Akt Protein Kinase Enhances Human Telomerase Activity through
Phosphorylation of Telomerase Reverse Transcriptase Subunit*
Sang Sun
Kang
§,
Taegun
Kwon
,
Do Yoon
Kwon
, and
Su Il
Do¶
From the
Shin Dong Bang R&D Center, Seoul 137-132 and
¶ Korea Research Institute of Bioscience and Biotechnology Korea
Institute of Science and Technology,
Taejon 305-333, Republic of Korea
 |
ABSTRACT |
With the amino acid sequences of all reported Akt
kinase physiological substrates, the possible Akt kinase substrate
specificity has been suggested. The serine/threonine residue to be
phosphorylated in these proteins is placed within stretches of amino
acids with homology, and the arginine residues on the
5 and
3
positions and a hydrophobic amino acid on the +2 position are conserved relative to those of serine/threonine residues
(XXRXRXXS/TXX). We
noticed two putative Akt kinase phosphorylation sites
(220GARRRGGSAS229)
and
(817AVRIRGKSYV826)
in human telomerase reverse transcriptase (hTERT) subunit. To demonstrate that hTERT is an Akt kinase substrate protein, we performed
the nonradioactive protein kinase assay with the fluorescein hTERT
peptide
(817AVRIRGKSYV826).
We observed the phosphorylation of hTERT peptide by the human melanoma
cell lysate or the activated recombinant Akt kinase proteins in
vitro. With the treatment of the growth factor deprivation or
okadaic acid, we also observed the up-regulation of both hTERT peptide
phosphorylation and the telomerase activity. We noticed that Wortmannin
down-regulates hTERT peptide phosphorylation and telomerase activity
together. In addition, we observed the enhancement of telomerase
activity with the pretreatment of Akt kinase in vitro.
Thus, these observations suggest that Akt kinase enhances human
telomerase activity through phosphorylation of hTERT subunit as one of
its substrate proteins.
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INTRODUCTION |
Akt protein kinase (a serine/threonine kinase, also called protein
kinase B or Akt kinase) was identified at first as an oncogene because
of its transformation ability of normal cells (1, 2). However, the role
of Akt kinase was characterized as the anti-apoptosis protein, which
protects the cell death induced by growth factor withdrawal and prompts
the cell proliferation or survival (3-6). Moreover, with cDNA
sequence homology search, the pleckstrin homology domain in Akt was
identified (7). The recognition of pleckstrin homology domain, which
binds to membrane with the phosphatidylinositol-3,4-diphosphate activation, provided a clue to explain the linkage between the activation of phosphatidylinositol 3-OH
(PI3)1 kinase and Akt kinase
activity. Furthermore, it was characterized that
phosphatidylinositol-3,4-diphosphate localizes Akt kinase to membrane
and alters its conformation, activating its kinase activity by
phosphorylation of two different kinases. PI3-dependent protein kinase 1 phosphorylates Thr308 in the activation
loop of the kinase domain of protein kinase B, and
PI3-dependent protein kinase 2 phosphorylates
Ser473 near the carboxyl terminus of protein kinase B
(7-9). The release of Akt from membrane was also known to be a key
regulatory step of Akt kinase. It was proposed that inositol
trisphosphate, presumably generated from
phosphatidylinositol-4,5-diphosphate by phospholipase C-
, could
release pleckstrin homology domain-containing proteins from membrane
(8-10). After releasing from membrane, the activated Akt kinase would
become available to phosphorylate its downstream targets until
inactivated by protein phosphatase 2A (PP2A). Therefore, this evidence
indicated that Akt kinase is a key effector of PI3 kinase signal
pathway, and the identification of Akt kinase authentic substrates
provides clues to understand how PI3 kinase signal pathway contributes
for the cell survival.
The first identified physiological substrate of Akt kinase was the
glycogen synthase kinase 3 (GSK3)
and
and the heart isoenzyme
6-phosphofructo-2-kinase (10, 11,). Phosphorylation of GSK3 by Akt
kinase was reported to stimulate glycogen synthesis with the
inactivation of GSK3 kinase and to affect other aspects of cellular
function (11). It was proposed that GSK3 plays a role in the regulation
of protein synthesis, modulation of transcription factors (AP-1 and
cAMP response element-binding proteins), the cell fate determination
(in Drosophila), and dorsal-ventral patterning (in
Xenopus). Other researchers also suggested that Akt is
upstream of the p70 ribosomal protein S6 kinase, but the connection is assumed to be indirect (11, 12). Recently, the inactivation of
pro-apoptotic proteins (such as BAD protein or caspase-9) by Akt kinase
phosphorylation was demonstrated (13-15). Besides BAD protein and
caspase-9, H2B histone was also identified as an Akt kinase authentic
substrate (16). From all reported Akt kinase substrate amino acid
sequences, including BAD, PEK2, GSK3, H2B and caspase-9, the putative
Akt kinase substrate consensus sequence was identified (10-16). The
consensus sequence is conserved within the amino acid stretches; the
arginine residues at positions
5 and
3 were positioned relative to
those of serine/threonine residues to be phosphorylated by Akt kinase
in these proteins
(XXRXRXXS/TXX; the underline is a hydrophobic amino acid).
