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. Author manuscript; available in PMC: 2008 Aug 1.
Published in final edited form as: FEBS Lett. 2006 Apr 27;580(13):3051–3058. doi: 10.1016/j.febslet.2006.04.051

Akt1 is dynamically modified with O-GlcNAc following treatments with PUGNAc and insulin-like growth factor-1

Johanna C Gandy 1, Abigail E Rountree 1, Gautam N Bijur 1,*
PMCID: PMC2493066  NIHMSID: NIHMS56278  PMID: 16684529

Abstract

The Ser/Thr kinase Akt1 is activated by growth factors subsequent to its phosphorylation on Thr308 and Ser473. In the present study, Akt1 was found to be constitutively modified with O-GlcNAc. Treatment of SH-SY5Y cells with O(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc), which inhibits the enzymatic removal of O-GlcNAc from proteins, increased cytosolic O-GlcNAc-Akt1 levels. Treatment of cells with insulin-like growth factor-1 (IGF-1) also increased O-GlcNAc-Akt1 levels and increased Akt1 phosphorylation. PUGNAc treatment did not attenuate IGF-1 induced Akt1 phosphorylation. These results indicate that Akt1 can be simultaneously modified with O-GlcNAc and phosphorylated. However, PUGNAc induced the nuclear accumulation of Akt1 suggesting that the O-GlcNAc-modification on Akt1 may play a role in Akt1 nuclear localization.

Keywords: Akt1, O-GlcNAc, PUGNAc, IGF-1, Wheat germ agglutinin, GSK3β

1. Introduction

The serine/threonine kinase Akt1 (protein kinase B) affects a broad range of cell functions. Activation of Akt1 increases glucose transport, regulates gene transcription, and promotes cell survival in the face of lethal conditions (reviewed extensively in [1–3]). Akt1 is activated by a variety of stimuli including growth factors such as insulin-like growth factor-1 (IGF-1) and hormones such as insulin [3,4]. Stimulation with IGF-1 activates phosphatidylinositol-3 kinase signaling which results in the dual phosphorylation of Akt1 on Thr308 and Ser473 and maximally increases Akt1 activity [5]. One well known substrate for Akt1 is glycogen synthase kinase-3β (GSK3β). Akt1 phosphorylates Ser9 of GSK3β which inhibits GSK3β activity [6].

Akt1 is predominantly in the cytosol of unstimulated cells [7,8], but subsequent to activation by IGF-1 a fraction of Akt1 accumulates in the nucleus [9,10]. However, compared to the rapid phosphorylation of Thr308 and Ser473 and activation of Akt1, which is evident within 3 min of IGF-1 treatment [8,9], Akt1 nuclear accumulation occurs relatively slowly with maximal nuclear accumulation of Akt1 occurring after 20 min of IGF-1 treatment [9,10]. The mechanism responsible for Akt1 nuclear transport is unclear.

Numerous proteins have recently been found to be post-translationally modified with O-linked β-N-acetylglucosamine (O-GlcNAc) moieties. The O-GlcNAc-modification can affect several biochemical processes including signal transduction, protein localization, and protein–protein interactions [11,12]. O-GlcNAc-modification of proteins occurs enzymatically when a single GlcNAc unit is covalently attached to Ser and Thr residues on cytosolic and nuclear proteins [13], and on some proteins O-GlcNAc occurs reciprocal to phosphorylation on the same Ser-Thr sites [13]. The source of GlcNAc for the O-GlcNAc-modification is the hexosamine biosynthetic pathway (HBP), with one of the end products of the HBP being UDP-GlcNAc [14], the donor sugar nucleotide for O-GlcNAc. Thus, increased glucose flux through the HBP can contribute to increased levels of O-GlcNAcmodified proteins. Accumulation of O-GlcNAc-modified proteins can also be achieved by inhibition of N-acetyl-β-Dglucosaminidase (O-GlcNAcase), the enzyme which catalyzes the removal of O-GlcNAc from proteins [15]. O(2-acetamido- 2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc) potently and selectively inhibits O-GlcNAcase [15–17], and has become a valuable tool for the identification of O-GlcNAc-modified proteins. It is unknown if Akt1 is directly modified with O-GlcNAc, and if this modification affects IGF-1-induced Akt signaling. The present study shows that in SH-SY5Y neuroblastoma cells a subfraction of cytosolic Akt1 is constitutively modified with O-GlcNAc, and PUGNAc treatment increased the level of O-GlcNAc-modified Akt1 in the cytosol and caused the accumulation of Akt1 in the nucleus.

