From the Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1 Kusong-Dong, Yusong, Taejon 305-701, Republic of Korea
Received for publication, February 23, 2000, and in revised form, January 25, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Akt is a protein serine/threonine kinase
that plays an important role in the mitogenic responses of cells to
variable stimuli. Akt contains a pleckstrin homology (PH) domain and is
activated by phosphorylation at threonine 308 and serine 473. Binding
of 3'-OH phosphorylated phosphoinositides to the PH domain results in the translocation of Akt to the plasma membrane where it is activated by upstream kinases such as
(phosphoinositide-dependent kinase-1 (PDK1). Over-expression of
constitutively active forms of Akt promotes cell proliferation and
survival, and also stimulates p70 S6 kinase (p70S6K). In many cells, an
increase in levels of intracellular cyclic AMP (cAMP) diminishes cell
growth and promotes differentiation, and in certain conditions cAMP is
even antagonistic to the effect of growth factors. Here, we show that
cAMP has inhibitory effects on the phosphatidylinositol
3-kinase/PDK/Akt signaling pathway. cAMP potently inhibits
phosphorylation at threonine 308 and serine 473 of Akt, which is
required for the protein kinase activities of Akt. cAMP also negatively
regulates PDK1 by inhibiting its translocation to the plasma membrane,
despite not affecting its protein kinase activities. Furthermore, when
we co-expressed myristoylated Akt and PDK1 mutants which constitutively
co-localize in the plasma membrane, Akt activity was no longer
sensitive to raised intracellular cAMP concentrations. Finally, cAMP
was also found to inhibit the lipid kinase activity of PI3K and to
decrease the levels of phosphatidylinositol 3,4,5-triphosphate in
vivo, which are required for the membrane localization of PDK1.
Collectively, these data strongly support the theory that the
cAMP-dependent signaling pathway inhibits Akt activity by
blocking the coupling between Akt and its upstream regulators, PDK, in
the plasma membrane.
The phosphatidylinositol 3-kinase
(PI3K)1-dependent
cell signaling pathway has emerged as a key regulatory pathway involved in a number of cellular events (1). Upon activation of growth factor
tyrosine kinase receptors, the p85 regulatory subunit of PI3K recruits
the p110 catalytic subunit to the plasma membrane (2). The p110
catalytic subunit increases the level of PtdIns-3,4,5-P3 and phosphatidylinositol 3,4-bisphosphate (PtdIns-3,4-P2),
which induce the membrane translocation of PDK1 and Akt (also called PKB or RAC-PK) by binding to the pleckstrin homology domain (3). In the
membrane, PDK1 phosphorylates and activates Akt in a
PtdIns-3,4,5-P3- or
PtdIns-3,4-P2-dependent manner (4, 5). By a
mechanism that involves phospholipase C (6), activated Akt is released from the membrane and phosphorylates various targets.
This complex and unique signaling pathway has been implicated in a
variety of cellular events such as cell proliferation and survival (1,
7). Previously, it has been shown that various survival factors, such
as nerve growth factor, require the activation of PI3K to prevent
various cell types from undergoing apoptosis (8, 9). The mechanism by
which the PI3K pathway protects cells from programmed cell death has
been studied intensively. Recently, it was shown that Akt can
phosphorylate serine 136 of BAD, a member of the pro-apoptotic Bcl-2
family, forming a binding site for 14-3-3 (10, 11). As BAD binds
14-3-3, it can no longer bind to Bcl-2 and Bcl-XL to
inhibit their pro-survival activity. Akt also phosphorylates other
important cellular factors involved in apoptosis, such as caspase-9
(12) and fork-head transcription factors (13), which results in the
inhibition of apoptosis.
Besides blocking apoptosis, the PI3K signaling pathway is involved in
glycogen synthesis (14, 15), glucose transport (16), and protein
synthesis (17). The activities of Akt especially are closely
correlated with these important biological activities. For example,
GSK-3 cAMP and cAMP-dependent protein kinase (PKA)
are regulators of development in many organisms and are involved in
many other cellular processes. For example, cAMP is a key regulator of
Drosophila development by a mechanism in which PKA inhibits
the Hedgehog-dependent activation of the Cubitus
interruptus transcription factor (22). In Dictyostelium
amoebae, extracellular cAMP regulates
G-protein-dependent and -independent pathways to control
aggregation as well as the activity of GSK-3 and the transcription
factors GBF (G-box binding factor) and STAT during multicellular
development (23, 24).