In our previous observation, we identified that human telomerase
activity in SK-MEL 28 (a melanoma cell line) cells was enhanced in the
serum-free condition without any activity change of tyrosine kinase
(17). Even though the mechanism by which telomerase is regulated in a
cell is not yet established, several researchers reported that
telomerase activity is effectively inhibited with the serine/threonine
kinase inhibitors than the tyrosine kinase inhibitors (18, 19). Other
researchers also observed that the incubation of cell nuclear
telomerase extracts with PP2A, inactivating the active Akt kinase with
its dephosphorylation activity, abolished the telomerase activity.
Thus, they suggested that the telomerase activity is regulated by the
serine/threonine phosphorylation (9, 19). Moreover, it was reported
that PI3 kinase plays a role for the induction of telomerase activity
in B cells (20). Therefore, we speculated that human telomerase is
regulated by Akt kinase in PI3 kinase pathway for the cell proliferation or survival.
To demonstrate our speculation, we inspected human telomerase amino
acid sequences with the putative Akt kinase substrate consensus
sequence. Two putative Akt kinase phosphorylation sites (all serine
residues)
(220GARRRGGSAS229
and
817AVRIRGKSYV826)
in human telomerase reverse transcriptase (hTERT, telomerase catalytic
subunit), not in human telomerase associate protein subunit, have been
identified (21, 22). We observed the phosphorylation of the
fluorescein-labeled hTERT peptide
(817AVRIRGKSYV826)
by the human melanoma cell lysate or the activated Akt kinase in
vitro. In addition, we observed the up-regulation of hTERT phosphorylation and the telomerase activity in a human melanoma cell
line with the treatment of okadaic acid (a PP2A inhibitor) or the
growth factor deprivation. With the treatment of wortmannin (a specific
PI3-Akt kinase inhibitor), however, we noticed the down-regulation of
hTERT phosphorylation and the telomerase activity. Moreover the
enhancement of telomerase activity with the pretreatment of Akt kinase
in vitro was also observed. Therefore, these observations strongly suggest that Akt kinase activates human telomerase through hTERT phosphorylation, as one of Akt kinase target proteins.
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EXPERIMENTAL PROCEDURES |
Cell Culture
SK-MEL 28 cell (a human melanoma cell
line) was purchased from ATCC (Manassas, VA). Media and supplements
were obtained from Life Technologies, Inc. The cell line was maintained in Dulbecco's modified essential medium (DMEM) containing 10% heat-inactivated (30 min at 56 °C) fetal bovine serum (FBS), 100 units of potassium penicillin/ml, 100 µg of streptomycin/ml, 2 mM glutamine, and 20 mM sodium bicarbonate. The
cells were incubated at 5% CO2, 95% humidity, and
37 °C chamber. The growth media was changed every 3 days.
Akt Protein Kinase Assay--
Akt kinase assay was performed
with the protocol provided by Promega (Madison, Wisconsin) PepTag
nonradioactive protein kinase C (PKC) assay system, except for the
substrate peptides. For Akt kinase substrates, the fluorescein
(fluorescein isothiocyanate was conjugated on the amino terminus of
peptide) H2B histone
(30RKRSRKESYS
39) and hTERT
(817AVRIRGKSYV826)
oligopeptides were purchased from Peptron Co. (Daejun, Korea). 5 µg
of fluorescein oligopeptide was incubated with 10 µl of differential treated cell lysates or the activated Akt kinase in 20 µl of protein kinase reaction mixture (20 mM HEPES, pH 7.2, 10 mM MgCl2, 10 mM MnCl2,
1 mM dithiothreitol, 0.2 mM EGTA, 20 µM ATP, 1 µg phosphatidylserine, protein kinase
activator) at 30 °C for 30 min. The reactions were stopped by
heating to 95 °C for 10 min. The phosphorylated peptide was
separated on 0.8% agarose gel at 100 V for 15 min. The phosphorylated products, which gained one more negative charge, were migrated to the
anode. After the gel was photographed on a transilluminator, the
phosphorylated peptide band was sliced out. The optical density (at 488 nm) of phosphorylated substrate was measured with the spectrophotometer, following the protocol provided by the assay kit manufacturer.
Activation of the Recombinant GST-Akt Kinase Protein--
Akt
kinase-agarose beads and PP2A were purchased from Upstate Biotechnology
Inc. (Lake Placid, NY). Akt kinase was activated with the protocol
provided by the manufacturer. SK-MEL 28 cells (107) grown
in the serum-free condition for 24 h were lysed with radioimmune precipitation buffer lysate buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1%
SDS, 1 mM sodium orthovanadate, 100 µg/ml
phenylmethylsulfonyl fluoride, 1 µg of aprotinin). The cell lysate
was precleaned with GST-agarose beads. Akt kinase-agarose beads (20 µg) were incubated with 50 µl of precleaned cell lysate and protein
kinase assay buffer (20 mM HEPES, pH 7.2, 10 mM
MgCl2, 10 mM MnCl2 1 mM
dithiothreitol, 0.2 mM EGTA, 20 µM ATP, 5 µg of phosphatidylserine, protein kinase activator) in a final volume
100 µl for 1 h at 30 °C. The beads were precipitated and
washed three times with the excess cell lysis buffer. The final pellet
was used for the Akt kinase assay.
