pi3k/akt signaling pathway involved in regulation of t lymphocyte activation and apoptosis mediated...
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PI3K/Akt signaling pathway
involved in regulation of T
lymphocyte activation and
apoptosis mediated by CD3
ZHANG Jing, LIU Yanxin, LIU Shilian & ZHENG Dexian
National Laboratory of Medical Molecular Biology, Institute of Basic
Medical Sciences, Chinese Academy of Medical Sciences, Beijing
100005, China
Correspondence should be addressed to Zheng Dexian (e-mail:
Abstract To study the expression and kinase activity of
phosphatidylinositol 3 -kinase (PI3K) and protein kinase B
(PKB or Akt) during activation and apoptosis of human Jur-
kat T lymphocytes (TJK) with stable expression of CD8 chi-
mera fused human CD8 extracellular and transmembrane
domains to intracellular domain of mouse CD3 Western blot,
kinase activities detection and immunoprecipitation were
carried out. It was shown that Jurkat cells with expression of
wild type chimera CD8 died by apoptosis after continuous
stimulation of anti-CD8 monoclonal antibody. The expres-
sions of PI3K and Akt, and the kinase activity of Akt re-
markably increased during the process. However, this phe-
nomenon did not occur in the Jurkat cells (T1JK) with ex-
pression of the mutant of CD8 chimera (Y170F), suggesting
that PI3K/Akt signaling pathway is involved in activation
and apoptosis of T lymphocyte mediated by CD3
Keywords: apoptosis, CD8 chimera, T lymphocyte, PI3K, Akt.
Phosphatidylinositol 3 - kinase (PI3K) and protein
kinase B (PKB or Akt) are very important to cell survival.
PI3K/Akt pathway is involved in cellular response to
growth factors, lymphokines, and responsible for glucose
translocation or glycogen and protein synthesis[1,2]
, activa-
tion of immune cells[3]
, and inhibition of apoptosis[4,5]
. Akt
is a necessary regulator for cell survival. PI3K/Akt path-
way is also required for the activation of T lymphocyte
mediated by T cell antigen receptor complex (TCR/CD3).
Stimulatory signals of TCR/CD3 complex are transduced
by immunoreceptor tyrosine-based activation motifs
(ITAMs) in the cytoplasmic tail of CD3. The consensus
sequence of the ITAMs is YXXL(X)7-8 YXXL/I. There are
three ITAMs in CD3 but only one in each of the CD3 ,
CD3 and CD3 cytoplasma tails[6]
. In the T cell activa-
tion process, two tyrosine residues in the ITAM are phos-
phorylated and followed by activation of protein kinase
cascades[7]
. If the activated T lymphocytes were stimu-
lated again, activation-induced cell death (apoptosis)
could occur. In the previous studies, a Jurkat cell line
(named TJK) with expression of a chimera molecule fused
extracellular-transmembrane domain of human CD8 to
the cytoplasmic domain of mouse CD3 was established.
Stimulation of the cells with anti-CD8 monoclonal anti-
body showed that TJK cells with expression of the wild
type chimera died by apoptosis, but not those cells with
mutations of Y170F, Y181F and Y170F/Y181F in
CD8 -ITAM (these cell lines were designated T1JK, T2JK
and T3JK, respectively). This indicated that the two tyro-
sines in CD3 -ITAM were required for the apoptotic sig-
nal transduction in T lymphocytes[8,9]
. However, the role
of PI3K/Akt signaling pathway in the course of apoptosis
of lymphocytes remains to be clarified. In the present
study, we explored the change of PI3K and Akt during the
activation-induced apoptosis in the cell lines stimulated
with anti-CD8 monoclonal antibody.
1 Materials and methods
( ) Cell culture and induction of cell activation and
apoptosis. TJK and T1JK cells were cultured in RPMI
1640 media containing 10% fetal calf serum (FCS). Cells
were serum-starved for 12 h by incubation in low-serum
RPMI 1640 media (containing 0.1% FCS and 10 mmol/L
HEPES), and adjusted to 106 cells/mL. When cell activa-
tion was carried out, the cells were washed with phos-
phate buffer saline (PBS), and then resuspended in 100 L
anti-CD8 mAb (20 g/mL in PBS) pre-warmed at 37 ,
followed by incubating for 30 s 6 h and stopped by add-
ing 1 mL ice-cooled PBS. The cells incubated for 30 min
without stimulation with anti-CD8 mAb were as the nega-
tive control. When induction of apoptosis was carried out,
5 mL cell suspension in low-serum RPMI 1640 media
containing 5×106 cells was added to a 60 mm cell culture
plates pre-coated with anti-CD8 mAb and cultured for 6
48 h. The negative control was the cells incubated for 30 h
without stimulation with anti-CD8 mAb.