More importantly, cAMP and its effector, PKA, are implicated in a
variety of cross-talks between intracellular signaling pathways. It was
reported that the exit from mitosis in Xenopus egg extracts required the MPF (maturation-promoting factor)-dependent
activation of the cAMP/PKA pathway (25). There is also evidence that
cross-talks between the Ca2+- and
cAMP-dependent pathways exist in the cytoplasm (26) and the
nucleus (27). Also, it was demonstrated that cAMP inhibits the Jak/STAT
pathway (28), which is important in cytokine signaling. cAMP-dependent protein kinase inhibits
G Recent findings suggest that there is also cross-talk between the PI3K
pathway and the cAMP-dependent pathway. For example, an
increased level of cAMP inhibits the
interleukin-2-dependent activation of p70S6K (34). In
addition, CREB (cAMP response element-binding protein), in which
transcriptional activities are induced by phosphorylation at serine
133, is phosphorylated at the same serine residue by both PKA and Akt
in vivo (35).
Here, we demonstrate that an increase in the level of intracellular
cAMP inhibits the activities of Akt, PI3K, and their downstream target,
p70S6K. Interestingly, PDK1 activity was not affected by cAMP
treatments, but its plasma membrane localization was dramatically reduced. Taken together, these results support that the
cAMP-dependent signaling pathway inhibits Akt through
inhibition of PI3K lipid kinase activity and the subsequent inhibition
of PDK1 localization at the plasma membrane.
Cell Culture and Transfection--
Swiss 3T3, HEK293, COS, and
Rat2 cells were grown in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum (Life Technologies, Inc.) at
37 °C in a humidified atmosphere with 5% CO2. Transient
transfection in COS cells was performed at 50% confluency by a
DEAE-dextran method (Promega) according to the manufacturer's instructions.
Preparation of Cell Lysates--
Serum-starved cells were
treated with various stimuli: 50 ng/ml EGF (Life Technologies, Inc.),
20 µM forskolin (Sigma or Calbiochem), 20 µM 1,9-dideoxyforskolin (Sigma), 100 ng/ml phorbol
12-myristate 13-acetate (Sigma), 10 nM calyculin A
(Calbiochem), 1 mM 8-bromo-cAMP (8-Br-cAMP, Calbiochem), 1 mM 8-bromo-cGMP (8-Br-cGMP, Calbiochem). Stimulation was
terminated by washing cells with ice-cold STE (consisting of 150 mM NaCl, 50 mM Tris-HCl, pH 7.4) and 1 mM EDTA. Cell lysates were prepared in Buffer A (containing
20 mM Tris-HCl (pH 7.5), 0.1% Nonidet P-40, 1 mM EDTA, 5 mM EGTA, 10 mM
MgCl2, 50 mM Protein Kinase Assays--
HA-tagged p70S6K and Akt were
immunoprecipitated by anti-HA monoclonal antibody coupled to protein
G-Sepharose (Amersham Phamacia Biotech). Myc-tagged PDK1 was
immunoprecipitated by anti-Myc monoclonal antibody coupled to protein
G-Sepharose. The samples were washed twice with Buffer A and then twice
with Buffer A containing 500 mM NaCl. Finally, the immune
complexes of p70S6K, Akt, and PDK1 were washed with Buffer C
(containing 20 mM Hepes (pH 7.2), 10 mM
MgCl2, 0.1 mg/ml bovine serum albumin, and 3 mM
PI3K p110 Lipid Kinase Assay--
HA epitope-tagged p110 PI3K
was immunoprecipitated with anti-HA monoclonal antibody coupled to
protein G-Sepharose. The samples were washed twice with 1% Nonidet
P-40 and 1 mM sodium orthovanadate in phosphate-buffered
saline, twice with washing buffer consisting of 100 mM
Tris-HCl (pH 7.5), 500 mM LiCl, and 1 mM sodium
orthovanadate, and finally twice with ST consisting of 150 mM NaCl and 50 mM Tris-HCl (pH 7.2). Then, the
samples were resuspended in PI kinase buffer containing 20 mM Hepes (pH 7.2), 100 mM NaCl, 10 µg/ml leupeptin, and 10 µg/ml pepstatin. Following the addition of a PI/EGTA mixture consisting of 1 mg/ml phosphoinositide and 2.5 mM EGTA, they were incubated at room temperature for 10 min. Then, a mixture consisting of 20 mM Hepes (pH 7.4), 5 mM MnCl2, 10 µM ATP, and 20 µCi
of [ Confocal Microscopic Analyses of PDK1--
Rat2 cells were grown
on coverslips and transfected with pEGFP-PDK1 by the LipofectAMINE
method (Life Technologies, Inc.). Quiescent cells stimulated with the
various stimuli were washed three times with cold phosphate-buffered
saline and fixed in 3.7% formaldehyde for 40 min. Fixed cells were
mounted on slide glasses with phosphate-buffered saline and
observed with a laser-scanning confocal microscope (Carl Zeiss LSM 510).