2. Materials and methods

2.1. Cell culture

SH-SY5Y neuroblastoma cells were cultured in RPMI 1640 medium as previously described [8]. Cells were plated at a density of 105 cells/60 mm dish and were cultured for 48 h prior to treatments. Adherent cells were rinsed twice with serum-free media supplemented with 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin and were cultured in serum-free media for 24 h prior to treatments with PUGNAc or IGF-1. HEK293 cells were grown in DMEM/F-12 supplemented with 5% fetal bovine serum, 100 μg/ml penicillin, and 100 μg/ml streptomycin (Gibco-Invitrogen).

2.2. Detection of glycosylated Akt1 with wheat germ agglutininconjugated beads

Cytosolic and nuclear extracts were prepared as described previously [18]. Cells were lysed in 100 μl lysis buffer (10 mM Tris, pH 7.5, 10 mM NaCl, 3 mM MgCl, 0.05% NP-40, 1 mM EGTA, 1 mM sodium orthovanadate, 50 mM sodium fluoride, 100 mM PMSF, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 5 μg/ml pepstatin A, 1 nM okadaic acid, and 50 μM PUGNAc (Toronto Research Chemicals)). Protein concentrations were determined using the bicinchoninic acid method (Pierce). Purity of the nuclear and cytosolic samples was determined by immunoblotting the two fractions with antibodies for the compartment-specific proteins tubulin (Sigma) a cytosolic protein and histone (Chemicon) a nuclear protein.

To detect O-glycosylation of Akt1, 50 μg of cytosolic protein in a total volume of 100 μl lysis buffer was first precleared with 30 μl of 50% v/v of unconjugated agarose beads for 2 h at 4 °C to eliminate non-specific interactions of proteins with the agarose beads. The agarose beads were collected by centrifuging in a microcentrifuge (20817 × g, 1 min). The supernatant was transferred to a new reaction tube and incubated with 30 μl of 50% v/v wheat germ agglutinin (WGA)-conjugated agarose beads (Vector Laboratories) for 20 h at 4 °C. The WGA-conjugated agarose beads were collected by centrifugation and the supernatant was saved to measure the amount of unbound Akt1. The unconjugated agarose beads and the WGA-conjugated agarose beads were washed three times in lysis buffer and mixed with 30 μl of Laemmli sample buffer, and placed in a boiling waterbath for 5 min. To determine the amount of unbound Akt1, the protein concentration was measured in the saved supernatant and 1 μg of protein was mixed with 30 μl of Laemmli sample buffer, boiled, and loaded onto a 7.5% SDS–polyacrylamide gel in parallel with the eluted proteins from the unconjugated beads and the WGA-conjugated beads. Proteins were immunoblotted as previously described [8] with Akt1 antibody (Sigma). Cytosolic extracts were also immunoblotted with antibodies for pThr308Akt, pSer473Akt, pSer9GSK3β (Cell Signaling), O-GlcNAc (RL2) (Affinity Bioreagents), GSKβ (BD Biosciences).

For peptide N-glycosidase F (PNGase F) (New England BioLabs) treatment, cells were lysed in 50 mM sodium phosphate buffer, pH 7.5, containing 0.05% NP-40, and protease inhibitors. The cytosolic extracts (100 μg of protein) were incubated with 20 units of PNGase F for 20 h in a 37 °C waterbath. The extracts were assayed for O-Glc- NAc-modification using the WGA-conjugated beads as described above. For β-N-acetyl-hexosaminidase (β-hex) (New England Bio- Labs) treatments, cytosolic extracts were incubated with WGA-conjugated beads as described above, then incubated with 10 units of β-hex in 50 mM sodium citrate buffer, pH 4.5, for 2 h in a 37 °C waterbath. The beads were washed and the eluted proteins were immunoblotted for Akt1. A 10 μl aliquot from samples incubated under identical conditions without the glycosidases were immunoblotted for Akt1 to verify that proteolysis of Akt1 had not occurred under these conditions. To disrupt protein complexes that may contribute to Akt1 association with WGA, cytosolic extracts were incubated with WGA-conjugated agarose beads in the presence of 0.3% SDS, and eluted proteins were immunoblotted for Akt1.