Telomerase Assay--
SK-MEL 28 cells (1 × 106) were plated onto a 100-mm2 plate and grown
in 10% FBS DMEM for 3 days. When the cell confluence became 70%,
media were changed with 10% FBS DMEM or DMEM without serum for the
control experiment. For the other set of plates containing 70%
confluent cells, media were changed with 10% FBS DMEM containing wortmannin or okadaic acid (Calbiochem-Novabiochem). After the plates
were incubated for the telomerase assay, the cells were harvested, and
the cell number was counted. The counted cells were centrifuged at
3000 × g for 10 min at 4 °C. For the each reaction, 2 × 106 cells were transferred into a fresh Eppendorf tube. The
telomerase reaction mixture with the cell extract (corresponding to
5 × 103 cells or 2 µg of protein) was amplified by
the telomeric repeat amplification protocol (17, 23). For the
pretreatment experiment, we used the activated recombinant Akt kinase,
the rat brain PKC (Promega), or PP2A. 20 µl of the reaction mixture
(12 µl of SK-MEL 28 cell lysate, 10 ng of enzyme, and 4 µl of 5×
assay buffer) was incubated at 30 °C for 30 min. 3 µl of the
pretreatment reaction mixture was used for the telomerase assay. The
assay procedure of telomerase followed the protocol provided by the
telomerase polymerase chain reaction enzyme-linked immunosorbent assay
manufacturer, Roche Molecular Biochemicals. The optical density of the
samples was measured with a microtiter plate reader at 450 nm within 30 min after addition of the stop solution. Alternatively, 12%
nondenatured acrylamide gel electrophoresis was performed with 20 µl
of polymerase chain reaction product and transferred on nitrocellulose
paper. The biotinylated telomerase product was probed with the
streptavidin-peroxidase conjugate (Amersham Pharmacia Biotech)
following the assay procedure of the manufacturer.
 |
RESULTS |
The Enhancement of Akt Kinase and Telomerase Activities in SK-MEL
28 Cells with the Treatment of Growth Factor Deprivation--
We
observed that the telomerase activity in SK-MEL 28 cells (human
melanoma cell line) is 2 -fold increased in the serum-free medium for
24 h of treatment (Fig. 1). Because
many cellular protein activities are enhanced by phosphorylation, we
speculated that the enhancement of telomerase activity in serum-free
conditions is also because of its phosphorylation by certain protein
kinase(s), resulting in the activation of telomerase. To determine
which protein kinase is responsible for the enhancement of telomerase activity in the growth factor deprivation condition, we assayed the
telomerase activity from the cells treated with several protein kinase
inhibitors. We observed that bisindolylmaleimide (a serine/threonine kinase inhibitor) inhibited the telomerase activity effectively in a
time- and dose-dependent manner, whereas genistein (a
tyrosine kinase inhibitor) did not efficiently inhibit the telomerase
activity (17, 18). Moreover, we observed that the tyrosine protein kinase activity is not enhanced with the treatment of growth factor deprivation (data not shown). Thus, we assumed that the increase of
telomerase activity in the serum-free condition is because of the
serine/threonine kinase(s). Recently, other researchers also suggested
that activation of Akt kinase is one mechanism to explain how PI3
kinase can mediate survival of H19-7 cells during serum deprivation or
differentiation (5). Therefore, based on this evidence, we speculated
that the activation of Akt kinase in SK-MEL 28 cells to resist
apoptosis induced by the growth factor deprivation enhances the
telomerase activity. For Akt kinase assay, we used the nonradioactive
protein kinase assay method with the fluorescein H2B, histone peptide
(30RKRSRKESYS
39), which is known as an Akt kinase substrate (13). As shown Fig. 1, Akt kinase activity was up-regulated with the treatment of
growth factor deprivation in a time-dependent manner. This result led us next to examine whether hTERT subunit or human
telomerase-associated protein subunit contains the putative target
amino acid sequences phosphorylated by Akt kinase (Table
I). In addition, we examined the
phosphorylation of hTERT in the serum-free medium with the fluorescein
hTERT
(817AVRIRGKSYV826)
peptide (see below). As shown in Fig. 1, the hTERT phosphorylation was
also enhanced with the treatment of growth deprivation in a
time-dependent manner. Thus, these results suggest that the increase of Akt kinase activity to protect from apoptosis induced by
the growth factor deprivation enhances human telomerase activity with
its phosphorylation in SK-MEL 28 cells.

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Fig. 1.
Human telomerase activity and Akt kinase in
SK-MEL 28 cell is enhanced with the growth factor deprivation. The
cells (1 × 106) were placed into 100-cm2
culture dish and grown in 10% FBS DMEM for 3 days. When the cells
became 70% confluent, media were changed to DMEM without FBS. The
telomerase activity ( ) of the 0-, 6-, 24-, and 48-h serum-free
culture cell extract (for the each reaction, 2 × 106
cells) was measured with polymerase chain reaction enzyme-linked
immunosorbent assay method (purchased from Roche Molecular
Biochemicals). The values are the mean of three replicates. Each
bar indicates the S.E. The telomerase activity is the
relative value to the maximum optical density at 450 nm. Akt kinase
activity ( ) of the 0-, 6-, 24-, and 48-h serum-free culture cell
extract was also measured with PepTag nonradioactive protein kinase
assay method (purchased from Promega). The fluorescein H2B histone
peptide
(30RKRSRKESYS39)
was used as a substrate of Akt kinase assay (13). With the fluorescein
hTERT peptide (see Fig. 2 and Table I), we also measured the hTERT
phosphorylation (×). Akt kinase activity and hTERT phosphorylation are
the relative value to the maximum optical density at 488 nm. Telomerase
( ), Akt kinase ( ), and hTERT phosphorylation (×) are enhanced
with the growth deprivation treatment in a time-dependent
manner.
