ORIGINAL PAPER

Glutamate Activates Protein Kinase B (PKB/Akt) through AMPAReceptors in Cultured Bergmann Glia Cells

Moises Morales Æ Martha E. Gonzalez-Mejıa ÆAlfonso Bernabe Æ Luisa C. R. Hernandez-Kelly ÆArturo Ortega

Accepted: 14 December 2005 / Published online: 3 May 2006

� Springer Science+Business Media, Inc. 2006

Abstract Glutamate is involved in gene expression

regulation in neurons and glial cells through the activation

of a diverse array of signaling cascades. In Bergmann glia,

Ca2+-permeable a-hydroxy-5-methyl-4-isoazole-propionic

acid (AMPA) receptors become tyrosine phosphorylated

after ligand binding and by these means form multiprotein

signaling complexes. Of the various proteins that associate

to these receptors, the phosphatidylinositol 3-kinase (PI-

3K) deserves special attention since D3-phosphorylated

phosphoinositides are docking molecules for signaling

proteins with a pleckstrin homology domain. In order to

characterize the role of PI-3K in AMPA receptors signal-

ing, in the present report we analyze the involvement of

the serine/threonine protein kinase B in this process. Our

results demonstrate an augmentation in protein kinase B

phosphorylation and activity after glutamate exposure.

Interestingly, the effect is independent of Ca2+ influx, but

sensitive to Src blockers. Our present findings broaden our

current knowledge of glial glutamate receptors signaling

and their involvement glutamatergic neurotransmission.

Keywords PKB Æ Glutamate receptors

phosphorylation Æ Bergmann glia Æ GSK3-b Æ Src

Introduction

Glutamate (Glu) is the main excitatory neurotransmitter in

the mammalian central nervous system (CNS) [1]. It trig-

gers a board variety of signaling events that result in gene

expression regulation both in neurons and in glial cells. Glu

acts through ionotropic and metabotropic receptors [2]. The

ionotropic Glu receptors subtypes are N-methyl-D-aspartate

(NMDA), a-amino-3-hydroxy-5-ethyl-isoxazole-4-propi-

onic acid (AMPA) and kainate (KA) receptors. Metabo-

tropic Glu receptors are coupled to G proteins and linked

either to phosphoinositide metabolism (Group I) or

inhibition of adenylate cyclase (Groups II and III).

Bergmann glia cells (BGC) surround parallel fiber-Pur-

kinje cell synapses in the cerebellum. The repertoire of

glutamatergic receptors in these cells include both iono-

tropic as well as metabotropic receptors [3, 4]. Glu released

from the parallel fibers depolarizes the BGC triggering not

only a significant Ca2+ influx but also a membrane to nuclei

cascade that is involved in transcriptional regulation [5].

Interestingly, AMPA receptors are involved in this process

and become tyrosine phosphorylated leading to the

recruitment and activation of transduction molecules

such as the focal adhesion kinase pp125FAK and the

phosphatidilinositol-3 kinase (PI-3K) [6].

PI-3K is composed of a regulatory subunit (p85) and a

catalytic subunit (p110). The p85 subunit contains two SH2

(Src homology 2) domains, which bind tyrosine phosphor-

ylated motifs present in the cytoplasmic domains of cell

surface receptors. Binding of the p85 to a phosphorylated

receptor promotes activation of p110 subunit. Activated

PI3K generates D3-phosphorylated phosphoinositides,

PtdIns [3, 4] P2 and PtdIns [3–5] P3, a motif recognized by

the pleckstrin homology domain present in several protein

kinases.