Immunoblot Analyses--
Cell lysates were boiled in SDS sample
buffer for 5 min. The samples were subjected to SDS-polyacrylamide gel
electrophoresis, and proteins were transferred to nitrocellulose
membranes (Schleicher & Schuell). Membranes were incubated for 15 min
in blocking solution (Tris-buffered saline containing 0.1% Tween 20, 2% bovine serum albumin, and 0.02% sodium azide) and further
incubated with the appropriate primary antibody for 1 h. 12CA5
anti-HA monoclonal antibody was purchased from Roche Molecular
Biochemicals. Phosphospecific Akt antibodies were obtained from New
England Biolabs. The membranes were then washed with blocking solution
and incubated for 30 min with either anti-mouse or anti-rabbit
secondary antibody conjugated to horseradish peroxidase. Bound
antibodies were detected with the enhanced chemiluminescence (Amersham
Phamacia Biotech).
Detection of in Vivo PtdIns Levels--
Determination of
PtdIns-3,4,5-P3 level in vivo was carried out as
described (36, 37). Briefly, 1 × 105 COS cells were
plated and subjected to serum starvation for 16 h. To
metabolically label cells, 32PO4 (obtained from
New England Nuclear, 50 µCi/plate) was added, and the cells were
incubated at 37 °C for 60 min. Excess 32PO4
was washed, and the appropriate stimuli were added. Then, lipids
were extracted and separated as described previously (36, 37).
Phosphorylated lipids were visualized and quantified by autoradiography.
cAMP Inhibits the Kinase Activity of Akt in Vivo--
To
understand how the cAMP-dependent cell signaling pathway
modulates Akt, we examined the effects of forskolin, an activator of
adenylyl cyclase, on the protein kinase activities of Akt induced by a
variety of agonists for cell proliferation including EGF, phorbol
12-myristate 13-acetate, calyculin A, and serum. We stimulated pCMV6-HA-Akt-transfected COS cells with various agonists followed by
treatment with forskolin as indicated in Fig.
1A. The protein kinase
activities of Akt were strongly stimulated by EGF and calyculin A, and
weakly by serum as previously reported (19, 38, 39). Interestingly, the
Akt activities induced by all of these agonists were strongly inhibited
by treatment with forskolin (Fig. 1A). Thus, we decided to
investigate further how cAMP-dependent signaling is
involved in the regulation of Akt activities as well as the other
signaling components downstream of PI3K.
To confirm whether cAMP was indeed responsible for inhibiting the Akt
activity following forskolin treatment, we treated cells with the
cell-permeable cyclic nucleotide analogues 8-Br-cAMP and 8-Br-cGMP. As
shown in Fig. 1B, 8-Br-cAMP, but not 8-Br-cGMP, specifically
inhibited the EGF-induced Akt activity. Although forskolin or 8-Br-cAMP
did not inhibit Akt activity as potently as wortmannin, they decreased
it to less than 20% of the EGF-induced activity (Fig. 1, A
and B). To clarify whether we observed a true cAMP effect or
a stress response to chemicals and whether the effect of forskolin was
dependent on the manufacturer, we examined the effects of
1,9-dideoxyforskolin, a forskolin analogue that cannot activate
adenylyl cyclase, and forskolins from two different manufacturers. As
shown in Fig. 1C, both forskolins strongly inhibited the
EGF-stimulated Akt activities. However, as expected, Akt activity was
not inhibited by 1,9-dideoxyforskolin. These results confirmed that
forskolin inhibited Akt specifically through cAMP.