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Table I
Alignment of the putative Akt kinase phosphorylation site of hTERT
subunit and the consensus sequence
We noticed two putative Akt kinase substrate sites in hTERT amino acid
sequences with its putative substrate consensus sequence. The
identified Akt kinase substrate proteins are GSK3 and (10),
heart isoenzyme 6-phosphofructo-2-kinase (PFK-2) (11), BAD protein
(13), caspase-9 (15), and H2B histone (16). The arginine residues on
the 5 and 3 positions and a hydrophobic amino acid on the +2
position (underline) are conserved relative to those of
serine/threonine residues
(XXRXRXXS/TXX).
The consensus sequence is not found in the human telomerase-associated
protein, another telomerase subunit. We used hTERT peptide
(817AVRIRGKSYV826) as
an Akt kinase assay substrate (in Figs. 1, 2, and
3).
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The Phosphorylation of hTERT Subunit by Akt Kinase--
Inspecting
the amino acid sequences of all known Akt kinase substrates (H2B, BAD,
PEK2, GSK3, and caspase-9), the serine residue is found within
stretches of amino acids with homology (Table I). The arginine residues
at positions
5 and
3 and the hydrophobic amino acid at the +2
position are conserved relative to the serine/threonine residues, which
are probably phosphorylated in these proteins (11-16). Therefore, the
peptide
(XXRXRXXS/TXX, a hydrophobic amino acid is underlined) seems to be conserved for Akt
kinase substrate. With this sequence
(XXRXRXXS/TXX), two putative Akt kinase phosphorylation sites
(220GARRRGGSAS229)
and
(817AVRIRGKSYV826)
(Table I), and two similar sites
(260PGRTRGPSDR269)
and
(977VLRLKCHSLF986)
are identified in hTERT (21, 22). However, Akt kinase substrate sequences
(XXRXRXXSXX)
were not found in human telomerase-associated protein subunit that
contain several PKC recognition sites (24).
With the fluorescein hTERT
(817AVRIRGKSYV826)
peptide, we observed that the hTERT peptide was phosphorylated by the
human melanoma cell lysate, which contains Akt kinase activity (Fig. 1). To further determine that the serine residue
(817AVRIRGKSYV826)
in hTERT is phosphorylated by Akt kinase, we used the activated Akt
kinase. GST-Akt recombinant kinase expressed in Escherichia coli was activated with human melanoma cell lysate. With the
increase of assay time, the amount of phosphorylated hTERT peptide was increased (Fig. 2A). As shown
in Fig. 2B, the increase of hTERT peptide phosphorylation
was also observed with the increase of the activated Akt kinase
concentration. For the control experiment, we used the inactive
recombinant GST-Akt kinase protein, which has no kinase activity (Fig.
2, A and B). Furthermore, we observed that the
other hTERT synthetic peptide (220
GARRRGGSAS
229) and GST-hTERT 670-843 recombinant
protein, which contains the putative phosphorylation site
(817AVRIRGKSYV829),
were phosphorylated by the activated Akt kinase (data not shown). Thus,
these results demonstrated that Akt kinase phosphorylates hTERT as one
of its target proteins.

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Fig. 2.
Phosphorylation of hTERT peptide by Akt
kinase. To demonstrate that Akt kinase phosphorylates the
fluorescein hTERT
(817AVRIRGKSYV826)
peptide (see Table I), a protein kinase assay was performed as
described in Fig. 1. With the activated recombinant Akt kinase, we
observed the increase of hTERT peptide phosphorylation, depending on
the reaction time (A) or the enzyme concentration
(B). For the control experiment, we also used the
inactivated recombinant Akt kinase. The phosphorylated product ( )
and unphosphorylated substrate ( ) are marked. The ratio of product
to substrate (P/S) was indicated below.
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The Regulation of Human Telomerase Activity and hTERT
Phosphorylation with Drugs Affecting Akt Kinase Activity--
For the
further demonstration that Akt kinase regulates the telomerase activity
as its substrate, we used wortmannin and okadaic acid to modulate Akt
kinase activity. It was reported that wortmannin is a
phosphatidylinositol-Akt kinase pathway inhibitor, resulting in the
specific Akt kinase inactivation (8, 9, 11). We assayed the telomerase
activity of human melanoma cell line with wortmannin treatment (0, 50, 100 nM for 2 h treatment). As shown in Fig.
3A, wortmannin inhibited human
telomerase activity in a dose-dependent manner. On the
other hand, okadaic acid, a PP2A inhibitor, is known to activate Akt
kinase with its phosphorylation (8, 9, 11). To address that the
activation of Akt kinase also enhances human telomerase activity, we
also performed human telomerase assay with okadaic acid treatment (0, 75, 150, 300 nM for 2 h), and we observed that the
treatment of okadaic acid enhanced telomerase activity in a
dose-dependent manner (Fig. 3A). To monitor Akt
kinase activity change with wortmannin or okadaic acid treatments, the
fluorescein H2B peptide (Fig. 3B) was used as Akt kinase
assay substrate. We observed the inhibition of Akt kinase activity with
wortmannin (100 nM for 2 h) and its activation with
okadaic acid (300 nM for 2 h). We also used the fluorescein hTERT peptide to monitor telomerase phosphorylation (Fig.
