bottom panel).

To determine more precisely whether Akt phosphoryla-tion is critical for its nuclear events, we employed the ade-novirus-expressing dominant-negative Akt-K179A,T308A, S473A. As compared to the cells infected withthe wild-type adenovirus, the dominant-negative Akt ormock virus-infected cells failed to show any significanttranslocation after 10 min of NGF treatment. Consistently,even in cells subjected to 30 min of NGF stimulation, weobserved a basal level of endogenous Akt translocation inthe dominant-negative Akt or mock virus-infected cells(Fig. 3B). We verified Akt expression in the adenovirus-in-fected cells (Fig. 3C). This shows that the activity of Aktkinase is involved, at least to some degree, in Akt nucleartranslocation, and that Akt phosphorylation appears tobe crucial for its nuclear events.

A) Cells transfected with HA-Akt wild-type, HA-Akt-K179A, HA-Akt-6 h prior to stimulation with or without 100 ng/ml NGF for 30 min. Thirtyspho-Akt Ser-473 antibody proteins (top and middle panels) and similarusing 10 lg of whole cell lysate (bottom panel). (B) and (C) PC12 cells wereressing adenovirus or mock adenovirus. Following NGF treatment for 0,

hospho-Akt antibody from both the nuclear and cytoplasmic fractions.y.

Fig. 4. Nuclear translocation of RFP-Akt and RFP-Akt mutants in PC12 cells. PC12 cells grown and transfected with (A) RFP-Akt, (B) RFP-Akt-KD*;K179A, T308D, S473D, (C) RFP-Akt-AAA; K179A, T308A, S473A, respectively, on coverslips were serum-starved for 16 h prior to stimulation with orwithout 100 ng/ml NGF for 30 min. After stimulation, the cells were fixed and stained with DAPI and visualized via fluorescence microscopy.

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Changes in Akt localization during NGF stimulationwere also monitored via immunofluorescence. RFP-Akt-wild-type was observed primarily within the cytoplasm ofthe unstimulated PC12 cells. Significant nuclear localiza-

tion was detected after 30 min of NGF stimulation(Fig. 4A). Similar alterations in the subcellular distributionpatterns were observed with RFP-Akt-KD*(K179A,T308D, S473D) (Fig. 4B), whereas RFP-Akt-

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AAA(K179A, T308A, S473A) was found to result in con-siderable cytoplasmic labeling, excluding the nucleus evenafter 30 min of NGF treatment (Fig. 4C). Collectively,these findings support the notion that the Akt phosphory-lation status could be correlated with the degree to whichAkt nuclear localization had taken place.

Sustained phosphorylation of nuclear Akt and its kinase

activity require the stimulation of PI3K activity

As exposure to growth factors has previously been dem-onstrated to trigger the nuclear translocation of PI3K, wehave attempted to determine whether PI3K might not onlyregulate Akt nuclear translocation, but also the retentionof active Akt within the nucleus. In order to assess thisnotion, we applied either wortmannin or PD98059 to thePC12 cells for 30 min, either separately or in combination.Later, we treated the PC12 cells with NGF for an addition-al 30 min, or left the cells untreated, and then preparednuclear fractions. Compared to NGF-treated control,wortmannin pretreatment substantially diminished NGF-triggered Akt phosphorylation, but not completely inhibit-ed it. However, PD98059 has no effect. We also notednegligible nuclear Akt phosphorylation levels in the sam-ples pretreated with the combination of wortmannin andPD98509 (Fig. 5A, top panel). The expression of PARP,which was employed as a control and as a nuclear marker

Fig. 5. Nuclear Akt activation is negatively regulated by PI3K inhibitor. (A) Nnot by treatment with PD98059 alone. (B) In vitro Nuclear Akt kinase assay. Telicited inhibitory effects on Akt kinase activity. PC12 cells were treated wwortmannin and PD98509 for 3 h, following 12 h of serum starvation. Kinasduplicate immunoprecipitates.

protein, remained unaffected (Fig. 5A, bottom panel).These results indicate that PI3K but not MAPK signalingis, to some degree, involved in the phosphorylation statusof Akt within the nucleus.