2.3. Measurement of Akt1 activity

Akt1 activity was measured using a non-radioactive Akt kinase assay kit (Cell Signaling) with some minor modifications. Briefly, 100 μg of cytosolic extracts were immunoprecipitated with 3.5 μg of Akt1 antibody in lysis buffer for 18 h at 4 °C. The immobilized immune complexes were washed three times with 500 μl lysis buffer and washed twice with 500 μl kinase buffer (25 mM Tris, pH 7.5, 5 mM β-glycerophosphate, 2 mM DTT, 0.1 mM sodium orthovanadate, and 10 mM MgCl2). The immune complexes were resuspended in 50 μl of kinase buffer containing 0.2 mM ATP and 1 μg of GSK3 fusion protein substrate. The reaction was incubated in a 30 °C water bath for 30 min. The reaction was stopped by the addition of SDS sample buffer followed immediately by boiling for 5 min. The samples were loaded onto a 12% SDS–polyacrylamide gel, transferred onto nitrocellulose and immunoblotted with a GSK3α/β-phospho-specific antibody to evaluate Akt1 activity. The blot was then stripped and reblotted with the Akt1 antibody to determine efficiency of immunoprecipitation.

2.4. Cell transfections

To express wild-type Akt (wtAkt) and mutated Akt (DDAkt), HEK293 cells (5 × 105 cells) were transfected in 35 mm dishes in DMEM/F-12 media supplemented with 1% fetal bovine serum, 100 μg/ml penicillin, and 100 μg/ml streptomycin (Gibco-Invitrogen) at 37°C. After achieving approximately 70% growth confluency, each dish was transfected with 2 μg of either wtAKT or DDAKT plasmids using Fugene6 (Roche Diagnostics Corporation) according to manufacturer’s instructions. After 24 h post-transfection, the cells were harvested as described previously.

2.5. Immunofluorescence

Cells were immunofluorescently labeled as described previously [18]. Cells cultured on poly-D-lysine-coated glass coverslips were treated with PUGNAc (50 μM, 30 min). The cells were washed with PBS and then fixed and permeabilized in cold methanol–acetone (1:1) for 10 min at −20 °C. The coverslips were washed twice with PBS and blocked with 2% BSA in PBS for 2 h. The coverslips were incubated with Akt1 antibody (28 μg/ml) for 18 h at 4 °C. The coverslips were thoroughly washed with PBS and incubated for 1 h at room temperature with 10 μg/ml fluorescein isothiocyanate-conjugated anti-mouse antibody (Jackson Immunoresearch). The coverslips were washed thoroughly with PBS and incubated with 1 ng/ml Hoechst 33342 for 10 min at room temperature. The coverslips were washed with PBS and a final rinse with water, and mounted onto glass coverslips and examined by a fluorescence microscope (Nikon) set at 400 × magnification.

3. Results

To examine the O-GlcNAc-modification of Akt1, cells were lysed and the cytosolic extract (50 μg of protein input) was incubated with either unconjugated agarose beads or WGAconjugated agarose beads. WGA-conjugated agarose beads bind O-GlcNAc-modified proteins with high affinity [19]. Akt1 binding to WGA was clearly evident (Fig. 1A) while only minimal non-specific binding of Akt1 was seen on the control unconjugated agarose beads. Abundant Akt1 was detected in a 1 μg sample of the unbound proteins (UB), indicating that a large fraction of the cytosolic Akt1 is not constitutively modified with GlcNAc. For all subsequent experiments the cytosolic extracts were first precleared with unconjugated beads. To test if the glucose concentration in the media impacts Akt1 glycosylation, cells were cultured in glucose-free media for 24 h, then treated with 20 mM glucose for 1 h. This resulted in a marked increase in the WGA binding of Akt1 (Fig. 1B), indicating that Akt1 glycosylation is affected by glucose content in the media. Changes in glucose concentration however did not affect basal or IGF-1-induced Akt1 phosphorylation at Thr308 and Ser473 (data not shown).

Fig. 1.