3C). The activated Akt kinase was used as the positive
control for the phosphorylated product hTERT peptide. The
heat-inactivated normal cell lysate was used as the negative control.
We noticed that the inhibition of hTERT peptide phosphorylation with
wortmannin (100 nM for 2 h) and the activation with
okadaic acid (300 nM for 2 h) paralleled with the Akt
kinase activity change (Fig. 3B). Thus, these results,
indicating that drugs affecting Akt kinase modulate both human
telomerase activity and the phosphorylation of hTERT, suggest that
human telomerase is activated by Akt kinase phosphorylation.

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Fig. 3.
Akt kinase regulates human telomerase
activity with hTERT phosphorylation. To determine whether human
telomerase activity is regulated by Akt kinase activity, telomerase
activity in human melanoma cell line with the treatment of wortmannin
or okadaic acid was observed. Akt kinase or telomerase activity assay
was performed with the protocol as described in Fig. 1. With the
treatment of Wortmannin, telomerase activity was down-regulated,
whereas it was up-regulated with the treatment of okadaic acid in a
dose-dependent manner (A). The values are the
mean of three replicates. Each bar indicates the S.E. The
positive control was 293 cell lysate, provided by the manufacturer. The
negative control was heat-treated (65 °C, 10 min) SK-MEL 28 cells.
Akt kinase activity (B) was enhanced with okadaic acid (300 nM, 2 h) or the serum-free (for 24 h) treatment,
whereas it was inactivated with wortmannin (100 nM, 2 h). The fluorescein hTERT peptide phosphorylation (C) was
also enhanced with okadaic acid (300 nM for 2 h) or
the serum-free (for 24 h) treatment, whereas it was inactivated
with wortmannin (100 nM, 2 h). The activated
recombinant Akt kinase (10 ng) was used as the positive control to
identify the phosphorylated product hTERT peptide. The heat-inactivated
SK-MEL 28 cell lysate was also used as the negative control. The
phosphorylated product ( ) and unphosphorylated substrate ( ) are
marked.
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The Enhancement of Telomerase Activity with the Pretreatment of Akt
Kinase--
To further confirm that Akt kinase activates human
telomerase with its phosphorylation, we performed the telomerase
activity assay with/without the pretreatment of Akt kinase in
vitro (Fig. 4). Recently, other data
suggested that PKC
or PP2A controls telomerase activity in human
breast cancer cells (19, 24). Thus, for the control experiment, we used
the rat brain PKC and PP2A. With the pretreatment of PKC and the
activated Akt kinase (10 ng), the human telomerase activity enhanced by
2-fold that of the normal control (Fig. 4), whereas the telomerase
activity decreased 2-fold with the pretreatment of PP2A (10 ng). Thus, these results also suggest that human telomerase is activated by Akt
kinase phosphorylation. Taken together, our data strongly suggest that
Akt kinase phosphorylates hTERT as one of its target proteins and
enhances human telomerase activity.

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Fig. 4.
The activation of telomerase with Akt
kinase. The telomerase activity of human melanoma cell lysate
after the pretreatment with 10 ng of the activated Akt kinase, rat
brain PKC, or PP2A was measured as described in Fig. 3. Human
telomerase activity was enhanced with Akt kinase or PKC, whereas it was
inhibited with PP2A. The positive control was 293 cell lysate, provided
by the manufacturer. Negative control was heat-treated (65 °C, 10 min) SK-MEL 28 cell lysate.
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DISCUSSION |
The activation or inactivation of Akt kinase in the cell is one of
the critical regulatory points to deliver either a survival or an
apoptotic signal. Thus, the upstream signal transduction pathway in
which Akt kinase is regulated in the cell becomes a research area
interested intensively (8-11). To understand how Akt kinase function
in the PI3 kinase pathway contributes to the cell
proliferation/survival, the identification of substrate protein that is
phosphorylated by Akt kinase and the characterization of how Akt kinase
phosphorylation modulates the protein function (either activation or
inhibition) also seem to be important.
In this article, we demonstrated that hTERT is one of Akt kinase
authentic substrate proteins with the nonradioactive Akt kinase assay
method, and human telomerase activity is enhanced through hTERT
phosphorylation by Akt kinase. With the growth factor deprivation, the
up-regulation of human telomerase activity was observed (Fig. 1).
