Inactivation of Integrin-linked Kinase Induces Aberrant Tau Phosphorylation via Sustained Activation of Glycogen Synthase Kinase 3
in N1E-115 Neuroblastoma Cells*
Toshiaki Ishii
,
Hidefumi Furuoka,
Yoshikage Muroi and
Masakazu Nishimura
From the
Department of Pathobiological Science, Obihiro University of Agriculture
and Veterinary Medicine, Obihiro Hokkaido 080-8555, Japan
Received for publication, April 18, 2003
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ABSTRACT
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Integrin-linked kinase (ILK) is a focal adhesion serine/threonine protein
kinase with an important role in integrin and growth factor signaling
pathways. Recently, we demonstrated that ILK is expressed in N1E-115
neuroblastoma cells and controls integrin-dependent neurite outgrowth in
serum-starved cells grown on laminin (Ishii, T., Satoh, E., and Nishimura, M.
(2001) J. Biol. Chem. 276, 4299443003). Here we report that
ILK controls tau phosphorylation via regulation of glycogen synthase
kinase-3
(GSK-3
) activity in N1E-115 cells. Stable transfection of
a kinase-deficient ILK mutant (DN-ILK) resulted in aberrant tau
phosphorylation in N1E-115 cells at sites recognized by the Tau-1 antibody
that are identical to some of the phosphorylation sites in paired helical
filaments, PHF-tau, in brains of patients with Alzheimer's disease. The tau
phosphorylation levels in the DN-ILK-expressing cells are constant under
normal and differentiating conditions. On the other hand, aberrant tau
phosphorylation was not observed in the parental control cells. ILK
inactivation resulted in an increase in the active form but a decrease in the
inactive form of GSK-3
, which is a candidate kinase involved in PHF-tau
formation. Moreover, inhibition of GSK-3
with lithium prevented aberrant
tau phosphorylation in the DN-ILK-expressing cells. These results suggest that
ILK inactivation results in aberrant tau phosphorylation via sustained
activation of GSK-3
in N1E-115 Cells. ILK directly phosphorylates
GSK-3
and inhibits its activity. Therefore, endogenous ILK protects
against GSK-3
-induced aberrant tau phosphorylation via inhibition of
GSK-3
activity in N1E-115 cells.
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INTRODUCTION
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Tau is a microtubule-associated protein that stabilizes microtubules within
neurites and axons (1). It is
hypothesized that tau hyperphosphorylation leads to the destabilization of
microtubules and aggregation of tau proteins, which impairs axonal transport
and eventually results in neuronal cell death
(13).
Indeed, tau hyperphosphorylation appears to be an early event preceding the
formation of paired helical filaments
(PHF)1 in the brains
of Alzheimer's disease patients
(4). On the other hand, tau
phosphorylation also seems to control microtubule dynamics during neurite
outgrowth and neuronal maturation, because embryonic and neonatal tau is much
more heavily phosphorylated than adult tau
(58).
Thus, the study of the regulation of tau phosphorylation in neurons is
important for understanding the neurofibrillary degeneration in Alzheimer's
disease, as well as the physiologic function of tau in neurite outgrowth and
neuronal development.
Integrin-linked kinase (ILK) is a cytoplasmic serine/threonine kinase
protein that serves as a mediator in integrin- and growth factor-mediated
signal transduction (9,
10). ILK is expressed in
N1E-115 cells and controls integrin-dependent neurite outgrowth
(11). In the present study,
stable transfection of a kinase-deficient mutant of ILK (DN-ILK), which
behaves as a dominant negative and inactivates endogenous ILK, resulted in
aberrant tau phosphorylation. DN-ILK overexpression increased the active form
and decreased the inactive form of GSK-3
. On the other hand, a selective
uncompetitive inhibitor of GSK-3
, lithium, inhibited aberrant tau
hyperphosphorylation. These results suggest that inactivation of ILK results
in sustained activation of GSK-3
and leads to aberrant tau
phosphorylation in N1E-115 neuroblastoma cells.
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EXPERIMENTAL PROCEDURES
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ReagentsLY294002 was obtained from Sigma. Rabbit polyclonal
anti-ILK IgG (UB 06550 and UB 06592) and myelin basic protein
were obtained from Upstate Biotechnology, Inc. (Lake Placid, NY).