Recently it was reported that the activity of Akt is biphasically
regulated in a time-dependent manner (40). Rittenhouse and
colleagues (40) revealed that in platelets, the generation of
PtdIns-3,4,5-P3 leads to the first phase of activation of
Akt, and subsequently the Ca2+-dependent and
wortmannin-sensitive accumulation of PtdIns-3,4-P2 causes
the second phase of activation of Akt. Therefore, we examined the
effects of cAMP on Akt activities throughout the activation time course
of the kinase. We stimulated pCMV6-HA-Akt-transfected COS cells with
forskolin for 15 min prior to EGF stimulation. We also completed
similar experiments with p70S6K-transfected COS cells. p70S6K is one of
the best characterized downstream targets of Akt (19-21) and is
negatively regulated by cAMP-dependent cell signaling in
immune cells (34). The EGF-induced activities of Akt (Fig.
2A) as well as p70S6K (Fig.
2B) were strongly inhibited by forskolin throughout the time
course of the activation. However, one interesting observation is that
the early activation peak (2 and 5 min stimulation) of Akt is more
resistant to forskolin than that of p70S6K (Fig. 2).
cAMP Inhibits the Upstream Regulators of Akt--
Immunoblot
analyses of endogenous p70S6K in Fig. 2B showed that the
cAMP-dependent signaling pathway interferes with the
EGF-induced phosphorylation of p70S6K, which is required for its
activation. This finding suggested that forskolin might either
induce the activities of protein serine/threonine phosphatases or
inhibit upstream kinases such as Akt or PDK1. First, we examined
whether protein serine/threonine phosphatases are involved in the
forskolin-mediated inhibition of Akt. As shown in Fig.
3, the phosphotransferase activities of
Akt were strongly induced by calyculin A, a cell-permeable inhibitor of
protein phosphatases 1 and 2A, and these induced activities were
inhibited by forskolin. These results imply that the
cAMP-dependent signaling pathway inhibits Akt mainly
through serine/threonine phosphatase-independent mechanisms.
Next, we examined the possibility that forskolin might inhibit the
mechanisms that activate Akt. The molecular mechanism for activation of
Akt is well characterized at a molecular level. As described in the
introduction, Akt must localize in the membrane to be phosphorylated
and activated by PDK (3, 41). Also, several findings support the
theory that Akt must be phosphorylated at threonine 308 by PDK1
and at serine 473 by yet unidentified PDK2 to be fully activated (4,
42, 43). To determine whether the mechanism of the inhibition of Akt by
cAMP is related to the membrane localization of Akt, we examined the
effect of cAMP on myristoylated Akt (myrAkt; kindly provided by Dr.
R. A. Roth). Because myrAkt has a myristoylation signal at
its N terminus, the protein is not only constitutively localized in the
plasma membrane but also maintains a constitutively active status in the cell (compared with the A2 mutant, which has an impaired
myristoylation signal) (20). Interestingly, the activity of myrAkt was
strongly inhibited by forskolin (~20% activity of the untreated
control; Fig. 4A). This
implies that cAMP inactivates Akt by some other mechanism besides
affecting its localization. Thus, we examined whether cAMP affects the
two phosphorylation events required for Akt activation. We found that
cAMP severely impaired the phosphorylation of threonine 308 and serine
473 of Akt (Fig. 4B).
To examine whether the inhibition of Akt activity by cAMP is COS
cell-specific, we completed experiments with different cell lines
originated from various species, as shown in Fig.