M. Morales Æ M. E. Gonzalez-Mejıa Æ L. C. R. Hernandez-Kelly ÆA. Ortega (&)

Departamento de Genetica y Biologıa Molecular,

Cinvestav-Zacatenco, Mexico , DF, Mexico

e-mail: arortega@cinvestav.mx

A. Bernabe

Unidad Academica Facultad de Ciencias Quımico-Biologicas,

Universidad Autonoma de Guerrero, Chilpancingo, Guerrero,

Mexico

Neurochem Res (2006) 31:423–429

DOI 10.1007/s11064-005-9034-2

123

Protein kinase B (PKB/Akt), a member of AGC protein

kinase family, is a well characterized effector of PI-3K

involved in signal transduction pathways activated by

growth factors and other ligands. It participates in several

cellular functions including cell growth, metabolism,

apoptosis and translational control [7]. Three PKB isoforms

have been described: PKB-a/Akt-1, PKB-b/Akt-2 and PKB-

c/Akt-3. PKB is composed of a N-terminal PH domain, a

central kinase domain in which Thr308 may be phosphory-

lated and a regulatory C-terminal hydrophobic domain that

also contains another phosphorylation site (Ser473). In most

of the systems studied thus far, PI-3K is necessary for PKB

activation and it is accepted that its products, phosphati-

dylinositol-3,4,5-triphosphates, recruit PKB to the plasma

membrane. Although, it is important to mention that PKB

can also be activated through PI-3K-independent pathways

[8]. PKB is phosphorylated on Thr308 in the activation loop

by the phosphoinositide-dependent kinase 1 (PDK1) and on

Ser437 by a yet-undefined kinase. Activated PKB is then

translocated to the cytosol and nucleus where it acts over its

substrates. PKB targets several key proteins in cell physi-

ology, including apoptosis regulators, transcription factors

and proteins involved in translational control.

The role of PKB in the stimulation of translation initiation

is mediated through the glycogen synthase kinase 3-b(GSK3-b) and the mammalian target of rapamycin (mTOR)

[9]. GSK3-b is a ubiquitously expressed protein–serine/

threonine kinase that is inhibited after PKB phosphorylation

in Ser9 [10]. In the other hand, PKB directly phosphorylates

the mammalian target of rapamycin (mTOR), which with its

partner raptor phosphorylates the eIF4E binding protein-1

(4EBP1) stimulating translation initiation [11].

The signaling events triggered by Glu within BGC have

been hypothesized to contribute to the plasticity of the

parallel fiber-Purkinje cell synapse [12]. Considering that

in cultured chick BGC, Ca2+-permeable AMPA receptors

become tyrosine-phosphorylated after Glu exposure and

thereby recruit PI-3K, we decided to explore a

Glu-dependent phosphorylation of two of the most char-

acterized PI-3K downstream effectors, PKB and GS3K-b.

We were able to detect an AMPA receptors-mediated in-

crease in PKB phosphorylation and a PKB-dependent

GS3K-b phosphorylation. These results reveal the com-

plexity of Glu signaling in BGC and favor their important

participation in glutamatergic neurotransmission.

Experimental procedure

Materials

Tissue cultures reagents were from Gibco (Grand Island,

NY). Plasticware was purchased from Costar (Cambridge,

MA). The antibodies used were rabbit polyclonal anti-

PKB, anti-phospho PKB (Ser-473) and anti-phospho-

GSK3-b (Ser-9) (Santa Cruz Biotechnology, Santa Cruz,

CA). Horseradish peroxidase-linked anti-rabbit antibodies

were purchased from Zymed Laboratories (San Francisco,

CA). The enhanced chemiluminescence (ECL) Western

blot detection reagents were obtained from Amersham

(Buckinghamshire, UK). The Bradford and SDS–PAGE

reagents were form Bio-Rad (Hercules, CA). All other

reagents were obtained from Sigma Chemical Co.

(St. Louis, Mo).

Cell cultures and stimulation protocol

Chick cerebellar BGC were prepared from 14 days old

chick embryos as described previously [13]. Cells were

grown for 5–6 days on plastic dishes in Dulbecco’s mod-

ified Eagle’s medium (DMEM) supplemented with 10%

fetal calf serum, 2 mM glutamine and 50 lg/ml gentami-

cin. The cultures contained >95% of anti-KBP positive

cells. Confluent monolayers were incubated for 2 h in

DMEM with 0.5% bovine serum albumin (BSA) to reduce

the basal PKB and GSK3 phosphorylation. Cells were

stimulated with the indicated ligands in solution A (25 mM

Hepes-Tris, 130 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2,

0.8 mM MgCl2, 33.2 mM glucose and 1 mM NaHPO4 pH

7.4). Total protein extracts were prepared from confluent

monolayers in 5 volumes of RIPA buffer (50 mM Tris–

HCl pH 7.2, 150 mM NaCl, 2% Triton X-100, 0.2% SDS).