Anti-GSK-3
antibody for immunoprecipitation was obtained from
Transduction Laboratories (Lexington, KY). Anti-Tau-1 was obtained from Roche
Applied Science. Anti-Tau,
anti-phospho(Ser199,Ser202)-Tau, and anti-GSK-3
were obtained from Calbiochem (La Jolla, CA).
Anti-phospho(Ser9)-GSK-3
and
anti-phospho(Tyr279/216)-GSK-3
/
were
obtained from ABA (Golden, CO). AlexaFluor (R)488 goat anti-rabbit IgG was
obtained from Molecular Probes (Eugene, OR). GSK-3
substrate (2B-SP) was
obtained from Takara (Custom Synthesis Service, Tokyo, Japan). All other
chemicals were of analytical grade and were obtained from Sigma or Wako Pure
Chemical Co. (Osaka, Japan) unless otherwise specified.
Construction and Transfection of cDNA Vectors and Cell
Culture Mouse N1E-115 neuroblastoma cells were maintained in
Dulbecco's modified Eagle's medium containing 20% fetal bovine serum (FBS;
Hyclone, Logan, UT). The kinase-deficient ILK (DN-ILK) was generated by
site-directed mutagenesis (Glu to Lys) at amino acid residue 359 within the
kinase domain of wild type ILK (GenBankTM accession number AF256520
[GenBank]
)
using PCR as described previously
(11). Wild-type ILK and DN-ILK
cDNAs were ligated into the polylinkers in two different mammalian expression
vectors, pTracerTM-CMV2 (V88501; Invitrogen) and pRc-CMV
(V75020; Invitrogen). The DN-ILK cDNA was transfected into N1E-115
cells (5 x 105 cells/100-mm culture dish) using the calcium
phosphate precipitation method as described by Graham and van der Eb
(12), and 48 individual
Zeocin-resistant cell lines were isolated over the next 4 to 5 weeks. Among
them, three different cell lines were selected based on the detection of green
fluorescent protein fluorescence and confirmation of gene transcription using
reverse transcriptase PCR. The cloned cell lines were maintained in Dulbecco's
modified Eagle's medium containing 20% FBS and Zeocin (0.5 mg/ml).
ILK AssayILK assay was performed as described by
Delcommenne et al.
(13). Cells were lysed in 50
mM Hepes buffer (pH 7.5) containing 150 mM NaCl, 1%
Nonidet P-40, 0.5% sodium deoxycholate, 10 µg/ml leupeptin, 2.5 µg/ml
aprotinin, 1 mM phenylmethylsulfonyl fluoride, 5 mM
sodium fluoride, and 1 mM sodium orthovanadate. The lysates were
incubated with anti-ILK antibody (UB 06592) at 4 °C for 12 h. After
incubation, the lysates were precleared, and immune complexes were collected
with protein A-Sepharose. After washing twice with lysis buffer and once with
50 mM HEPES (pH 7.0) buffer containing 1 mM EDTA, the
immunoprecipitated ILK was incubated for 20 min at 30 °C in the presence
or absence of 10 µg of the exogenous substrate myelin basic protein in a
total volume of 50 µl kinase reaction buffer (50 mM HEPES (pH
7.0), 10 mM MnCl2, 10 mM MgCl2, 2
mM NaF, 1 mM Na3VO4) containing 6
µM [
-32P]ATP (10 µCi; NEG-502Z; PerkinElmer
Life Sciences). The reaction was stopped by the addition of an equal volume of
2x SDS-PAGE sample buffer. The kinase reaction products were analyzed
using SDS-PAGE (520% polyacrylamide) and autoradiography. For detection
of the immunoprecipitated ILK and DN-ILK proteins, the precipitated proteins
were released from the immunobeads by boiling in 80 µl of SDS-PAGE sample
buffer for 5 min. Equal volumes of the samples were loaded onto SDS-PAGE.
Total ILK and DN-ILK proteins were detected by immunoblotting with an anti-ILK
antibody (UB 06550) that recognized both ILK and DN-ILK proteins.
GSK-3
GSK-3
assay was performed
essentially as described by Cross
(14). Cells were lysed in 50
mM Hepes buffer (pH 7.5) containing 150 mM NaCl, 1%
Nonidet P-40, 5 mM sodium fluoride, 1 mM sodium
orthovanadate, 5 mM EDTA, 100 nM okadaic acid, and
protease inhibitors (Complete; Roche Diagnostics). The lysates were incubated
with anti-GSK-3
antibody (Transduction Laboratories, Lexington, KY) at 4
°C for 1 h followed by overnight incubation with protein G-Sepharose.