5: Swiss 3T3 mouse fibroblast, COS monkey
kidney cell, Rat2 fibroblast, and HEK293 human embryonic kidney cells.
cAMP almost completely blocked the EGF-stimulated phosphorylation of
endogenous Akt in all of the selected cell lines (Fig. 5). These
results strongly suggested that components upstream of Akt must be
affected by cAMP.
cAMP Does Not Inhibit PDK1 Activity but Does Inhibit Its Membrane
Localization--
We examined whether the kinase activity of PDK1, the
immediate upstream kinase for Akt, is inhibited by cAMP. Myc-tagged
PDK1 was transiently expressed in COS cells, and its phosphotransferase activities were measured. As previously reported by others (4, 43), the
autophosphorylation and Akt threonine 308 phosphotransferase activities
of PDK1 were highly active in quiescent cells and slightly (~10%)
increased by EGF stimulation (Fig. 6,
inset and upper panels, respectively).
Interestingly, forskolin has no effect on the PDK1 activities (Fig. 6,
inset and upper panels).
Upon mitogen stimulation, PI3K increases the levels of
PtdIns-3,4,5-P3 and PtdIns-3,4-P2 that recruit
Akt as well as PDK to the membrane (3). To determine whether cAMP
affects the membrane localization of PDK1, we transiently expressed
pEGFP-PDK1 in Rat2 cells and examined the changes in subcellular
localization of the kinase. Surprisingly, the EGF-induced membrane
localization of PDK1 was drastically reduced by treatments with
wortmannin and forskolin (Fig. 7). This
suggests that cAMP down-modulates Akt activities by blocking the
membrane localization of PDK1 and the consequent coupling between PDK1
and Akt in the plasma membrane.
To confirm this inhibitory mechanism of cAMP on PDK1, we co-transfected
pECE-HA-myrAkt and pBJ5-FLAG-myrPDK1 in COS cells. The transfected
cells were treated with forskolin for 20 min, and the Akt activities
were measured. As previously shown in Fig. 4A, forskolin
treatments reduced myrAkt activity in cells expressing myrAkt alone
(Fig. 8, compare lanes 2 and
3). However, forskolin could not inhibit the myrAkt
activities from cells co-expressing myrPDK1 (Fig. 8, compare
lanes 4 and 5). Consistent with this finding, the
faster migrating (inactive and dephosphorylated) form of myrAkt was
increased by forskolin treatment only in cells expressing myrAkt alone
(Fig. 8, third panel). These results confirm that inhibition
of the membrane localization of PDK is the molecular mechanism behind
the inhibition of Akt by the cAMP-dependent pathway.
cAMP Inhibits PI3K Lipid Kinase Activity and in Vivo Production of
PtdIns-3,4,5-P3--
As the subcellular localization of
PDK1 is regulated by the level of PtdIns-3,4,5-P3, we
examined whether cAMP affects the lipid kinase activities of PI3K. We
transiently transfected COS cells with pSR We have demonstrated that cAMP down-modulates Akt activity by
interfering with the membrane localization of PDK1 and inhibiting the
lipid kinase activity of PI3K. When we observed that the kinase activity of myristoylated Akt was strongly inhibited by forskolin, we
first suspected that PKA might directly phosphorylate Akt and consequently inhibit the phosphotransferase activity of Akt, as Akt has
a putative PKA phosphorylation site in its catalytic domain. However,
the catalytic subunit of PKA was unable to phosphorylate Akt in
vitro, and an Akt mutant that lacks the putative PKA
phosphorylation site was still strongly inhibited by forskolin in
vivo (data not shown). Furthermore, the phosphorylations required
for the activity of Akt were greatly diminished by forskolin treatment
in vivo (Figs. 4B and 5). These results led us to
study the effects of cAMP on PDK1 and PI3K, two known upstream
regulators of Akt.