To insight into the functional consequences of Akttranslocation, we evaluated the activity of Akt kinase inthe nuclear fractions of cells treated for 3 h with PI3Kinhibitors or MEK1 inhibitors, either separately or in com-bination, followed by exposure to NGF. NGF elicited anearly fourfold augmentation in Akt activity. Wortmanninpretreatment resulted in a �50% reduction in Akt activity.Wortmannin and PD98509 mixture also reduced Akt activ-ity. By contrast, PD98509 alone elicited a minimal reduc-tion in Akt kinase activity (Fig. 5B), indicating that thenuclear activation of Akt is unlikely to be significantlyinvolved in MAPK signaling. These findings show thatthe activity of PI3K may be a prerequisite for the mainte-nance of nuclear Akt kinase activity.

A mutant form of the p85 regulatory subunit of PI3K(Dp85), which neither binds nor activates the p110 catalyticsubunit, has been demonstrated to function as a dominant-negative mutant of this protein [18]. As was previouslyreported [19], the dominant-negative Dp85 (deletion ofamino acids 478–513) has been determined to dramaticallyinhibit PI3K activity in both the cytosol and the nucleus,whereas constitutively active p110*(K227E) significantlyenhanced it (data not shown).

uclear Akt phosphorylation was affected by wortmannin pretreatment, butreatment with wortmannin or a combination of wortmannin and PD98509ith wortmannin (100 nM) or PD98059 (100 lM) or a combination ofe activity is shown as the average (±SD) of two experiments, each with

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To determine more precisely whether PI3K activity isindeed required for the maintenance of Akt phosphoryla-tion within the nuclei of cells stimulated with NGF, weinfected PC12 cells with an adenovirus that expressed con-stitutively active or dominant-negative PI3K, followed bythe treatment of these cells with or without NGF for30 min. We conducted immunofluorescent staining to char-acterize the subcellular localization of infected PI3K. Myc-p110* is distributed throughout both the cytoplasm and thenucleus, and NGF triggers further nuclear translocation(Fig. 6A. right). However, Dp85 is predominantly foundwithin the cytoplasm, and NGF treatment elicits only amodest amount of p85 nuclear translocation (Fig. 6A. left).Regardless of NGF status, the nuclear fraction infected bythe constitutively active PI3K evidenced substantial Akt

Fig. 6. PI3K activity is implicated in the maintenance of nuclear Akt activatioDp85 PI3K-expressing adenovirus, after which the cells were treated for 30 mfollowed by NGF treatment. (B) Western blotting analysis of phosphorylated Ap110* and Dp85 PI3K on Akt kinase activity determined. Kinase activityimmunoprecipitates. (D) The expression of infected viruses was verified by W

phosphorylation, a finding similar to that observed in thecytosolic fraction. In contrast, almost no phosphorylatedAkt could be observed within the dominant-negativePI3K-infected nuclear fraction in the absence of NGF,whereas a considerable quantity of phosphorylated Aktwas detected both within the cytosolic fraction and nuclearfraction in the presence of NGF (Fig. 6B). This suggeststhat functional PI3K is necessary for the maintenance ofAkt phosphorylation in the nucleus and may play a rolein mediating the translocation of phosphorylated Akt intothe nucleus.

The overexpression of constitutively active p110* result-ed in enhanced nuclear Akt kinase activity, whereas theoverexpression of Dp85 led to almost complete abolitionof NGF-induced Akt activation, as compared to what

n. PC12 cells were infected with constitutively active or dominant-negativein with or without NGF. (A) The localization of p110* and Dp85 PI3K,kt from each nuclear and cytoplasmic fraction, as indicated. (C), Effects of

is expressed as the average (±SD) of an experiment with triplicateestern blotting against anti-myc antibody and anti-p85 antibody.

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was observed in the mock virus-infected cells, althoughDp85 overexpression exerted no significant effects on Aktactivity in the unstimulated cells (Fig. 6C). The expressionsof infected viruses were confirmed via Western blottingagainst anti-myc antibody or anti-p85 antibody, respective-ly (Fig. 6D).