After washing twice with lysis buffer and once with 50 mM HEPES
buffer (pH 7.0) containing 1 mM EDTA, the immunoprecipitated
GSK-3
was incubated for 30 min at 30 °C in the presence or absence
of 4 µM specific substrate peptide 2B-SP
(Ac-RRAAEELDSRAGS(p)PQL) in a total volume of 50 µl kinase reaction buffer
(50 mM HEPES (pH 7.0), 10 mM MnCl2, 10
mM MgCl2, 2 mM NaF, 1 mM
Na3VO4) containing 10 µM
[
-32P]ATP (0.2 µCi; NEG-502Z; PerkinElmer Life Sciences).
The reaction was stopped by placing the samples on ice. After brief
centrifugation, 25 µl of the reaction supernatant was spotted onto P81
phosphocellulose paper filters (Whatman, Maidstone, UK), washed with 75
mM phosphoric acid, and rinsed with acetone. The amount of
radioactive phosphate incorporated into substrate peptides was determined by
scintillation counting.
Immunofluorescent StainingCells grown on Lab-Tek®
chamber slides (Nunc, Tokyo, Japan) were fixed in 1% neutral buffered
formaldehyde solution for 10 min and then permeabilized with 0.25% saponin in
Hanks' balanced salt solution for 20 min. Permeabilized cells were incubated
for 1 h in rabbit anti-phospho(Ser199,Ser202)-Tau
antibody (Calbiochem; final dilution 1:100 in phosphate-buffered saline).
After rinsing in phosphate-buffered saline, the cells were incubated for 1 h
in AlexaFluor (R)488 goat anti-rabbit IgG (1:100). Images were obtained by
fluorescent microscopy (Olympus, Tokyo, Japan) and confocal laser scanning
microscopy (Nikon, Tokyo, Japan).
AntibodiesAnti-Tau (recognizes both native and
phosphorylated forms of tau), anti-Tau-1 (recognizes tau dephosphorylated at
Ser195, Ser198, Ser199, Ser202,
and Thr205 (15,
16)), and anti-GSK-3
antibody for immunoprecipitation (Transduction Laboratories) were mouse
monoclonal antibodies. All the other antibodies, antiphospho
(Ser199,Ser202)-Tau (recognizes tau phosphorylated at
Ser199 and Ser202), anti-GSK-3
(recognizes both
native and phosphorylated forms of GSK-3
),
anti-phospho(Ser9)-GSK-3
(Affinity Bioreagents; recognizes
GSK-3
phosphorylated at Ser9),
anti-phospho(Tyr279/216)-GSK-3
/
(Affinity
Bioreagents; recognizes GSK-3
and GSK-3
phosphorylated at
Tyr279 and Tyr216, respectively), and anti-ILK (Upstate
Biotechnology, Inc.; UB 06550 for immunoblotting and UB 06593
for immunoprecipitation), were rabbit polyclonal antibodies.
Western Blot AnalysisCells were solubilized in 100 µl of
sample buffer containing 2% SDS, 10% glycerol, 50 mM
dithiothreitol, 0.1% bromphenol blue, and 62.5 mM Tris-HCl (pH 6.8)
after washing once with phosphate-buffered saline. For detection of ILK
expression, cells were solubilized in 5 volumes of buffer containing 1% Triton
X-100, 150 mM NaCl, 50 mM Tris-HCl (pH 7.4), 5
mM EGTA, and 2 mM phenylmethylsulfonyl fluoride at 4
°C. The solubilized materials were subjected to SDS-PAGE (520%
gradient, 6.5 or 10% polyacrylamide) and transferred onto nitrocellulose
membranes at 4 °C in 25 mM Tris-HCl (pH 8.4), 192 mM
glycine, 20% methanol, and 0.025% SDS. After blocking, the blots were probed
with appropriate primary antibodies in Tris-buffered saline containing 0.05%
Tween 20, followed by goat anti-rabbit or goat anti-mouse IgG conjugated to
horseradish peroxidase. The final protein·IgG complexes were visualized
following the reaction to 3,3'-diaminobenzidine tetrahydrochloride.