However, any increase of tyrosine kinase activity was not observed in
the growth factor-free condition, except Akt kinase. Thus, we
speculated that Akt kinase, which was induced to resist apoptosis with
the treatment of growth factor deprivation, also enhances human
telomerase activity, and inspected human telomerase amino acid
sequences with the putative Akt kinase substrate consensus sequence
(XXRXRXXS/TXX;
the underline represents a hydrophobic amino acid). We noticed two
putative Akt kinase phosphorylation sites
(220GARRRGGSAS229
and
817AVRIRGKSYV826)
in hTERT (21). To demonstrate whether hTERT is phosphorylated by Akt
kinase, we used the fluorescein hTERT peptide
(817AVRIRGKSYV826)
as a substrate for the human melanoma cell lysate or the activated Akt
kinase protein. As shown in Figs. 1, 2, and 3, both the positive control substrate, H2B peptide
(30RKRSRKESYS39)
and hTERT peptide were phosphorylated by the activated Akt kinase and
the cell lysate, respectively. In addition, we observed the down-regulation of the telomerase activity and hTERT phosphorylation with the treatment of wortmannin, a specific Akt kinase inhibitor from
a fungus metabolite, and the up-regulation of these with the treatment
of okadaic acid, an inhibitor of protein phosphatase 2A, in the human
melanoma cell line (Fig. 3). Moreover, we observed the telomerase
activity enhancement with Akt kinase pretreatment in vitro
(Fig. 4). Thus, these observations strongly suggest that human
telomerase is activated through hTERT phosphorylation by Akt kinase as
one of its target proteins. The other possible Akt kinase
phosphorylation site peptide
(220GARRRGGSAS229)
in hTERT was also phosphorylated by Akt kinase (data not shown). However, we do not know which site is more frequently phosphorylated and how the differential phosphorylation affects the telomerase activity in vivo. Thus, it remains to be determined how the
phosphorylation is regulated by Akt kinase, depending on the cell cycle
or the outside signal. Considering Akt kinase substrate specificity and the kinase assay with peptide, we speculate that the order of phosphorylation in vivo is
(817AVRIRGKSYV826) > (220GARRRGGSAS229).
Two sites
(260PGRTRGPSDR269)
and
(977VLRLKCHSLF986),
which are similar to Akt kinase consensus sequence, are also found in
hTERT. However, it remains to be characterized whether the
phosphorylation at these sites also affects the telomerase activity
in vivo with the phosphorylation site mapping. We are now
investigating with a series of these site mutants.
The abnormally active telomerase in cancer cells has become one of the
most interesting targets to cure many cancers (20-22). Moreover, the
expression of hTERT subunit containing two putative phosphorylation
sites by Akt kinase is only up-regulated in the cancer cells, whereas
other components of telomerase are unchanged (21, 22, 25). Even though
it is still unclear why hTERT expression is only up-regulated in the
cancer, it is interesting that two Akt kinase phosphorylation sites are
exclusively located in hTERT, not any in other components of
telomerase. Thus we speculate that because telomerase activity is
regulated through hTERT phosphorylation by Akt kinase, only hTERT
expression among other components of telomerase is tightly regulated in
the cells.
Recently, Akt kinase family proteins (Akt1, Akt2, and Akt3) have been
characterized (26-28). Because of their amino acid sequence homology,
each Akt kinase-specific function seems to be for the redundancy. Thus,
we suppose that Akt2 and 3 are also the responsible kinase for the
telomerase phosphorylation. However, it remains to be determined
whether Akt2 and Akt3 also have the same substrate specificity.
Interestingly, in inspecting the amino acid sequences of Akt1, 2, and
3, we identify the consensus phosphorylation sites (TERPRPNTFY) that can
explain why Akt kinase was autophosphorylated in other research data
(13). Until now, two different Akt kinase kinases and their
phosphorylation sites on Akt kinase are characterized. They are
PI3-dependent protein kinase 1, which phosphorylates Thr308 in the activation loop of the kinase domain of
protein kinase B, and PI3-dependent protein kinase 2. which
phosphorylates Ser473 near the carboxyl terminus. Thus, it
is necessary to characterize how the autophosphorylation of Akt kinase
on (TERPRPNTFY) regulates
its function in vivo.
Several biological important proteins, including BAD protein,
caspase-9, H2B histone, PEK2, and GSK3, have been identified as the
substrates of Akt kinase (10-16). The arginine residues at positions
5 and
3 were conserved relative to those of serine residues to be
phosphorylated in these proteins. With the replacement either
3 or
5 arginine with alanine in the Akt kinase substrate consensus
sequences, the serine phosphorylation by Akt kinase was abolished
completely (16).2 Thus, it
seems to be that Akt kinase substrate specificity
(XXRXRXXS/TXX) is
conserved well in the other proteins as Akt kinase substrate. Therefore, the inspection of these consensus amino acid sequences may
help to determine whether these proteins are the possible Akt kinase
substrate. With Akt kinase substrate consensus sequences (XXRXRXXS/TXX;
the underline is a hydrophobic amino acid), we noticed several possible
Akt kinase substrate proteins, including the apoptosis-related
proteins, RIP-like kinase (30), and caspase-7 (31). Recently, it was
reported that caspase-9 is inactivated by Akt kinase phosphorylation
for the cell survival or proliferation (15). Thus, we speculate that
RIP-like kinase and caspase-7 activities seem to be inactivated by Akt
kinase phosphorylation, similar to the regulation mechanism of
caspase-9. Moreover, we noticed the consensus sequence in the several
GTPase proteins, including CDC42 and rac1 (32). These consensus
sequences are also noticed in human I
B-related protein (33) and
MEKK3 (34). Even though the relationships between Akt kinase and these proteins are presently unknown, Akt kinase phosphorylation sites of
these proteins may contribute the signal cross-talk between two
different signal pathways. Interestingly, GLUT4, a glucose transport
protein, also contains the consensus Akt kinase phosphorylation site
(29). We assume that because glucose is the basic energy source for the
cell survival, Akt kinase is also adopted to involve the cell glucose
metabolism with the phosphorylation of GSK3, 6-phosphofructo-2-kinase,
and GLUT4.