Previous studies have suggested that PDK1 is constitutively active and
that its activity is regulated mainly by membrane localization via
binding to PtdIns-3,4,5-P3 (3, 4, 40). In support of
this hypothesis, a PDK1 mutant containing an N-terminal
myristoylation signal constitutively activates co-expressed Akt protein
in vivo (3). These results suggest that the pleckstrin
homology domain-mediated localization of PDK1 and Akt in the plasma
membrane is critical for the functional coupling between the two
protein kinases. Interestingly, our results strongly support the
theory that cAMP signaling interferes with this process by
inhibiting the lipid kinase activity of PI3K and the in vivo
production of PtdIns-3,4,5-P3 (Fig. 9, A and
B, respectively). However, we do not currently understand
how cAMP inhibits PI3K in the cell. Our preliminary data suggest at
least that a direct phosphorylation of the p110 and p85 subunits of PI3K by PKA is not a mechanism behind the inhibition of the PI3K activity.2 We also examined
the changes in tyrosine phosphorylation status of the p85 subunit of
PI3K by cAMP but found that basal and EGF-stimulated tyrosine
phosphorylation of the p85 protein was not significantly changed by
cAMP under our experimental conditions.2 In addition, we
found that cAMP does not affect the expression of
PTEN.2 Thus, although we have excluded several
possibilities for the mechanism of PI3K inhibition by cAMP, further
studies are needed to elucidate this matter fully.
Although we consistently observed an inhibitory role of cAMP for Akt in
various cultured cells (Fig. 5), Van Obberghen and colleagues (44)
reported on the activation of Akt by PKA through a PI3K-independent
pathway. However, not only was the activation of Akt by cAMP and PKA
only minor, cAMP rather inhibited phosphorylation at serine 473 of Akt
(44). This finding does not agree with studies by other groups
demonstrating that phosphorylation at serine 473 of Akt is necessary
for its activity (4, 42, 43). On the other hand, we demonstrated that
the EGF-stimulated phosphorylation of endogenous Akt at both threonine
308 and serine 473 was inhibited to basal levels by forskolin
treatments (Fig. 5). We also demonstrated the inhibition of PI3K
activity (Fig. 9A), the in vivo production of
PtdIns-3,4,5-P3 (Fig. 9B), translocation of PDK1
to the plasma membrane (Fig. 7), and p70S6K activity (Fig.
2B) by cAMP. We believe these findings reflect the
general nature of the inhibitory effect of cAMP on the PI3K pathway, as
it would be illogical for cAMP to activate Akt while inhibiting its
upstream regulators. Furthermore, our findings were consistent in a
variety of cell lines. While we were revising this manuscript, others
also revealed, in agreement with our data, that cAMP cannot induce the
phosphorylations of Akt at threonine 308 and serine 473 and cannot
activate Akt activity (45, 46). They also mentioned that cAMP decreases
the activity of Akt (45). Therefore, we believe that we have
demonstrated without a doubt the inhibition of Akt and other components
of the PI3K pathway by cAMP under more relevant and natural
experimental conditions. This conclusion is further supported by other
groups' results that cAMP indirectly inhibits p70S6K, a downstream
target of Akt and PI3K, in vivo (34) and that cell-permeable
cAMP fails to activate Akt in vivo (47).
Akt plays important roles in protecting cells from various apoptotic
pressures. Recent studies have shown that Akt exerts its anti-apoptotic
activities by phosphorylating important regulators for apoptosis such
as BAD (10, 11), caspase-9 (12), Forkhead transcription factors (13),
etc. However, activation of the cAMP-dependent pathway can
promote apoptosis. Lanotte and colleagues (48) have shown that
activation of the cAMP pathway induces apoptosis within 4-6 h via
CREM (cAMP-responsive element modulator) activation in a
leukemia cell. In the v-Abl-transformed cell, cAMP induces programmed
cell death via Raf-1 inhibition (49). In addition, retinoic acid and
cAMP act in a synergistic fashion to induce apoptosis via caspase-3
activation (50). However, we believe that these findings have yet to
link the cAMP-dependent pathway directly to the known
molecular mechanisms for regulating apoptosis. With our present
findings, we now propose that inhibition of the Akt activity may be one
of the major mechanisms behind the induction of apoptosis.
In the present study, we have shown that cAMP inhibits the in
vivo production of PtdIns-3,4,5-P3 and the activities
of PI3K, PDK1, Akt and p70S6K, which suggests that other downstream
targets of the PI3K pathway are also regulated by cAMP. As the PI3K-
and cAMP-dependent pathways are deeply involved in various
cellular events including cell growth, life span, metabolism, mobility and development, our findings may provide important clues to
understanding the cAMP-PI3K cross-talk network and its role in the cell.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(glycogen synthase kinase-3
) is phosphorylated at serine 9, and its activities are down-modulated by Akt (14). Meanwhile, Tor
(target of rapamycin) and p70S6K, which phosphorylate a translation
initiation factor, 4E-BP (eIF4E-binding protein), and a ribosomal
protein, S6, respectively, are positioned as downstream targets of Akt
(18-21).