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RESULTS
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DN-ILK Inhibits Basal ILK Activity and Prevents Stimulation of ILK
Activity after Cell Adhesion on Laminin under Serum-free
ConditionsMouse N1E-115 neuroblastoma cells grown on a laminin
matrix exhibit neurite outgrowth in response to serum deprivation
(11,
17). We have demonstrated
previously (11) that ILK is
expressed in N1E-115 neuroblastoma cells and controls integrin-dependent
neurite outgrowth in serum-starved cells grown on laminin. To inactivate
endogenous ILK, cells were stably transfected with DN-ILK, which behaves as a
dominant negative (11,
13). Based on the results
obtained from immunoblotting (Fig.
1), the expression level of DN-ILK protein in DN-ILK-transfected
cells was estimated to be at least twice that of endogenous ILK protein, and
neither the ILK nor DN-ILK expression level changed under differentiating
conditions. The ILK activity in the parental cells under serum-free conditions
was transiently activated after seeding on the laminin matrix, whereas that in
the DN-ILK-transfected cells was not. Also, weak basal ILK activity was
detected only in the parental cells under non-differentiating conditions
(Fig. 1). These findings are
consistent with our previous observations
(11). Thus, DN-ILK inactivates
endogenous ILK under both differentiating and non-differentiating
conditions.

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FIG. 1. Inactivation of endogenous ILK induces aberrant tau phosphorylation.
Cells were cultured for 16 h under either differentiating (seeding on
laminin-coated plates in the absence of FBS) or non-differentiating (seeding
on non-coated plates in the presence of FBS) conditions. Aberrant
phosphorylation level of tau in parental and DN-ILK-transfected cells was
detected by immunoblotting with an anti-Tau-1 antibody, which recognizes only
a non-phosphorylated epitope of tau, and an
anti-phospho(Ser199,Ser202)-Tau antibody, which
recognizes tau phosphorylated at Ser199,Ser202. Total
tau was detected by an anti-Tau antibody, which recognizes both native and
phosphorylated tau. For the ILK assay and detection of ILK and DN-ILK
proteins, cells were cultured for 60 min, which was required for maximal
activation of ILK activity
(11), under either
differentiating or non-differentiating conditions and then lysed. ILK was
immunoprecipitated from cell extracts. ILK activity was determined using
myelin basic protein as an exogenous substrate, as described under
"Experimental Procedures." ILK and DN-ILK expression levels in
cell lysates (20 µg) were analyzed using immunoblotting with an
affinity-purified polyclonal anti-ILK antibody.
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Inactivation of Endogenous ILK Induces Aberrant Tau
PhosphorylationTo examine whether tau phosphorylation is involved
in neurite outgrowth in N1E-115 cells, we analyzed the tau phosphorylation
level in parental and DN-ILK-transfected cells using phosphorylation- and
dephosphorylation-dependent anti-Tau antibodies. Western blots of tau from
both parental and DN-ILK-transfected cells were probed with three different
tau antibodies, anti-Tau-1, which recognizes an epitope of tau only when it is
not phosphorylated, antiphospho (Ser199,Ser202)-Tau,
which recognizes tau phosphorylated at Ser199 and
Ser202, and anti-Tau, which recognizes both native and
phosphorylated forms of tau. In non-transfected parental cells, tau was
recognized by anti-Tau-1 but not
anti-phospho(Ser199,Ser202)-Tau antibody, under both
normal and differentiating conditions. Thus, tau in the parental cells was not
phosphorylated at sites recognized by those antibodies in either condition. On
the other hand, cells stably transfected with a DN-ILK to inactivate the
endogenous ILK had dramatically decreased anti-Tau-1 immunoreactivity but an
increased anti-phospho(Ser199,Ser202)-Tau
immunoreactivity (Fig. 1).
Thus, tau was phosphorylated in DN-ILK-transfected cells, and the tau
phosphorylation level did not change even under differentiating conditions.
Total tau detected with anti-Tau antibody migrated as several bands in the 40-
to 70-kDa range, but some of the protein bands, which migrate relatively
slower on SDS-PAGE, were weaker when tau was phosphorylated
(Fig. 1). These results suggest
that inactivation of endogenous ILK results in aberrant hyperphosphorylation
of tau, at least at Ser199 and Ser202.