Fig. 1

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Akt1 is modified with O-GlcNAc. (A) Cytosolic extracts from SH-SY5Y cells were incubated with either unconjugated agarose beads or WGA-conjugated agarose beads. The proteins bound to the unconjugated agarose beads (beads) and WGA-conjugated agarose beads (WGA) were eluted by boiling, and together with the unbound proteins (UB) were immunoblotted with Akt1 antibody. (B) SH-SY5Y cells were cultured in glucose-free media for 24 h, then treated with 20 mM glucose for 1 h. Cytosolic extracts were analyzed for O-GlcNAc-modification of Akt1 using WGA-conjugated agarose beads and cytosolic extracts were immunoblotted for total levels of Akt1. (C) Cytosolic extracts (100 μg of protein) were incubated with 20 units of PNGase F for 20 h in a 37 °C waterbath, and then incubated with WGA-conjugated beads. For β-N-acetyl-hexosaminidase (β-hex) treatments, cytosolic extracts were incubated with WGA-conjugated beads as described above, then incubated with 10 units of β-hex for 2 h in a 37 °C waterbath. The eluted proteins were immunoblotted for Akt1. An aliquot from samples without the glycosidases were immunoblotted for Akt1 to verify that proteolysis of Akt1 had not occurred under these conditions. To confirm that β-hex did not affect Akt1 phosphorylation, Akt1 was immunoprecipitated from 100 μg of cytosolic extracts from cells treated with and without IGF-1 (50 ng/ml, 30 min), then incubated in the absence and presence of β-hex, and then immunoblotted with pSer473Akt antibody. The blot was stripped and then reblotted with Akt1 antibody. (D) Cells were treated with 50 μM PUGNAc for 45 min and cytosolic extracts were analyzed for O-GlcNAc-modification of Akt1 using WGA-conjugated beads. Protein complexes were disrupted by the addition of 0.3% SDS during the incubation of the cytosolic extracts with the WGA-conjugated agarose beads. The eluted proteins were immunoblotted for Akt1.

WGA binds terminal GlcNAc residues, thus several methods were used to determine if the Akt1 binding to WGA was mediated by O-GlcNAc. Lysates were incubated with excess PNGase F, which removes N-linked GlcNAc [20]. PNGase F treatment did not affect Akt1 binding to WGA (Fig. 1C) however incubation with β-N-acetylhexosaminidase (β-hex), which removes terminal O-linked glycosidic modifications [21], markedly decreased Akt1 binding to WGA. Thus the WGA-Akt1 interaction is due primarily to O-GlcNAc on Akt1. To test if β-hex treatment affected Akt1 phosphorylation, cells were treated with and without IGF-1 (50 ng/ml, 30 min) and Akt1 was immunoprecipitated from cytosolic fractions. The immunoprecipitated Akt1 was reacted in the absence or presence of β-hex and then immunoblotted for pSer473. β-hex treatment did not affect basal or IGF-1-induced Akt1 Ser473 phosphorylation (Fig. 1C), nor did it affect Akt1 Thr308 phosphorylation (data not shown). Thus, β-hex treatment selectively removed Akt O-glycosylation.

To further establish O-GlcNAc-modification of Akt1, cells were treated with PUGNAc (50 μM for 45 min) to increase the level of O-GlcNAc-modified proteins. PUGNAc treatment resulted in a marked increase in Akt1 binding to WGA (Fig. 1D). To confirm that the precipitation of Akt1 with WGA was due to O-GlcNAc on Akt1 and not due to an O-glycosylated Akt1-binding protein, cytosolic extracts were incubated with WGA in the presence of 0.3% SDS, a method to disrupt protein complexes which has been used previously for O-GlcNAc analysis [22]. The presence of SDS did not prevent Akt1 binding to WGA and it did not affect the PUGNAc-mediated increase in Akt1 binding to WGA. Thus, the binding of Akt1 to WGA is due to a direct interaction between Akt1 and WGA and not due to an intermediary O-glycosylated protein bound to Akt1. Total cytosolic Akt1 levels were not increased with PUGNAc treatment indicating that PUGNAc did not affect Akt1 synthesis or stability. Taken together, these results show that a limited pool of cytosolic Akt1 is constitutively modified with O-GlcNAc and that the O-GlcNAc-modification of Akt1 is dynamic.