In summary, the phosphorylation site of Akt kinase, which triggers a
plethora of potential biological outcomes, seems to be highly conserved
as Akt kinase substrate consensus sequences
(XXRXRXXS/TXX). Thus, the inspection of protein sequences with this consensus sequence
may help to identify Akt kinase substrate protein. However, because
some of its substrate proteins are activated or inactivated by Akt
kinase phosphorylation for the cell proliferation/survival or the
signal cross-talk, it is also necessary to determine whether Akt kinase
phosphorylation activates or inactivates each Akt kinase substrate
protein function. Thus, the identification of Akt kinase substrate
protein and the characterization of its functional modification by Akt
kinase phosphorylation may provide clues to understanding how Akt
kinase functions contributes to protect the cell apoptosis or promote
the cell proliferation collectively. In this article, we demonstrate
that Akt kinase activates human telomerase activity, which plays an
important role in keeping the telomere length for the cell
proliferation through the phosphorylation of hTERT subunit, as one of
Akt kinase target proteins.
 |
ACKNOWLEDGEMENTS |
We thank Jung Hyun Lee, Yong Tae Jung, and
Sok Kuon Lee for their help in preparing this manuscript. We also thank
Drs. Jae Sun Chun, Pan Gil Suh, and Jong Suk Ahn for their comments.
 |
FOOTNOTES |
*
This work was supported by Research and Development Fund
from Shin Dong Bang Inc. and by the grants of Biotech 2000 NB1010 and
G7 project HS-2150 from the Korean Ministry of Science and Technology.The costs of publication of this
article were defrayed in part by the
payment of page charges. The 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: Shin Dong Bang R&D
Center, 363, Yangjae 2-dong, Seocho gu, Seoul 137-132, Republic of
Korea. Tel.: 82-2-573-6100; Fax: 82-2-571-5058; E-mail: 95324JIN{at}mail.hitel.net.
2
S. S. Kang, T. Kwon, D. Y. Kwon, and
S. I. Do, unpublished data.
 |
ABBREVIATIONS |
The abbreviations used are:
PI3, phosphatidylinositol 3-OH;
PP2A, protein phosphatase 2A;
GSK3, glycogen
synthase kinase 3;
hTERT, human telomerase reverse transcriptase;
DMEM, Dulbecco's modified essential medium;
FBS, fetal bovine serum;
PKC, protein kinase C;
GST, glutathione S-transferase.
 |
REFERENCES |
-
Chang, H. W.,
Aoki, M.,
Fruman, D.,
Auger, K. R.,
Bellacosa, A.,
Tsichlis, P. N.,
Cantly, L. C.,
Roberts, T. M.,
and Vogt, P. K.
(1997)
Science
276,
1848-1850[Abstract/Free Full Text]
-
Khwaja, A.,
Rodriguez-Viciana, P.,
Wennstrom, S.,
Warne, P. H.,
and Downward, J.
(1997)
EMBO J.
16,
2783-2793[Abstract/Free Full Text]
-
Franke, T. F.,
Kaplan, D. R.,
Cantly, L. C.,
and Toker, A.
(1997)
Science
275,
665-668[Abstract/Free Full Text]
-
Crowder, R. J.,
and Freeman, R. S.
(1998)
J. Neurosci.
18,
2933-2943[Abstract/Free Full Text]
-
Eves, E. M.,
Xiong, W.,
Bellacosa, A.,
Kennedy, S. G.,
Tsichlis, P. N.,
Rosner, M. R.,
and Hay, N.
(1998)
Mol. Cell. Biol.
28,
2143-2152
-
Ulrich, E.,
Duwel, A.,
Kauffman-Zeh, A.,
Gilbert, C.,
Lyon, D.,
Rudkin, B.,
Evan, G.,
and Martin-Zanca, D.
(1998)
Oncogene
16,
825-832[CrossRef][Medline]
[Order article via Infotrieve]
-
Datta, K.,
Franke, T. F.,
Chan, T. O.,
Makkris, A.,
Yang, S. I.,
Kaplan, D. R.,
Morrison, D. K.,
Golemis, E. A.,
and Tsichlis, P. N.
(1995)
Mol. Cell. Biol.
15,
2304-2310[Abstract]
-
Hemmings, B. A
(1997)
Science
277,
534[Free Full Text]
-
Hemmings, B. A.
(1997)
Science
275,
628-630[Free Full Text]
-
Franke, T. F.,
Kaplan, D. R.,
and Cantly, L. C.
(1997)
Cell
88,
435-437[Medline]
[Order article via Infotrieve]
-
Toker, A.,
and Cantley, L. C.
(1997)
Nature
387,
673-676[CrossRef][Medline]
[Order article via Infotrieve]
-
Pullen, N.,
Dennis, P. B.,
Andjelkovic, M.,
Dufner, A.,
Kozma, S. C.,
Hemmings, B. A.,
and Thomas, G.