Immunofluorescent Staining of Aberrantly Phosphorylated
TauTo examine the intracellular localization of aberrantly
phosphorylated tau, cells were stained with antibody against phosphorylated
tau. Immunofluorescent staining of cell mono-layers with
anti-phospho(Ser199,Ser202)-Tau antibody is shown in
Fig. 2. DN-ILK-transfected
cells were strongly stained with the antibody against phosphorylated tau under
normal and differentiating conditions. On the other hand, parental cells were
not significantly stained under differentiating conditions, but very weak
dot-like structures were observed only in the non-differentiated cells,
suggesting that a small minority of the tau, which could not be detected by
the immunoblotting analysis, might be phosphorylated under non-differentiating
conditions. In the DN-ILK-transfected cells, intracellular cytoplasm, except
the nucleus, was stained with strong immunofluorescent intensity, and
microtubule-like structures were observed. Further analysis of the
DN-ILK-transfected cells using confocal laser scanning microscopy indicated
that microtubule-like structures spread and cover right under the whole plasma
membrane of the cells and form basket-like structures
(Fig. 2, top, left and
right).

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FIG. 2. Subcellular localization of aberrantly phosphorylated tau at
Ser199 and Ser202. After cells were cultured for 16
h under either differentiating (a and b) or
non-differentiating (c and d) conditions, cells were stained
with anti-phospho(Ser199,Ser202)-Tau antibody, as
described under "Experimental Procedures." In DN-ILK-transfected
cells, the intracellular cytoplasm, except for the nucleus, was strongly
stained with the antibody, and microtubule-like structures were observed under
normal and differentiating conditions (b and d). On the
other hand, parental cells were not significantly stained under
differentiating conditions (c), but very weak dot-like
structures were observed in the non-differentiated cells (a).
Further analysis of the DN-ILK-transfected cells using the confocal laser
scanning microscopy. Microtubule-like structures spread and covered
immediately under the whole plasma membrane of the cells and formed
basket-like structures (top, left and right).
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Active Form of GSK-3
Increases in DN-ILK-transfected
CellsTo examine signal pathways involved in tau phosphorylation,
we analyzed the activation status of GSK-3
, because GSK-3
is one
of the candidate kinases that can phosphorylate tau at both Ser199
and Ser202 (18) and
also has an important role in the ILK-mediated signal pathway
(13,
19,
20). Activation of GSK-3
is dependent on Tyr216 phosphorylation
(21). On the other hand,
GSK-3
activity is inhibited by direct phosphorylation at Ser9
by ILK (20,
22) and by protein kinase
B/Akt, which is also activated via ILK
(13,
23). We therefore examined
whether the levels of these phosphorylated GSK-3
forms are different
between parental and DN-ILK-transfected cells and are changed under different
culture conditions by Western blot analysis using
anti-phospho(Ser9)-GSK-3
and
anti-phospho-(Tyr279/216)-GSK-3
/
antibodies. Tyr216 in GSK-3
was highly phosphorylated in
DN-ILK-transfected cells but was very weakly phosphorylated in parental cells
(Fig. 3A). In
contrast, Ser9 in GSK-3
was highly phosphorylated in parental
cells but not in DN-ILK-transfected cells. Moreover, these phosphorylation
levels were not significantly different between non-differentiating and
differentiating conditions. The expression level of GSK-3
was not
different among all cells tested. These results suggest that ILK inactivation
results in Ser9 dephosphorylation and increased Tyr216
phosphorylation in GSK-3
, thereby activating the enzyme.
GSK-3
Activity Is Activated by Inactivation of
Endogenous ILK in DN-ILK-transfected CellsTo test whether the
level of GSK-3
activity increases in DN-ILK-transfected cells, we
performed a GSK-3
kinase assay on proteins immunoprecipitated from cell
lysates with a monoclonal GSK-3
antibody. The level of GSK-3
activity in DN-ILK-transfected cells was approximately three times more than
that in parental cells (Fig.
3B). The level of GSK-3
activity was not different
between differentiating and non-differentiating conditions in either
DN-ILK-transfected or parental cells (Fig.
3B). Thus, inactivation of endogenous ILK results in
increased GSK-3
activity. This result is consistent with the fact that
ILK inactivation induced an increase in the levels of active GSK-3
formed via Tyr216 phosphorylation
(Fig. 3A).
Lithium Reduces Tau Phosphorylation in a Dose-dependent
MannerLithium is an uncompetitive GSK-3
inhibitor
(24). We therefore examined
the effect of lithium on tau phosphorylation. Cells were treated with varying
concentrations of LiCl for 16 h under non-differentiating conditions.