-activated PLC
2
activity by phosphorylating PLC
2 at a serine residue
in vivo (29). In addition, the Ras-mediated
mitogen-activated protein kinase pathway is strongly inhibited
by cAMP-dependent signaling in various mechanisms
(30-33).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-glycerophosphate, 1 mM sodium orthovanadate, 2 mM dithiothreitol, 40 µg/ml phenymethylsulfonyl fluoride, and 10 µg/ml leupeptin). For
the p110 PI3K lipid kinase assay, we used Buffer B (containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 10% glycerol, 1% Nonidet P-40, and 500 µM sodium orthovanadate).
-mercaptoethanol), Buffer D (containing 20 mM Hepes (pH
7.2), 10 mM MgCl2, 10 mM MnCl2, 1 mM dithiothreitol, and 0.2 mM EGTA), and Buffer E (containing 50 mM Tris
(pH 7.5), 10 mM NaCl, 1 mM dithiothreitol, and
10% glycerol), respectively. S6 phosphotransferase activities were assayed in a reaction mixture consisting of 1× Buffer C, 1 µg of S6
protein, 20 µM ATP, and 5 µCi of
[
-32P]ATP at 30 °C for 20 min. Akt activities were
assayed in a reaction mixture consisting of 1× Buffer D, 1 µg
histone of H2B, 2 µg of PKI, 5 µM ATP, and 5 µCi of
[
-32P]ATP at 30 °C for 20 min. PDK1 kinase
activities were assayed in a reaction mixture consisting of 1× Buffer
E, 1 µg of (histidine)6-Akt protein, 10 mM
MgCl2, 3 µM ATP, and 5 µCi of
[
-32P]ATP at 30 °C for 20 min. Then, assay samples
were subjected to SDS-polyacrylamide gel electrophoresis.
Phosphorylated proteins were visualized by autoradiography and
quantified with a phosphorimager (BAS1500, Fuji).
-32P]ATP was added, and the samples were further
incubated at 30 °C for 20 min. The reactions were stopped by the
addition of 1 M HCl, and phospholipids were extracted with
CHCl3. Dried samples were separated by thin-layer
chromatography (TLC). Phosphorylated lipids were visualized by autoradiography.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (20K):
[in a new window]
Fig. 1.
Inhibition of Akt activity by cAMP. COS
cells were transiently transfected with HA-tagged wild-type Akt in
pCMV6 and serum-starved for 16 h. A, cells were
pretreated with (+) or without ( ) forskolin (F) for 15 min
and further treated with various agonists, as indicated, for an
additional 5 min. Then, the cells were lysed and assayed for Akt
activity as described under "Experimental Procedures."
Phosphorylated substrates were visualized by autoradiography
(middle panel) and quantified with phosphorimager analyses
(top panel). Anti-HA immunoblot analyses were completed from
the same cell lysates (bottom panel). PMA,
phorbol 12-myristate 13-acetate; Cal A, calyculin A;
FBS, fetal bovine serum. B, cells were pretreated
without (
) or with (+) 8-Br-cAMP, 8-Br-cGMP, or wortmannin
(wort) for 15 min and treated with EGF for an additional 5 min as indicated. Protein kinase assays for Akt (top and
middle panels) and immunoblot analyses for Akt (bottom
panel) were completed as described under "Experimental
Procedures." C, cells were treated with (+) or without (
) forskolin
purchased from Sigma (F1) or Calbiochem (F2), or
1,9-dideoxyforskolin (ddF) for 15 min, followed by EGF
treatment for 5 min. Akt assays (top and middle
panel) and immunoblot analyses (bottom panel) were
completed as described. The values in the top panels
represent the mean of three independent cell preparations ± S.D.
View larger version (24K):
[in a new window]
Fig. 2.