The effects of PUGNAc treatment on Akt1 O-GlcNAc-modification and Akt1 phosphorylation were tested. Cells were time-dependently (0–60 min) treated with PUGNAc and cytosolic extracts were immunoblotted with an antibody, RL2, that detects O-GlcNAc on proteins. PUGNAc treatment caused an overall increase in the level O-GlcNAc-modified proteins in the cytosol (Fig. 2A) and increased O-GlcNAc-modification of Akt1 as early as 15 min following PUGNAc treatment (Fig. 2B). Maximum, 203% of control, Akt1 O-GlcNAc-modification occurred after 30 min of PUGNAc treatment, and this level was maintained after 60 min of PUGNAc treatment. There was little or no decrease in basal Akt1 phosphorylation at Thr308 and Ser473 following PUGNAc treatment, and PUGNAc did not decrease basal Akt1 activity (Fig. 2C). Thus, O-GlcNAc-modification of Akt1 did not attenuate any of the activity-associated indices of Akt1.

Fig. 2.

Fig. 2

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PUGNAc and IGF-1 increase cytosolic O-GlcNAc levels and O-GlcNAc-modification of Akt1. (A) SH-SY5Y cells were time-dependently treated with 50 μMPUGNAc and 50 ng/ml IGF-1 for the indicated times. Cytosolic extracts were immunoblotted for O-GlcNAc with RL2 antibody. (B) Cytosolic extracts from PUGNAc and IGF-1 treated cells were incubated with WGA-conjugated beads for analysis of O-GlcNAc-modification of Akt1. O-GlcNAc modified Akt1 bands were quantitated by scanning densitometry. Means ± S.E., n = three experiments; *P < 0.05 (ANOVA) compared to values from untreated cells. Cytosolic extracts were immunoblotted with pThr308Akt1, pSer473Akt, and Akt1 antibodies. (C) For measurement of Akt1 activity, cells were time-dependently treated with 50 μM PUGNAc or treated with IGF-1 (50 ng/ml, 30 min). The cytosolic extracts were immunoprecipitated with Akt1 antibody and incubated with GSK3 fusion protein substrate. The reaction mixture was immunoblotted with a GSK3 phospho-specific antibody. To determine efficiency of immunoprecipitation the blot was stripped and reblotted with Akt1 antibody.

IGF-1 potently induces phosphorylation of Akt1 at Thr308 and Ser473. The effect of IGF-1 treatment was tested on Akt1 phosphorylation and O-GlcNAc-modification. IGF-1 treatment resulted in a robust and rapid increase in Thr308 and Ser473 phosphorylation of Akt1 (Fig. 2B). Maximum phosphorylation occurred in less than 15 min of IGF-1 treatment and remained at maximum level for the entire 60 min IGF-1 time course. Interestingly, IGF-1 treatment also time-dependently increased O-GlcNAc on several cytosolic proteins (Fig. 2A) and also increased Akt1 binding to WGA (Fig. 2B), indicating that a subfraction of Akt1 is modified with O-GlcNAc following IGF-1 stimulation. Compared to Akt1 phosphorylation the increase in O-GlcNAc-modification of Akt1 was markedly delayed. O-GlcNAc-modified Akt1 level increased after 15 min of IGF-1 treatment to 135% of control. Maximum O-GlcNAc-modified Akt1 was seen between 30 and 45 min of IGF-1 treatment (198% and 206%, respectively, of control). Thus, IGF-1 treatment caused both robust phosphorylation and activation of Akt1 and increased the level of O-GlcNAc-modified Akt1.