Treatment of DN-ILK-transfected cells with LiCl reduced
anti-phospho(Ser199,Ser202)-Tau immunoreactivity but
increased anti-Tau-1 immunoreactivity in a dose-dependent manner
(Fig. 4). Moreover, reaction of
slowly migrated tau bands with anti-Tau, which were supposed to be
non-phosphorylated tau, was recovered after treatment with LiCl in a
dose-dependent manner. The same results were obtained in DN-ILK-transfected
cells under differentiating conditions (data not shown). These results suggest
that LiCl inhibited tau phosphorylation at Ser199 and
Ser202 and also at the sites recognized by anti-Tau-1. On the other
hand, the same treatment of parental cells with 25 mM LiCl did not
affect either anti-Tau-1 or
anti-phospho(Ser199,Ser202)-Tau immunoreactivity. These
results suggest that GSK-3
activation induced by ILK inactivation is
directly involved in tau phosphorylation at Ser199 and
Ser202 and also at the sites recognized by anti-Tau-1.

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FIG. 4. Aberrant tau phosphorylation was prevented by treatment of cells with
LiCl, an uncompetitive inhibitor of GSK-3 Cells were treated with
varying concentrations of LiCl for 16 h under non-differentiating conditions.
The level of aberrant tau phosphorylation was analyzed by immunoblotting with
both anti-Tau-1 and anti-phospho(Ser199,Ser202)-Tau
antibodies. Total tau proteins were detected by immunoblotting with anti-Tau
antibody, which recognizes both native and phosphorylated tau.
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Aberrant Tau Phosphorylation Is Partly Involved in Inhibition of
Neurite Outgrowth in DN-ILK-transfected CellsWe demonstrated
previously (11) that
activation of p38 mitogen-activated protein (MAP) kinase is involved in
ILK-mediated signal transduction leading to integrin-dependent neurite
outgrowth in N1E-115 cells. In our previous report
(11), we suggested that
signaling pathways other than p38 MAP kinase, which is also activated via ILK
activation, might be involved in integrin-dependent neurite outgrowth, because
a p38 MAP kinase inhibitor, SB203580, blocked
80% of the ILK-dependent
neurite outgrowth (see Fig. 5). To examine whether aberrant tau phosphorylation is involved in the inhibition
of neurite outgrowth in DN-ILK-transfected cells, we treated the cells with 10
mM LiCl, a dose that prevents aberrant tau phosphorylation, under
serum-free conditions. Treatment of the DN-ILK-transfected cells with LiCl
partially recovered neurite outgrowth, in the presence or absence of SB203580,
to the levels of that in the SB20358-treated parental cells
(Fig. 5). On the other hand,
treatment of the parental cells with LiCl did not affect neurite outgrowth in
either the presence or absence of SB203580
(Fig. 5). These results suggest
that aberrant tau phosphorylation is partly involved in the inhibition of
neurite outgrowth in DN-ILK-transfected cells.

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FIG. 5. Effects of aberrant tau phosphorylation on neurite outgrowth in
DN-ILK-transfected cells. Cells were seeded on laminin-coated plates in
serum-free medium in the presence of SB203580, LiCl, or LiCl plus SB203580.
Treatment with only SB203580 was conducted for 3 h after seeding the cells by
removing and changing the culture medium. The number of cells possessing
neurites greater than twice the length of a cell body was assessed 16 h after
plating the cells. Values are the means ± S.D. of four separate
experiments. Statistical significance was determined using Student's
t test; a indicates p < 0.01 (versus
non-treatment), b indicates p < 0.01 (versus 10
mM LiCl), c indicates p < 0.01
(versus non-treatment), and d indicates p < 0.05
(versus 10 µM SB203580).
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DISCUSSION
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The present study demonstrates that ILK inactivation induces aberrant tau
phosphorylation via GSK-3
activation in N1E-115 cells, which is partly
involved in the inhibition of neurite outgrowth, based on the following
observations: 1) tau was phosphorylated at Ser199 and
Ser202 and at the sites recognized by anti-Tau-1 in DN-ILK
transfected cells but not in parental cells, 2) ILK inactivation increased the
active form but decreased the inactive form of GSK-3
, leading to
increased GSK-3
activity, and 3) treatment of the DN-ILK-transfected
cells with LiCl, an uncompetitive inhibitor of GSK-3
, blocked aberrant
tau phosphorylation and partly recovered the levels of neurite outgrowth in
DN-ILK-transfected cells.