Discussion

It is now clear that Akt migrates to the nucleus as theresult of treatment with growth factors. However, themechanism by which Akt translocates into the nucleusremains unclear. An interesting mechanism inherent tothe nuclear translocation of Akt has been discovered inhuman mature T-cell leukemia, and involves participationin the PI3K-dependent Akt1 signaling pathway [20]. AsPI3K has been detected in both the cytoplasmic and nucle-ar fractions, it has been suggested to function as a physio-logical regulator of Akt within the nucleus, as well as thecytoplasm. In the present study, we showed that Akt phos-phorylation is coupled to its nuclear translocation underconditions of NGF stimulation, which is modulated byPI3K. We have also determined that PI3K activity is nec-essary for the maintenance of Akt activation within thenucleus.

Growth factor-induced activation of Akt is mediated byPI3K, and that PI3K inhibitors prevent Akt phosphoryla-tion on Thr-308 and Ser-473 in the cytoplasm, To charac-terize the ability of PI3K to regulate the migration of Aktto the nucleus in PC12 cells, we employed a broadly usedPI3K inhibitor, namely wortmannin, followed by the over-expression of GFP-tagged Akt. The GFP-tagged Akt wasshown to have been predominantly located within the cyto-plasm after being introduced into the cells. However, inresponse to NGF stimulation, we noted a marked nucleartranslocation, which was blocked by the application ofwortmannin, as shown by a comparison with cells treatedwith PD98509, a selective MEK1 inhibitor (Fig. 2). ThusAkt nuclear translocation may be attributable to an activa-tion of PI3K, rather than to an MAPK-dependent mecha-nism. Surprisingly wortmannin treatment cannotcompletely abrogate NGF-induced activation of nuclearAkt, and we found that nuclear Akt kinase activity wasinhibited by no more than 50–70% by wortmannin(Fig. 5). However, we showed that PI3K activity is requiredfor retaining Akt in its phosphorylated active state (Fig. 6).

The maintenance of Akt activation in the nucleus doesnot strictly occur in a PI3K-dependent manner. Rather, itcan also apparently be activated in a PI3K-independentmanner, such as by a cAMP-elevating agent directly, viathe actions of PKA [21] or Ca2+/calimodulin-dependentkinases [22]. Nevertheless, the nuclear translocation ofAkt appears to exclusively occur in a PI3K-dependentmanner in PC12 cells, although the mechanism regulatingthe nuclear localization of Akt remains somewhat enigmat-ic. This suggests that PI3K plays a dual role in the regula-tion of Akt within the nucleus. One of these roles involves

the facilitation or induction of nuclear translocation fol-lowing its phosphorylation by NGF, and the second is atleast a partial role in the maintenance of phosphorylationand kinase activity in the nucleus, although we have beenunable to dismiss the possibility of the existence of aPI3K-independent mechanism for the maintenance ofnuclear Akt activity.

With regard to PI3K, and specifically to ongoing PI3Kactivity, PI(3, 4,5)P3 production appears to constitute aprerequisite for its intranuclear accumulation. It hasrecently been reported that treatment with PI3K inhibitorscan result in the abolition of PI3K nuclear translocation inMC3T3-E1 cells, and this phenomenon has also been notedin HL-60 cells. As intranuclear Akt is active, its phosphor-ylation must occur on the threonine and serine residues.There is a growing body of convincing evidence that Thr-308 may be phosphorylated by PDK1. Although the nucle-ar translocation of PDK1 has yet to be elucidated in detail[23], it is currently thought that Akt may be activated with-in the cytoplasm and may then migrate into the nucleus[24]. However, our data do not militate toward the dismiss-al of the possibility that, for the sustained intranuclear per-manence and activation of Akt, as is induced by NGF,PI3K activity in the nucleus is also required. Despite thelarge body of evidence that suggests the intranuclear pres-ence of PI3K, the manner in which PI3K is activated andinactivated within the nucleus has yet to be precisely eluci-dated. Moreover, we currently possess no useful informa-tion with regard to the manner in which PI (3,4,5)P3

signaling is attenuated within the nucleus, and this is clear-ly a matter that warrants further attention.