After a 15 min incubation at 4�C, the cell suspension was

centrifuged for 5 min at 10,000 · g and the pellet dis-

carded. The protein concentration of the solubilized

material was determined by the Bradford method.

SDS–PAGE and Western blots

Equal amounts of protein (approximately 20 lg) were

denaturated in Laemmli’s sample buffer and resolved

through 10% sodium dodecyl sulfate (SDS) polyacryl-

amide gels and electroblotted to nitrocellulose membranes.

Blots were stained with Ponceau S stain to confirm that

protein content was equal in all lanes. Membranes were

soaked in PBS to remove the Ponceau S and incubated in

Tris-buffered saline (TBS) containing 5% dried skimmed

milk and 0.1% Tween 20 for 90 min to block the excess of

nonspecific protein binding sites. Membranes were then

incubated overnight at 4�C with the primary antibodies

(1:1,000 dilution), followed by secondary antibodies.

Immunoreactive polypeptides were detected using the ECL

chemiluminescence Kit (Amersham). Densitometric anal-

ysis was performed analyzed with the Prism program

(GraphPad). The optical densities of phospho-PKB and

phospho-GSK3b were normalized to PKB densities.

424 Neurochem Res (2006) 31:423–429

123

Statistical analysis

Data are expressed as the mean � standard error (SE). A

one-way analysis of variance (ANOVA) was performed to

determine significant differences between the different

conditions. When this analysis indicated significance (at

the 0.05 level), post-hoc Student–Newman–Keuls test

analysis was used to determine which conditions were

significantly different from each other (Prism, GraphPad

Software, San Diego, CA).

Results

Glu induces PKB and GSK3-b phosphorylation

In order to evaluate if Glu exposure resulted in PKB

phosphorylation, confluent BGC cultures were exposed for

30 min to 1 mM Glu. The results are shown in Fig. 1, a

significant increase in PKB Ser473 phosphorylation is

present after Glu treatment. As expected, exposure to the

phosphatase inhibitors, LiCl (3 mM) or Na3VO4 (1 mM)

also resulted in PKB phosphorylation [14]. The

time-dependency of the Glu effect was established, and as

depicted in panel A of Fig. 2, a gradual increase in PKB

phosphorylation is present after Glu treatment, reaching a

maximal value after 30 min. Note that after 60 min of Glu,

PKB phosphorylation is significantly reduced, suggesting

the involvement of a phosphatase activity since the total

PKB levels are not reduced. Although Ser473 phosphory-

lation has been correlated with PKB activity, we decided to

explore its activity. For this purpose, we investigated the

phosphorylation of one of the best characterized PKB

C Glu LiCl

p-PKB (Ser473)

PKB

% o

f co

ntr

ol

C Glu LiCl Na3VO40

100

200

300

400 *

* *

Na3VO4

Fig. 1 Glu induces PKB phosphorylation in cultured BGC. Confluent

monolayers were stimulated for 30 min with 1 mM Glu, 3 mM LiCl or

1 mM Na3VO4. Total protein extracts were obtained and Western

blots were carried out (20 lg of protein per lane). The membrane was

incubated with either anti-PKB or anti-phospho PKB (Ser473)

antibodies. The results of at least three independent experiments were

normalized after scanning of autorradiograms and the mean � SE

is shown.*P < 0.001 (ANOVA)

C 5 10 15 30 60 min

p-GSK3-β (Ser9)

PKB

C 5 10 15 30 60 min

p-PKB (Ser473)

PKB

C 5 10 15 30 60Time (min)

% o

f co

ntr

ol

C 5 10 15 30 60

Time (min)

% o

f co

ntr

ol

***

**

* *

**

**

0

50

100

150

200

250

300

0

50

100

150

200

250

(a)

(b)

Fig. 2 Kinetics of PKB and GSK3-b phosphorylation. Confluent

BGC monolayers were stimulated with 1 mM Glu for the indicated

periods. Total protein extracts were obtained and Western blots were

done as in Fig. 1. Panel A: The membrane was exposed to either

anti-PKB or anti-phospho PKB (Ser473) antibodies. Panel B: The

membrane was incubated with either anti-PKB or anti-phospho

GSK3-b (Ser9) antibodies. The results of at least three independent

experiments were normalized after scanning of autorradiograms and

the mean � SE is shown. *P < 0.001, **P < 0.01 (ANOVA)