Integrin signaling is required for neurogenesis in serum-starved N1E-115
cells (25). N1E-115 cells
grown on a laminin matrix exhibit neurite outgrowth in response to serum
deprivation (11,
17). This neurite outgrowth
depends on an integrin-dependent signal pathway, because pre-treatment of
cells with anti-
1 integrin antibody inhibited neurite outgrowth
(11,
17). We demonstrated recently
(11) that ILK is expressed in
N1E-115 cells and controls integrin-dependent neurite outgrowth in
serum-starved cells grown on laminin. In the present study, stable
transfection of DN-ILK, which behaves as a dominant negative and inactivates
endogenous ILK, resulted in aberrant tau phosphorylation. On the other hand,
the aberrant tau phosphorylation was not observed in the parental cells under
either normal or differentiating conditions. ILK activity in the parental
cells was increased after seeding on the laminin matrix under serum-free
conditions, whereas that in the DN-ILK-transfected cells was not. Thus, both
cell adhesion to laminin and serum deprivation were necessary for full
activation of ILK. On the other hand, weak basal ILK activity was detected in
the parental cells under non-differentiating normal conditions
(Fig. 1). It remains unknown
how basal ILK activity is maintained under non-differentiation. These results
suggest that endogenous ILK prevents aberrant tau phosphorylation. Moreover,
aberrant tau phosphorylation did not significantly occur in the parental cells
under normal conditions, suggesting that weak basal ILK activity is sufficient
to protect tau from aberrant phosphorylation.
To examine whether aberrantly phosphorylated tau participates in
microtubule formation, we stained cells with antibody against phosphorylated
tau (Fig. 2). Intracellular
cytoplasm, except for the nucleus, was stained with strong immunofluorescent
intensity, and microtubule-like structures were observed only in
DN-ILK-transfected cells. Moreover, analysis of the DN-ILK-transfected cells
using confocal laser scanning microscopy indicated that microtubule-like
structures spread and cover right under the whole plasma membrane of the cells
and form basket-like structures (Fig.
2, top, left and right). Thus, aberrantly
phosphorylated tau participates in microtubule-like structures but is located
only immediately under the plasma membrane in the cytosol without being able
to form neurites in DN-ILK-transfected cells. On the other hand, parental
control cells under non-differentiating conditions were very weakly stained
with antibodies against phosphorylated tau, which could not be detected by the
immunoblotting analysis, but neurite-bearing control cells under
differentiating conditions were not (Fig.
2, a and c). These results suggest that tau
phosphorylation at Ser199 and Ser202 might negatively
control microtubule rearrangement necessary for neurite outgrowth via
microtubule instability.
Tau is phosphorylated at Ser199 and Ser202 and at the
sites recognized by anti-Tau-1, which correspond to Ser195,
Ser198, Ser199, Ser202, and
Thr205. Ser199 and Ser202 are phosphorylated
by GSK-3
(18), which is
a candidate kinase involved in PHF-tau formation
(26,
27). Moreover, GSK-3
has
an important role in the ILK-mediated signal pathway
(10,
19). We therefore examined the
involvement of GSK-3
in aberrant tau phosphorylation in
DN-ILK-transfected cells. Activation of GSK-3
is dependent upon the
Tyr216 phosphorylation
(21). On the other hand,
GSK-3
activity is inhibited by direct Ser9 phosphorylation by
ILK (19,
22) and by protein kinase
B/Akt whose activity is also activated via ILK
(13,
23). Tyr216 in
GSK-3
was highly phosphorylated in DN-ILK-transfected cells but was very
weakly phosphorylated in parental cells. In contrast, Ser9 in
GSK-3
was highly phosphorylated in parental cells but not in
DN-ILK-transfected cells. These phosphorylation levels were not significantly
different between non-differentiating and differentiating conditions
(Fig. 3A). Thus,
GSK-3
was phosphorylated at both Ser9 and Tyr216
in parental cells, whereas Tyr216 phosphorylation was considerably
lower (Fig. 3A).
Recently, Bhat et al.
(28) suggested that
Ser9 phosphorylation is sufficient to override the
Tyr216 phosphorylation-induced activation of GSK-3
.
Therefore, the level of GSK-3
activity seems to maintain lower via
Ser9 phosphorylation in parental cells. These results suggest that
ILK inactivates GSK-3
via phosphorylation at Ser9 and
prevents activation. In contrast, ILK inactivation results in Ser9
dephosphorylation and increased Tyr216 phosphorylation in
GSK-3
, thereby activating the enzyme. Indeed, the level of GSK-3
activity in DN-ILK-transfected cells was significantly higher than that in
parental cells (Fig.
3B).