In the next set of experiments the relationship between O-Glc- NAc-modification and phosphorylation of Akt1 was investigated. Cytosolic extracts from control and IGF-1-treated cells were incubated with WGA. The WGA-bound proteins were eluted and immunoblotted with pThr308 and pSer473Akt antibodies. Large increases in Thr308 and Ser473 phosphorylation were seen on the WGA-bound Akt1 compared to the total level of bound Akt1 (Fig. 3A), indicating that nearly all of the O-Glc- NAc-modified Akt1 is also phosphorylated on Thr308 and Ser473. To further confirm if Akt1 is phosphorylated and modified with O-GlcNAc simultaneously, HEK293 cells were transiently transfected with wild-type (wt) Akt and with a mutant, constitutively active Akt with both Thr308 and Ser473 mutated to Asp (DD). These mutations mimic phosphorylation at these sites. Both the wtAkt and DDAkt were tagged with hemagglutinin (HA) to differentiate between the expressed Akt and endogenous Akt1. Cytosolic extracts from the transfected cells were incubated with WGA-conjugated beads and the eluted proteins were immunoblotted with HA antibody. Both the wtAkt and DDAkt boundWGAequally well (Fig. 3B), indicating that Akt1 can be modified with O-GlcNAc and phosphorylated on Thr308 and Ser473 simultaneously.

Fig. 3.

Fig. 3

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Akt1 can be simultaneously modified with O-GlcNAc and phosphorylated on Thr308 and Ser473. (A) SH-SY5Y cells were stimulated with 50 ng/ml IGF-1 for 30 min. Cytosolic extracts were incubated with WGA-conjugated agarose beads, and the eluted proteins were immunoblotted with pThr308Akt1, pSer473Akt1, and Akt1 antibodies. (B) HEK293 cells were transiently transfected with wild-type HA-tagged Akt1 or an HA-tagged Akt1 mutant in which the Thr308 and Ser473 were dually mutated to Asp (DDAkt). Cytosolic extracts were incubated with WGA-conjugated agarose beads and eluted proteins were immunoblotted with HA antibody. (C) SH-SY5Y cells were treated with 50 μM PUGNAc 30 min prior to treatment with IGF-1 (50 ng/ml, 30 min). Cytosolic extracts were immunoblotted with pThr308Akt1, pSer473Akt1, Akt1, pSer9GSK3β, and GSK3β antibodies. (D) Cells were treated with 20 μM LY294002 30 min prior to treatment with 50 ng/ml IGF-1 for 30 min or 50 μM PUGNAc for 30 min. Cytosolic extracts were incubated with WGA-conjugated beads for analysis of O-GlcNAc-modification of Akt1, and cytosolic extracts from IGF-1 and LY294002-treated cells were also immunoblotted for pThr308Akt and Akt1.

PUGNAc treatment has previously been reported to reduce the insulin-induced phosphorylation at Thr308 of Akt1 and Ser9 of GSK3β [23]. We wanted to determine if an elevation of O-GlcNAc levels by PUGNAc would also block IGF-1- mediated Akt1 signaling, thus the effect of PUGNAc treatment on IGF-1-induced Akt1 and GSK3β phosphorylation was tested. Cells were treated with PUGNAc 30 min prior to treatment with IGF-1 for 30 min. PUGNAc treatment did not cause any deficits in Akt1 Thr308 and Ser473 phosphorylation, and Ser9 phosphorylation of GSK3β (Fig. 3C). Thus, unlike the reported detrimental effects of PUGNAc on insulin-induced Akt1 signaling [23], in the present study PUGNAc treatment did not attenuate IGF-1-induced Akt1 signaling.

IGF-1 induces the activation of PI3K signaling. Since IGF-1 treatment was found to increase O-GlcNAc-modification of Akt1, the effect of the PI3K inhibitor LY294002 was tested on IGF-1-induced Akt1 O-glycosylation. IGF-1 treatment increased Akt1 O-glycosylation (Fig. 3D) which was mostly blocked by pretreatment of cells with LY294002 prior to stimulation with IGF-1. The blockade of IGF-1-induced Akt1 OGlcNAc- modification corresponded with the inhibition of Akt1 phosphorylation at Thr308. By comparison, O-Glc- NAc-modification of Akt1 by PUGNAc treatment was independent of PI3K signaling (Fig. 3D), as LY294002 pretreatment did not block PUGNAc-induced Akt1 O-glycosylation. Thus, IGF-1-induced Akt1 O-glycosylation is dependent on activation of PI3K signaling.