( ) Determination of cell apoptosis. Cells were
collected and washed with PBS, then stained with An-
nexin-V-FLOUS (Boehringer Mannham) according to the
manufacturers’ protocol. The apoptotic cells with green
color were examined and calculated under a fluorescence
microscope. The total cell number was calculated under
normal light in the same sight fields. The differences be-
tween TJK and T1JK groups were examined by chi-square
test, P 0.05.
( ) Western blot. Cells were collected and lysed
with routine procedure. Proteins were separated on
SDS-PAGE and transferred to PVDF Immobilon mem-
brane. Membranes were probed with anti-PI3K mAb
(Transduction Laboratories) and anti-Akt pAb (Santa
Cruz), respectively, then visualized with enhanced
chemiluminescence (ECL, Amersham/Pharmacia).
( ) Determination of Akt kinase activity. Cells
were collected and lysed with lysis buffer (1% NP-40, 100
mmol/L NaCl, 10 mmol/L Tris. Cl, pH 8.0, 1 g/mL pro-
568 Chinese Science Bulletin Vol. 46 No. 7 April 2001
Fig. 1. Dynamic change of apoptotic percentage of TJK and T1JK cells stimulated by anti-CD8 mAb.
Fig. 2. Expression of PI3K and Akt in
TJK and T1JK cell lines stimulated by
anti-CD8 mAb. Normal: TJK and T1JK
cells incubated in RPMI 1640 media
containing 10% fetal calf serum. Cont.
1: TJK and T1JK cells without stimula-
tion of anti-CD8 mAb for 30 min; Cont.
2: TJK and T1JK cells without stimula-
tion of anti-CD8 mAb for 30 h.
tease inhibitors, 1 mmol/L PMSF, 10 mmol/L NaF, 1
mmol/L NaVO3). Cell lysates were clarified by centrifu-
gation at 12000×g for 15 min at 4 . For immunoprecipi-
tation, the lysates were incubated with 10 L anti-Akt-
protein A-Sepharose CL-4B (Pharmacia).
The immunoprecipitate was washed with lysis buffer
twice, then washing buffer (500 mmol/L LiCl2, 100
mmol/L TrisCl, pH 7.5, 1 mmol/L EDTA) twice and
kinase buffer (500 mmol/L Tris.Cl, pH 7.5, 10 mmol/L
MgCl2, 1 mmol/L DTT) once. The pellet was suspended
in 15 L reaction buffer (kinase buffer containing 50
mol/L ATP, 3 Ci -32
P-ATP, and 3 g histone H2B was
used as the exogenous substrate), and incubated at 37
for 20 min. The reaction was stopped by adding the same
volume of 2×sample buffer and the labeled proteins were
separated in 15% SDS-PAGE gels and visualized by
autoradiography.
2 Results
( ) Apoptotic percentages of TJK and T1JK cells
stimulated by anti-CD8 mAb. The apoptotic percentages
of TJK and T1JK cells stimulated by anti-CD8 mAb in
different time (1, 6, 12, 18, 24, 30, 36 and 48 h) are shown
in fig. 1. The apoptotic percentage was about 4% in TJK
and T1JK cells stimulated with anti-CD8 mAb for 1 and 6
h. The percentage of apoptotic TJK was increased to
11.6% and T1JK to 9.4% respectively after the cells above
were stimulated for 12 h. Afterwards, the latter maintained
similar apoptotic percentage from 10% to 15% even
stimulating continually. But the percentage of TJK was
gradually increased and had a swift increase after stimula-
tion for 30 h, and reached 37.9% for 48 h. These results
suggested that anti-CD8 mAb induce cell death in TJK but
not in T1JK remarkably.