Lithium, a selective uncompetitive inhibitor of GSK-3
(24), inhibited tau
phosphorylation at Ser199 and Ser202 and also at the
sites recognized by anti-Tau-1 in a dose-dependent manner
(Fig. 4). These results suggest
that GSK-3
activation induced by ILK inactivation is directly involved
in tau phosphorylation at Ser199 and Ser202 and also at
the sites recognized by anti-Tau-1. As both Ser199 and
Ser202 are phosphorylated by GSK-3
(18), GSK-3
at least
directly phosphorylates these Ser residues in the DN-ILK-transfected cells.
Although a specific tyrosine kinase, which should be activated by ILK
inactivation, is probably involved in the Tyr216 phosphorylation in
GSK-3
, we have not yet determined the kinase involved in this study. To
understand the ILK-mediated regulatory mechanisms of GSK-3
, the specific
tyrosine kinase that is activated by ILK inactivation and phosphorylates
Tyr216 in GSK-3
must be determined.
We demonstrated previously
(11) that p38 MAP kinase in
the ILK-mediated signal pathway has an important role in integrin-dependent
neurite outgrowth in N1E-115 cells. On the other hand, a p38 MAP kinase
inhibitor, SB203580, blocked
80% of the ILK-dependent neurite outgrowth
but not to the levels of the DN-ILK-transfected cells (see
Fig. 5). In the previous study
(11), we suggested that
signaling pathways other than p38 MAP kinase, which are also activated via ILK
activation, are involved in integrin-dependent neurite outgrowth. We therefore
examined whether aberrant tau phosphorylation is involved in the inhibition of
neurite outgrowth in DN-ILK-transfected cells. Treatment of the
DN-ILK-transfected cells with 10 mM LiCl completely prevented
aberrant tau phosphorylation but only partially recovered neurite outgrowth to
the levels of that in the SB20358-treated parental cells. These results
suggest that aberrant tau phosphorylation is partly involved in the inhibition
of neurite outgrowth in DN-ILK-transfected cells. Furthermore, the results
also suggest that a p38 MAP kinase pathway is the sole downstream pathway in
ILK-dependent neurite outgrowth.
Tau hyperphosphorylation decreases the association of tau with microtubules
(29) and inhibits total
neurite number
(3032).
The inhibitory effect of aberrant tau phosphorylation on neurite outgrowth was
as expected. The effect of aberrant tau phosphorylation, however, is
considered to depend on the tau phosphorylation level and also on the number
of phosphorylated tau molecules. Although we could not estimate how many
molecules in total tau are phosphorylated in the DN-ILK-transfected cells in
this study, it is possible that the aberrant tau phosphorylation induced by
ILK inactivation at least affects microtubule stability or dynamics and leads
to inhibition of neurite outgrowth. On the other hand, endogenous ILK protects
tau from aberrant phosphorylation and probably maintains a kind of equilibrium
status responsible for microtubule reorganization. Thus, ILK is not only
involved in p38 MAP kinase activation but might also control microtubule
dynamics via regulation of GSK-3
activity during neurite outgrowth in
N1E-115 cells (Fig. 6). A
recent study (33) of nerve
growth factor-induced neurite outgrowth using pheochromocytoma (PC12) cells
demonstrated that ILK is involved in nerve growth factor-induced neurite
outgrowth via inhibition of tau hyperphosphorylation. This recent report using
PC12 cells and our results obtained using N1E-115 cells suggest that ILK is an
important regulator of both integrin- and growth factor-mediated signaling in
neurons and controls neurite outgrowth. Moreover, ILK might be critical for
the regulation of microtubule stability and rearrangement necessary for
integrin- and growth factor-mediated neurite outgrowth.
 |
FOOTNOTES
|
---|
* This work was supported in part by a grant-in-aid for scientific research
(C) (to T. I.) from the Japanese Ministry of Education, Science and Culture,
by The Naito Foundation (to T. I.), and by The Akiyama Foundation (to T. I.).
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact. 
To whom correspondence should be addressed. Tel.: 81-155-49-5366; Fax:
81-155-49-5369; E-mail:
ishii{at}obihiro.ac.jp.
1 The abbreviations used are: PHF, paired helical filaments; ILK,
integrin-linked kinase; GSK-3
, glycogen synthase kinase 3
; DN,
dominant negative; MAP, mitogen-activated protein; FBS, fetal bovine
serum. 
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. M. Niinobe (Institute for Protein Research, Osaka University,
Japan) for providing the N1E-115 cells.
 |
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