Neurochem Res (2006) 31:423–429 425

123

substrates, GSK3-b. As depicted in panel B of Fig. 2, Glu

increases Ser9 GSK3-b phosphorylation as early as 5 min

after Glu treatment reaching its maximal effect after

15 min. Similarly to PKB phosphorylation, longer expo-

sure results in almost basal phosphorylation levels sup-

porting the participation of a phosphatase. In order to fully

demonstrate a receptor-mediated effect, we exposed the

cultured cells to increasing concentrations of Glu and

evaluated both PKB and GSK3-b phosphorylation. As

clearly shown in Fig. 3, in both cases, a dose-dependency

could be established. Furthermore, when the EC50 values

for both effects were established in order suggest a com-

mon signaling pathway, an almost identical value was

obtained (95 and 93 lM). These results indeed point out

that the Glu-dependent increase in GSK3-b phosphorylation

is mediated through PKB.

AMPA receptors mediate PKB and GSK-bphosphorylation

In order to gain insight of the identity of the Glu receptors

involved in the described increases in PKB and GSK3-bphosphorylation, confluent BGC cultures were stimulated

with a fixed 1 mM concentration of Glu and the agonists,

NMDA (plus 10 lM glycine), KA and (�)-1-aminocycl-

opentane-trans-1,3-dicarboxylic acid (t-ACPD) for 15 min.

From the results presented in Fig. 4, it is quite evident that

KA is as effective as Glu in promoting PKB and GSK3-bphosphorylation. In this context, the pre-exposure to a non-

NMDA receptors antagonist such as 6-cyano-7-nitroqui-

noxaline-2,3-dione (CNQX) at a 50 lM concentration,

should be able to block the Glu effect. Indeed it is the case as

it is demonstrated in Fig. 5, pointing out the involvement of

p-GSK3-β (Ser9)

PKB

p-PKB (Ser473)

PKB

C 1 10 100 1000500

% o

f co

ntr

ol

% o

f co

ntr

ol

µM

C 1 10 100 1000500 µM

Log [Glu]-7 -6 -5 -4 -3 -2

0

50100

150200

250300

350

-7 -6 -5 -4 -3 -20

50100

150200

250300

350

Log [Glu]

EC50 = 93 µM

EC50 = 93 µM

(a)

(b)

Fig. 3 Dose-dependence of PKB and GSK3-b phosphorylation.

Confluent monolayers were treated with the indicated Glu concen-

trations for 15 min and the levels of phospho PKB and phospho

GSK3-b were measured as in Fig. 2. *P < 0.001, **P < 0.01

(ANOVA)

C Glu KA NMDA t-ACPD

p-PKB (Ser473)

PKB

% o

f co

ntr

ol

C Glu KA NMDA t-ACPD

p-GSK3-β (Ser9)

PKB

% o

f co

ntr

ol

C Glu KA NMDA t-ACPD

AMPA

AMPA

AMPA

C Glu KA NMDA t-ACPDAMPA

00

50

100

150

200

250

0

50

100

150

200

250

*

**

*

**

*

(a)

(b)

Fig. 4 Pharmacological characterization of PKB and GSK3-bphosphorylation. Cells we exposed to a fixed 1 mM concentration

of the indicated Glu analogs for 15 min and the levels of phospho

PKB and phospho GSK3-b were measured as in Fig. 2. *P < 0.001,

**P < 0.01 (ANOVA)

426 Neurochem Res (2006) 31:423–429

123

AMPA receptors. As already mentioned, in BGC, AMPA

receptors once activated, become tyrosine phosphorylated

and by these means interact and activate PI-3K [6].

Therefore, it was critical to evaluate if the increase in PKB

and GSK3-b phosphorylation was dependent on PI-3K

activity. For this purpose, we pre-treated the cells with

100 nM Wortmannin. Figure 5 shows the results, inhibi-

tion of PI-3K activity completely prevents the Glu-depen-

dent PKB and GSK3-b phosphorylation, demonstrating the

involvement of PI-3K in the effect.