( ) Expressions of PI3K and Akt in the TJK and
T1JK cell line during activation and apoptosis. As
shown in fig. 2, PI3K and Akt were expressed in the TJK
and T1JK cells cultured under normal condition. The ex-
pression levels of Akt and PI3K were decreased to the
lowest level when the cells were incubated overnight in
low-serum RPMI 1640 media, and rapidly boosted and
maintained at a high level in TJK cells stimulated by
anti-CD8 mAb. In contrast, there was not any change of
Akt and PI3K expression in T1JK cells. The change of
Akt was parallel to PI3K during the course of activation
and apoptosis in TJK cells. The results above indicated
that the expression of Akt and PI3K quickly increased
when TJK cells were activated, and the expression levels
were maintained at rather high level when the cells un-
derwent cell death. However, there was no obvious
change in T1JK cells under the same condition, suggest-
ing that Akt/PI3K pathway is involved in the regulation of
activation and apoptosis of T lymphocytes.
( ) Kinase activities of Akt in TJK and T1JK cell
line at the course of activation and apoptosis. As illus-
trated in fig. 3, kinase activity of Akt remarkably in-
creased in TJK cells stimulated with anti-CD8 antibody
for 30 s and maintained at a high level for 30 h. However,
the kinase activity of Akt in TJK cells without stimulation
was at a low stable level. And there was no significant
change of the kinase activity of Akt in T1JK cells under
the same condition.
Chinese Science Bulletin Vol. 46 No. 7 April 2001 569
NOTES
Fig. 3. Kinase activities of Akt in TJK and T1JK cells stimulated by
anti-CD8 mAb. Cont. 1: TJK and T1JK cell lines without stimulation of
anti-CD8 mAb for 30 min; Cont. 2: TJK and T1JK cell lines without
stimulation of anti-CD8 mAb for 30 h.
3 Discussion
Previous study showed that CD8 , a chimera mole-
cule fused by the extracellular-transmembrane domain of
human CD8 to the cytoplasmic domain of CD3 induced
apoptosis of CD8+ Jurkat T lymphocytes (TJK) stimu-
lated with anti-CD8 monoclonal antibody[8]
. When TJK
cells were transiently stimulated by anti-CD8 mAb, Y170
and Y181 in CD8 - ITAM were phosphorylated. The ty-
rosine phosphorylation was correlated with increased
binding of p85 subunit of PI3K and recruitment of PI3K
enzymatic activity. But in those cells stably transfected
with CD8 mutants of Y170F, Y181F and Y170F/Y181F,
PI3K could not be activated after stimulation with
anti-CD8 mAb[10]
, indicating that the two tyrosines of
CD3 -ITAM play an important role during the processes
of activation and apoptosis of T lymphocytes. The signal
transduction would be blocked by mutation of either of
the two tyrosines in CD3 cytoplasmic tail.
In the present study, TJK cells underwent apoptosis
after stimulation with anti-CD8 mAb for 36 h, but this
phenomenon did not occur in T1JK cells with expression
of CD8 mutants of Y170F under the same conditions.
This is consistent with the previous results mentioned
above. Most important of all, we observed at the first time
that PI3K and Akt expressed at a high level and the kinase
activity of Akt, similar to PI3K, increased during the
processes of activation and apoptosis of TJK cells stimu-
lated with antibody. However, the expression and kinase
activity of Akt and PI3K had no significant changes in the
T1JK cells under the same condition. This indicated that
the signals mediated by CD8 were transferred to down-
stream molecule PI3K and Akt and resulted in the expres-
sion and activation of the two kinases in the TJK cells.
The failure of signaling in T1JK cells implicated that the
tyrosines in the CD8 -ITAM are necessary for signal
transduction induced by anti-CD8 mAb. Furthermore, the
data show that the PI3K/Akt pathway is involved in the
death signal transduction in T lymphocytes.
Ward et al.[11]
reported that the activation of PI3K
and Akt was only related with the cellular activation or
anti-apoptosis. For example, the active form of PI3K
could inhibit apoptosis of COS7 cells mediated by CD95
(Fas), while the specific inhibitor of PI3K, wortmannin or
LY29002, could enhance the apoptosis of COS7 cells[12]
.
The apoptosis of PC12 was inhibited after stimulation
with nerve growth factor and the activation of PI3K was
closely related with this inhibition[13]
. In this report, we
show that the expression of PI3K and Akt in the phases of
apoptosis and activation was at rather a high level. When
the TJK cells were treated with wortmannin, the apoptotic
percentage was remarkably increased (data not shown).
Thus, we presume that there would be a crosstalk between
the death and activation signals after stimulation. The cell
survival or apoptosis was determined by signal dynamic
balance.
Acknowledgements This work was supported by the National Natural
Science Foundation of China (Grant No. 39870727).
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(Received July 7, 2000)
570 Chinese Science Bulletin Vol. 46 No. 7 April 2001