(1998)
Science
276,
707-710[Abstract/Free Full Text]
-
del Peso, L.,
Gonzalez-Garcia, M.,
Page, C.,
Herrera, R.,
and Nunez, G.
(1997)
Science
278,
687-689[Abstract/Free Full Text]
-
Datta, S. R.,
Dudek, H.,
Tao, X.,
Masters, S.,
Fu, H.,
Gotoh, Y.,
and Greenberg, M. E.
(1997)
Cell
91,
231-241[Medline]
[Order article via Infotrieve]
-
Cardone, M. H.,
Roy, N.,
Stennicke, H. R.,
Salvesen, G. S.,
Franke, T. F.,
Stanbrige, E.,
Frisch, S.,
and Reed, J. C.
(1998)
Science
282,
1318-1321[Abstract/Free Full Text]
-
Alessi, D. R.,
Caudwell, F. B.,
Andjelkovic, M.,
Hemmings, B. A.,
and Cohen, P.
(1996)
FEBS Lett.
399,
333-338[CrossRef][Medline]
[Order article via Infotrieve]
-
Kang, S. S.,
and Lim, S. E.
(1998)
J. Biochem. Mol. Biol.
31,
339-344
-
Ku, W. C.,
Cheng, A. J.,
and Wang, T. C.
(1997)
Biochem. Biophys. Res. Commun.
241,
730-736[CrossRef][Medline]
[Order article via Infotrieve]
-
Li, H.,
Zhao, L. L.,
Funder, J. W.,
and Liu, J. P.
(1997)
J. Biol. Chem.
272,
16729-16732[Abstract/Free Full Text]
-
Igarashi, H.,
and Sakaguchi, N.
(1997)
Blood
89,
1299-1307[Abstract/Free Full Text]
-
Nakamura, T. M.,
Morin, G. B.,
Chapman, K. B.,
Weinrich, S. L.,
Anderson, W. H.,
Lingner, J.,
Harley, C. B.,
and Cech, T. B.
(1997)
Science
277,
955-959[Abstract/Free Full Text]
-
Harrington, L.,
McPhail, T.,
Mar, V.,
Zhou, W.,
Oulton, R.,
Bass, M. B.,
Arruda, I.,
and Robinson, M. O.
(1997)
Science
275,
973-977[Abstract/Free Full Text]
-
Kim, N. W.,
and Wu, F.
(1997)
Nucleic Acids Res.
25,
2595-2597[Abstract/Free Full Text]
-
Li, H.,
Zhao, L.,
Yang, Z.,
Funder, J. W.,
and Liu, J. P.
(1998)
J. Biol. Chem.
273,
33436-33442[Abstract/Free Full Text]
-
Feng, J.,
Funk, W. D.,
Wang, S.-S.,
Wenrich, S. L.,
Avilion, A. A.,
Chiu, C.-P.,
Adoms, R. R.,
Chang, E.,
Allsopp, R. C., Yu, J.,
Le, S.,
West, M. D.,
Harly, C. B.,
Andrews, W. H.,
Greider, C. W.,
and Villeponteau, B.
(1995)
Science
269,
1236-1241[Medline]
[Order article via Infotrieve]
-
Chen, J. Q.,
Godwin, A. K.,
Bellacosa, A.,
Taguchi, T.,
Franke, T. F.,
Hamilton, T. C.,
Tsichlis, P. N.,
and Testa, J. R.
(1992)
Proc. Natl. Acad. Sci. U. S. A.
89,
9267-9271[Abstract]
-
Staal, S. P.
(1987)
Proc. Natl. Acad. Sci. U. S. A.
84,
5034-5037[Abstract]
-
Barthel, A.,
Nakatani, K.,
Dandekar, A. A.,
and Roth, R. A.
(1998)
Biochem. Biophys. Res. Commun.
243,
509-513[CrossRef][Medline]
[Order article via Infotrieve]
-
Fukumoto, H.,
Kayano, T.,
Buse, J. B.,
Edwards, Y.,
Pilch, P. F.,
Bell, G. I.,
and Seino, S.
(1989)
J. Biol. Chem.
264,
7776-7779[Abstract/Free Full Text]
-
Thome, M.,
Hofmann, K.,
Burns, K.,
Martinon, F.,
Bodmer, J.-L.,
Mattmann, C.,
and Tschopp, J.
(1998)
Curr. Biol.
8,
885-888[Medline]
[Order article via Infotrieve]
-
Lippke, J. A.,
Gu, Y.,
Sarnecki, C.,
Caron, P. R.,
and Su, M. S.-S.
(1996)
J. Biol. Chem.
271,
1825-1828[Abstract/Free Full Text]
-
Munemitsu, S.,
Innis, M. S.,
Clark, R.,
McCormick, F.,
Ullirich, A.,
and Polakis, P.
(1990)
Mol. Cell. Biol.
10,
5977-5982[Medline]
[Order article via Infotrieve]
-
Ray, P.,
Zhang, D. H.,
Elias, J. A.,
and Ray, A.
(1995)
J. Biol. Chem.
270,
10680-10685[Abstract/Free Full Text]
-
Ellinger-Ziegelbauer, H.,
Brown, K.,
Kelly, K.,
and Siebenlist, U.
(1997)
J. Biol. Chem.
272,
2668-2674[Abstract/Free Full Text]
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.