Because it has been suggested that O-GlcNAc may play a role in nuclear localization of proteins [24] we tested the possibility that PUGNAc treatment would lead to a redistribution of Akt1 in the nucleus. PUGNAc treatment timedependently increased Akt1 nuclear accumulation within 15 min of PUGNAc treatment (Fig. 4A) and marked accumulations of Akt1 in the nucleus were evident 30 and 60 min following PUGNAc treatment. In addition, immunofluorescence staining of Akt1 revealed that little Akt1 was detectable within the nucleus in control cells (Fig. 4C), but increased nuclear staining of Akt1 as well as perinuclear staining of Akt1 was evident following PUGNAc treatment. PUGNAc did not increase the nuclear levels of GSK3β (Fig. 4A), a protein which also shuttles into the nucleus [18], indicating that PUGNAc did not elicit a generalized accumulation of proteins into the nucleus. The level of histone, a resident nuclear protein, was not altered by PUGNAc treatment. Tubulin, a highly abundant cytosolic protein was not evident in the nuclear fraction (Fig. 4B), indicating that the nuclear extract was essentially devoid of contaminating cytosolic proteins. Thus, at least one functional outcome of increased O-GlcNAc levels on Akt1 signaling is the facilitation of Akt1 entry into the nucleus.

Fig. 4.

Fig. 4

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PUGNAc induces nuclear accumulation of Akt1. (A) SH-SY5Y cells were treated with 50 μM PUGNAc for the indicated times. Nuclear and cytosolic extracts were immunoblotted with Akt1 antibody and nuclear extracts with histone antibody and GSK3β antibody. (B) Complete separation of the cytosolic and nuclear fractions was confirmed by immunoblotting the cytosolic and nuclear extracts with tubulin and histone antibodies. (C) Control and PUGNAc-treated (50 μM, 30 min) cells were immunofluorescently labeled with Akt1-fluorescein isothiocyanateconjugated antibody, upper panels. Nuclei were stained with Hoechst 33342, lower panels.

4. Discussion

Much interest has recently focused on identifying proteins in the PI3K/Akt signaling pathway that are modified with O-Glc- NAc, because an elevation of O-GlcNAc-modified proteins has been associated with insulin resistance partially due to major defects in PI3K/Akt signaling [23,25]. Although several proteins upstream of Akt have been shown to be modified with O-GlcNAc, O-GlcNAc-modification of Akt1 has not been reported. In the present study the role of O-GlcNAc on IGF-1- induced Akt1 signaling was examined. A subfraction of the cytosolic pool of Akt1 was found to be constitutively modified with O-GlcNAc. In addition, treatment of cells with the selective O-GlcNAcase inhibitor PUGNAc and treatment with IGF-1 significantly increased O-GlcNAc-modification of the cytosolic subfraction of Akt1. Thus, this is the first demonstration that a portion of the cytosolic Akt1 is dynamically modified with O-GlcNAc.

WGA is commonly used for the separation of O-GlcNAcmodified proteins. Since WGA binds any terminal GlcNAc residue, several methods were used to confirm that Akt1 bears the O-GlcNAc-modification [26]. Akt1 binding to WGA was markedly reduced after incubation with β-hex but not with PNGase F. Treatment of cells with PUGNAc increased Akt1 binding to WGA, and the dissociation of protein complexes with SDS did not disrupt PUGNAc-induced Akt1 binding to WGA. Together, these results indicate that Akt1 is subject to modification by O-GlcNAc. Several antibodies have been developed which recognize O-GlcNAc moieties, but these antibodies, and WGA, have inherent limitations [27]. For example, proteins such as GSK3β can be modified with O-GlcNAc in vitro [28], however the O-GlcNAc-modification of GSK3β was below the threshold of detection by an O-GlcNAc-specific antibody [23]. O-GlcNAc antibodies were also evaluated for the detection of O-GlcNAc on Akt1 with mixed results (data not shown). Thus, the WGA lectin-based method may be more useful for analysis of the O-GlcNAc-modification of Akt1.