The subcellular distribution of the wild-type kinase isknown to be regulated rather tightly. Phosphorylated Akttranslocates to the nucleus, and this can be detected as ear-ly as 10 min after NGF stimulation, with maximal effectsbeing seen at 30 min, coupled with a simultaneous decreasein the levels of its cytoplasmic counterpart (Fig. 1), demon-strating that kinase activity is not required, instead itsphosphorylation status regulates its nuclear residence.HA-Akt-K179A cannot be detected in the nucleus of stim-ulated PC12 cells (Fig. 3A); by contrast HA-Akt-K179A,T308D, S473D was detected in the nuclei of stimulatedPC12 cells (Figs. 3A and 4B). Moreover, Akt-179A,T308A, S473A or RFP-Akt-AAA also failed to move intothe nucleus of stimulated PC12 cells (Figs. 3B and 4C).Collectively, it seems likely, then, that Akt phosphorylationis sufficient for the retention of Akt in the nucleus.

We are also only beginning to understand the manner inwhich the activity of Akt is turned on/off within the nucle-us. In this study, we have determined that Akt phosphory-lation plays a critical role in its nuclear retention, and thatthe NGF-induced nuclear translocation of Akt is partiallymaintained in a PI3K-dependent manner. The accurateand thorough elucidation of the mechanisms underlyingthe nuclear regulation of PI3K and Akt may provide uswith considerable insight into NGF-mediated signaltransduction.

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Acknowledgments

This work was supported by Grants from the Korea Re-search Foundation (KRF-2005-204-E00015) and the Kor-ea Science and Engineering Foundation (RO1-2006-000-10222-0) to J.-Y. Ahn.

Appendix A. Supplementary data

Supplementary data associated with this article can befound, in the online version, at doi:10.1016/j.bbrc.2006.08.120.

References

[1] D. Brodbeck, P. Cron, B.A. Hemmings, A human protein kinaseBgamma with regulatory phosphorylation sites in the activation loopand in the C-terminal hydrophobic domain, J. Biol. Chem. 274 (1999)9133–9136.

[2] A. Brunet, S.R. Datta, M.E. Greenberg, Transcription-dependentand -independent control of neuronal survival by the PI3K-Aktsignaling pathway, Curr. Opin. Neurobiol. 11 (2001) 297–305.

[3] D.P. Brazil, B.A. Hemmings, Ten years of protein kinase B signalling:a hard Akt to follow, Trends Biochem. Sci. 26 (2001) 657–664.

[4] S.R. Datta, H. Dudek, X. Tao, S. Masters, H. Fu, Y. Gotoh, M.E.Greenberg, Akt phosphorylation of BAD couples survival signals tothe cell-intrinsic death machinery, Cell 91 (1997) 231–241.

[5] L. del Peso, M. Gonzalez-Garcia, C. Page, R. Herrera, G. Nunez,Interleukin-3-induced phosphorylation of BAD through the proteinkinase Akt, Science 278 (1997) 687–689.

[6] K. Du, M. Montminy, CREB is a regulatory target for the proteinkinase Akt/PKB, J. Biol. Chem. 273 (1998) 32377–32379.

[7] M.H. Cardone, N. Roy, H.R. Stennicke, G.S. Salvesen, T.F. Franke,E. Stanbridge, S. Frisch, J.C. Reed, Regulation of cell death proteasecaspase-9 by phosphorylation, Science 282 (1998) 1318–1321.

[8] N.N. Ahmed, T.F. Franke, A. Bellacosa, K. Datta, M.E. Gonz-alez-Portal, T. Taguchi, J.R. Testa, P.N. Tsichlis, The proteinsencoded by c-akt and v-akt differ in post-translational modification,subcellular localization and oncogenic potential, Oncogene 8 (1993)1957–1963.

[9] R. Meier, D.R. Alessi, P. Cron, M. Andjelkovic, B.A. Hemmings,Mitogenic activation, phosphorylation, and nuclear translocation ofprotein kinase Bbeta, J. Biol. Chem. 272 (1997) 30491–30497.

[10] L.M. Neri, D. Milani, L. Bertolaso, M. Stroscio, V. Bertagnolo, S.Capitani, Nuclear translocation of phosphatidylinositol 3-kinase inrat pheochromocytoma PC 12 cells after treatment with nerve growthfactor, Cell Mol. Biol. (Noisy-le-grand) 40 (1994) 619–626.