At this stage, an obligated experiment was the removal

of external Ca2+ from the stimulation media. As shown in

Fig. 6, both PKB and GSK3-b phosphorylation are still

present in a Ca2+-free medium that contains 500 lM eth-

ylenediaminotetraacetic acid (EDTA). These results are

compatible with the fact that AMPA receptors activate the

non-receptors tyrosine kinases Lyn and FAK and even

become tyrosine phosphorylated in an ion-influx-indepen-

dent manner [6, 15]. Although the pharmacological char-

acterization of the Glu response suggested an AMPA

receptors-mediated effect, we decided to explore the

possibility of a Glu-dependent release of Ca2+ from intra-

cellular stores. The inclusion of 25 lM 1,2-bis(o-amino-

phenoxy)ethane-N,N,N¢,N¢-tetraacetic acid (BAPTA) in

Ca2+-free medium did not prevent either PKB or GSK3-bphosphorylation (Fig. 6).

Although the identity of the tyrosine kinase involved in

AMPA receptors phosphorylation has not been fully

C Glu CNQX

p-GSK3-β (Ser9)

PKB

CNQX+

% o

f co

ntr

ol

CGlu

GluGlu

CNQXCNQX+

C Glu Glu CNQX Glu

p-PKB (Ser473)

PKB

CNQX+%

of

con

tro

l

C GluGluGlu

GluGlu

CNQXCNQX+

Wort+Wort

Wort

Wort

Wort

Wort+

Wort+

Wort+

*

**

020406080

100120140160180200

020406080

100120140160180200

(a)

(b)

Fig. 5 Effect of CNQX and Wortmannin on PKB and GSK3-bphosphorylation. Cultured cells were incubated with 50 lM CNQX or

100 nM Wortmannin during 30 min. After that, 1 mM Glu was added

and incubation continued for 15 min. Phospho PKB and phospho

GSK3-b were measured as in Fig. 2. *P < 0.001, **P < 0.01 (ANOVA)

C lu Glu Glu GluGlu BAPTA

p-PKB (Ser473)

PKB

EDTA+ BAPTA+ EDTA+BAPTA+

% o

f co

ntr

ol

C GluGlu

GluGluBAPTAEDTA+ BAPTA+

EDTA+BAPTA+

C Glu Glu Glu Glu BAPTA

p-GSK3- (Ser9)

PKB

EDTA+ BAPTA+ EDTA+BAPTA+

% o

f co

ntr

ol

C GluGlu Glu

Glu

BAPTAEDTA+ BAPTA+EDTA+

BAPTA+

*

****

* ***

0

50

100

150

0

50

100

150

200

(a)

(b)

Fig. 6 PKB and GSK3-b phosphorylation are Ca2+-independent.

BGC were incubated with 500 lM EDTA, 25 lM BAPTA or both

EDTA and BAPTA for 30 min. Thereafter the cells were stimulated

with 1 mM Glu for 30 min. Phospho PKB and phospho GSK3-b were

measured as in Fig. 2. *P < 0.001, **P < 0.01 (ANOVA)

Neurochem Res (2006) 31:423–429 427

123

established, a role for Src has been suggested [6]. Therefore,

we exposed the cultured cells to a 10 nM concentration

of the Src inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-

butyl)pyrazolol[3,4-d]pyrimidine (PP2), previous to Glu.

The inclusion of the Src inhibitor was enough to prevent

PKB and GSK3-b phosphorylation (Fig. 7). All the data

presented thus far, indicated that GSK3-b phosphorylation

is a result of a Glu-dependent PKB activation, nevertheless

it was important to show that PKB inhibition indeed

prevents GSK3-b is phosphorylation. For this purpose we

decided to use the PKB inhibitor 1L-6-hydroxymethyl-

chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate at

a 10 lM concentration [16]. The results depicted in Fig. 8,

show that GSK3-b phosphorylation is reduced in the

presence of the PKB blocker.

Taken together our results demonstrate the involvement

of PKB and GSK3-b in the Glu elicited transactions

through AMPA receptors in BGC.

Discussion

Increasing evidence accumulates regarding a plausible role

of glial cells in neuronal communication [17]. Cultured

chick cerebellar BGC are a suitable preparation to study the

molecular mechanisms triggered after Glu exposure. In

fact, several membrane to nuclei signaling cascades that

results in transcriptional regulation have been described

taking advantage of this preparation [18].