In the present study the ramification of increased O-GlcNAc on IGF-1-induced Akt1 signaling was examined. Elevations in O-GlcNAc levels by PUGNAc treatment did not affect basal Akt1 phosphorylation at Thr308 and Ser473, alter basal Akt1 activity, or result in the attenuation of IGF-1-induced Akt1 signaling. Surprisingly, IGF-1 treatment itself increased the levels of O-GlcNAc-modified proteins in the cytosol, increased the level of O-GlcNAc-modified Akt1, and robustly activated Akt1. The Akt1 bound to WGA was also found to be highly phosphorylated on Thr308 and Ser473 following IGF-1 stimulation, and an Akt1 mutant devoid of the Thr308 and Ser473 sites bound WGA equivalently well as the wild-type Akt1. Furthermore, IGF-1-induced Akt1 O-gly- cosylation was blocked by LY294002 indicating the dependence of IGF-1-induced Akt1 O-glycosylation on PI3K signaling. Thus, these observations indicate that Akt1 can be simultaneously modified with O-GlcNAc and phosphorylated on Thr308 and Ser473. A high glucose concentration was also found to substantially increase the level of O-GlcNAc-modified Akt1, however decreases in basal Akt1 phosphorylation, or attenuation of IGF-1 signaling under hyperglycemic conditions were not observed (data not shown). The reported effects of PUGNAc on insulin-induced Akt signaling are mixed. For example, Vosseller et al. [23] reported that treatment of 3T3-L1 adipocytes with 100 μM PUGNAc for 16 h resulted in insulin resistance due to increased O-GlcNAc-modification of proteins and the partial blockade of insulin-induced Akt phosphorylation at Thr308. A concomitant decrease in Ser9 phosphorylation of GSK3β was also reported. Conversely, Arias et al. [29] found that treatment of rat skeletal muscle tissue with 100 μmol/L of PUGNAc for 19 h did not result in any noticeable diminution of insulin-induced phosphorylations at Thr308 and Ser473 of Akt or Ser9 of GSK3β, although insulin resistance and O-GlcNAc-modification of proteins was evident. Mechanistically speaking, insulin and IGF-1 activate PI3K/Akt1 signaling in essentially the same manner. In the present study acute treatment of 50 μM PUGNAc, total exposure time of 60 min, did not impede the full IGF-1-induced activation of Akt1, in concordance with Arias et al. using insulin [29]. However we cannot rule out the possibility that greater accumulations of O-GlcNAc-modified proteins and O-Glc- NAc-Akt1 may occur by chronic PUGNAc treatment which could deleteriously affect Akt1 signaling.

Having shown that Akt1 is modified with O-GlcNAc, the potential effect of this modification on the nuclear distribution of Akt1 was examined, as there is some evidence to suggest that O-GlcNAc may function as a signal for nuclear transport [24]. Akt1 is known to enter the nucleus subsequent to its activation. Interestingly, PUGNAc treatment was found to increase the level of Akt1 in the nucleus independent of phosphorylation of Thr308 and Ser473. The elevation in the nuclear level of Akt1 paralleled the increase in O-GlcNAc-modified Akt1 by PUGNAc. These results must be interpreted with caution because the mechanisms underlying Akt1 nuclear translocation remain unclear. Akt1 has been shown to increase in the nucleus subsequent to its phosphorylation at Thr308 [10], and by a process which requires the binding of Akt1 with the protein product of the TCL1 oncogene [30]. Thus, it is possible that the O-GlcNAc moiety on Akt1 may directly increase Akt1 binding to the nuclear pore complex, as indicated by the perinuclear staining of Akt1 following PUGNAc treatment (Fig. 4C), to facilitate the entry of Akt1 into the nucleus, or the O-GlcNAc-modification may increase the association of Akt1 with a nuclear transport protein.

It has been widely speculated that Akt1 is modified with O-GlcNAc, but this has never been reported, until now. We have shown that a subfraction of Akt1 is constitutively and dynamically modified with O-GlcNAc. Further investigations will be necessary to determine the sites on Akt1 modified with O-Glc- NAc, the effects of O-GlcNAc on Akt1 function, and its role in Akt1 nuclear transport.

Acknowledgments

We thank Paramita Mookherjee for providing the HA-Akt constructs. This research was supported by National Institutes of Health Grant NS044853.

Abbreviations

O-GlcNAc

O-linked β-N-acetylglucosamine

O-GlcN-Acase

N-acetyl-β-D-glucosaminidase

GSK3β

glycogen synthase kinase-3β

HBP

hexosamine biosynthetic pathway

β-hex

β-N-acetyl- hexosaminidase

IGF-1

insulin-like growth factor-1

PNGase F

peptide N-glycosidase F

PUGNAc

O(2-acetamido-2-deoxy-D-glucopyranosylidene) amino-N-phenylcarbamate

Footnotes

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