[11] W.H. Biggs 3rd, J. Meisenhelder, T. Hunter, W.K. Cavenee, K.C.Arden, Protein kinase B/Akt-mediated phosphorylation promotes

nuclear exclusion of the winged helix transcription factor FKHR1,Proc. Natl. Acad. Sci. USA 96 (1999) 7421–7426.

[12] A. Brunet, A. Bonni, M.J. Zigmond, M.Z. Lin, P. Juo, L.S. Hu, M.J.Anderson, K.C. Arden, J. Blenis, M.E. Greenberg, Akt promotes cellsurvival by phosphorylating and inhibiting a Forkhead transcriptionfactor, Cell 96 (1999) 857–868.

[13] G.J. Kops, B.M. Burgering, Forkhead transcription factors: newinsights into protein kinase B (c-akt) signaling, J. Mol. Med. 77 (1999)656–665.

[14] A. Yamaguchi, M. Tamatani, H. Matsuzaki, K. Namikawa, H.Kiyama, M.P. Vitek, N. Mitsuda, M. Tohyama, Akt activationprotects hippocampal neurons from apoptosis by inhibiting tran-scriptional activity of p53, J. Biol. Chem. 276 (2001) 5256–5264.

[15] N. Masuyama, K. Oishi, Y. Mori, T. Ueno, Y. Takahama, Y. Gotoh,Akt inhibits the orphan nuclear receptor Nur77 and T-cell apoptosis,J. Biol. Chem. 276 (2001) 32799–32805.

[16] P. Borgatti, A.M. Martelli, G. Tabellini, A. Bellacosa, S. Capitani,L.M. Neri, Threonine 308 phosphorylated form of Akt translocatesto the nucleus of PC12 cells under nerve growth factor stimulationand associates with the nuclear matrix protein nucleolin, J. CellPhysiol. 196 (2003) 79–88.

[17] P. Borgatti, A.M. Martelli, G. Tabellini, A. Bellacosa, S. Capitani,L.M. Neri, Translocation of Akt/PKB to the nucleus of osteoblast-like MC3T3-E1 cells exposed to proliferative growth factors, FEBSLett. 477 (2000) 27–32.

[18] R. Dhand, I. Hiles, G. Panayotou, S. Roche, M.J. Fry, I. Gout, N.F.Totty, O. Truong, P. Vicendo, K. Yonezawa, PI 3-kinase is a dualspecificity enzyme: autoregulation by an intrinsic protein–serinekinase activity, EMBO J. 13 (1994) 522–533.

[19] J.Y. Ahn, R. Rong, X. Liu, K. Ye, PIKE/nuclear PI3K signalingmediates the antiapoptotic actions of NGF in the nucleus, EMBO J.23 (2004) 3995–4006.

[20] Y. Pekarsky, A. Koval, C. Hallas, R. Bichi, M. Tresini, S. Malstrom,G. Russo, P. Tsichlis, C.M. Croce, Tcl1 enhances Akt kinase activityand mediates its nuclear translocation, Proc. Natl. Acad. Sci. USA 97(2000) 3028–3033.

[21] C.L. Sable, N. Filippa, B. Hemmings, E. Van Obberghen, cAMPstimulates protein kinase B in a Wortmannin-insensitive manner,FEBS Lett. 409 (1997) 253–257.

[22] M.J. Perez-Garcia, V. Cena, Y. de Pablo, M. Llovera, J.X. Comella,R.M. Soler, Glial cell line-derived neurotrophic factor increasesintracellular calcium concentration. Role of calcium/calmodulin inthe activation of the phosphatidylinositol 3-kinase pathway, J. Biol.Chem. 279 (2004) 6132–6142.

[23] R.A. Currie, K.S. Walker, A. Gray, M. Deak, A. Casamayor, C.P.Downes, P. Cohen, D.R. Alessi, J. Lucocq, Role of phosphatidylin-ositol 3,4,5-trisphosphate in regulating the activity and localization of3-phosphoinositide-dependent protein kinase-1, Biochem. J. 337(1999) 575–583.

[24] E. Astoul, S. Watton, D. Cantrell, The dynamics of protein kinase Bregulation during B cell antigen receptor engagement, J. Cell Biol. 145(1999) 1511–1520.