The reported physical interaction of PI3-K with iono-

tropic Glu receptors in neurons and glial cells [6, 19] as

well as its Glu-dependent activation, prompted us to

evaluate if the PKB/GS3K-b signaling pathway was

being activated. A time and dose-dependence PKB phos-

phorylation could be unequivocally established after Glu

p-PKB (Ser473)

PKB

C GluGlu PP2PP2+

CGlu

GluGlu

Glu

PP2PP2+

% o

f co

ntr

ol

p-GSK3-β (Ser9)

PKB

C Glu PP2PP2+

CGlu

Glu PP2PP2+

% o

f co

ntr

ol

*

*

020406080

100120140160180200

020406080

100120140160180200

(a)

(b)

Fig. 7 Inhibition of PKB and GSK3-b phosphorylation by the Src

inhibitor PP2. Confluent monolayers of Bergmann glia cells were

incubated with 10 nM PP2 for 30 min. After, 1 mM Glu was added

and incubation continued for 30 min. Phospho PKB and phospho

GSK3-b were measured as in Fig. 2. *P < 0.001, **P < 0.01 (ANOVA)

C GluGlu

p-GSK3-β (Ser9)

PKB

Akt I+Akt I

% o

f co

ntr

ol

CGlu

Glu Akt I+ Akt I

*

*

0

50

100

150

200

250

300

350

Fig. 8 Inhibition of GSK3-b phosphorylation by the PKB inhibitor

(Akt I) IL-6-Hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-

octadecylcarbonate. BGC monolayers were incubated with 10 lM

of Akt I. Thereafter the cells were stimulated with 1 mM Glu for

30 min. Phospho PKB and phospho GSK3-b were measured as in

Fig. 2. *P < 0.001, **P < 0.01 (ANOVA)

428 Neurochem Res (2006) 31:423–429

123

exposure (Figs. 1–3). The kinetic data strongly suggests

that Glu not only promotes PKB and GSK3-b phosphory-

lation, but also their dephosphorylation. Although the

identity of the phosphatase activity involved is beyond the

scope of this communication, it is tempting to speculate

that phosphatase 2A (PP2A) is responsible for the effect.

This suggestion is based upon the fact that exposure to

3 mM LiCl, an inhibitor PP2A, leads to an augmentation in

PKB phosphorylation (Fig. 1). Moreover, PP2A activity

depends on Ca2+ [20], an event linked to AMPA receptors

stimulation in cultured chick BGC [5].

Glu has been repeatedly reported to activate PKB in

neuronal cells [21] but much less is known about its

activity in glial cells. In fact, the mechanisms of activation/

inactivation of PKB through glial Glu receptors are not

completely understood. A concentration-dependence of the

Glu effect could be established and the EC50 for PKB and

GSK3-b phosphorylation is the same, therefore it is must

likely that PKB phosphorylates GSK3-b (Fig. 3). It is

important however to mention that the EC50 values re-

ported are biased by the signaling machinery involved in

the phosphorylation processes and are only indicative of a

receptor-mediated effect [22]. The use of a PKB inhibitor

allowed us to demonstrate that PKB phosphorylates GSK3-

b (Fig. 8). Through the use of Glu analogues we conclude

that the Glu receptors involved are ionotropic of the AMPA

subtype (Figs. 4, 5). In terms of the signaling cascade, the

effect is Ca2+-independent but sensitive to the Src inhibitor

PP2, results which are in line with our previous findings

regarding AMPA receptors tyrosine phosphorylation [6].

As expected then, both PKB and GSK3-b phosphorylation

were sensitive to the PI-3K inhibitor wortmanin (Fig. 5).

In summary, we provide here evidence for a Glu-

dependent, PI-3K-mediated PKB activation that results in

GSK3-b phosphorylation. These results broaden our

understanding of Glu signaling in glial cells and favor the

hypothesis that glial cells play a role in synaptic activity.

Acknowledgments The authors acknowledge the technical assis-

tance of Luis Cid and Blanca Ibarra. This work was supported by a

grant from Conacyt-Mexico (43164-Q) to A.O. Conacyt-Mexico

supports M.M. and M.E.G.M. through doctoral fellowships.

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