Time-independent inhibition of
Akt by cAMP. COS cells were transiently transfected with HA-tagged
Akt in pCMV6 (A) or HA-tagged p70S6K in pJ3H (B).
Following serum starvation for 16 h, cells were pretreated with
(+, open square) or without ( , closed square)
forskolin (F) for 15 min and further stimulated with 50 ng/ml EGF for the indicated time periods. Prepared cell lysates were
assayed for Akt and p70S6K activity as described under "Experimental
Procedures." Phosphorylated substrates were visualized by
autoradiography (top panel) and quantified with
phosphorimager analyses (bottom panel). Immunoblot analyses
showed that similar protein amounts of Akt or p70S6K were used in the
assays (middle panel). The values in the bottom
panel represent the mean of three independent cell
preparations ± S.D.
View larger version (30K):
[in a new window]
Fig. 3.
Phosphatases are not involved in the
inhibition of Akt by cAMP. COS cells were transiently transfected
with HA-tagged Akt in pCMV6 and serum-starved for 16 h. Cells were
pretreated with (+) or without ( ) forskolin (F) for 15 min
and further treated with calyculin A (Cal A) for 30 min or
EGF for 5 min. When both calyculin A and EGF were added, EGF was added
for the final 5 min of the 30-min calyculin A stimulation.
Phosphorylated substrates were visualized by autoradiography
(middle panel) and quantified with phosphorimager analyses
(top panel). Anti-HA immunoblot analyses were completed from
the same cell lysates (bottom panel). The values in the
top panel represent the mean of three independent cell
preparations ± S.D.
View larger version (34K):
[in a new window]
Fig. 4.
cAMP inhibits the activity of Akt by
preventing phosphorylation at Thr-308 and
Ser-473. A, COS cells transiently transfected with
myrAkt (myr) or its A2 mutant (A2) in pECE vector
were serum-starved for 16 h and treated with (+) or without ( )
forskolin (F) for 20 min. Phosphotransferase assays
(top and middle panels) and immunoblot analyses
(bottom panel) were performed as described under
"Experimental Procedures." The values in the top panel
represent the mean of three independent cell preparations ± S.D.
B, COS cells were transfected with HA-tagged wild-type Akt
and serum-starved for 16 h. Cells were pretreated with forskolin
(F) or wortmannin (wort) for 15 min and further
stimulated with EGF for 5 min. Cell lysates were immunoprecipitated
with anti-HA monoclonal antibody, and the immune complexes were
subjected to immunoblot analyses as described under "Experimental
Procedures." Analyses were completed using the same cell lysates with
anti-phosphothreonine 308-specific Akt antibody (top panel),
anti-phosphoserine 473-specific Akt antibody (middle panel),
or anti-HA monoclonal antibody (bottom panel).
View larger version (73K):
[in a new window]
Fig. 5.
Phosphorylation at Thr-308 and Ser-473 of
endogenous Akt is potently inhibited by cAMP. Serum-starved cells
were treated with (+) or without ( ) forskolin (F) for 15 min and
further stimulated with EGF for 5 min. Phospho-Akt-specific immunoblot
analyses (top and middle panels) were completed
as described under "Experimental Procedures." Anti-Akt immunoblot
analyses were also completed from the same cell lysates (bottom
panel) to show that the same amount of endogenous Akt protein was
used for each experiment.
View larger version (33K):
[in a new window]
Fig. 6.
cAMP does not inhibit PDK1 phosphotransferase
activity. COS cells expressing Myc-tagged PDK1 were serum-starved
for 16 h. Cells were pretreated with (+) or without ( ) forskolin
(F) or wortmannin (wort) for 15 min and further
stimulated with EGF for 5 min. The phosphotransferase activities of
immune-complex PDK1 were determined as described under "Experimental
Procedures." Incorporation of 32P into recombinant Akt
protein substrate was quantified with phosphorimager analyses
(upper panel). The values in the upper panel
represent the mean of three independent cell preparations ± S.D.
PDK1 autophosphorylation activities (inset) and immunoblot
analyses (lower panel) were also completed.
View larger version (98K):
[in a new window]
Fig. 7.
cAMP blocks the membrane localization of PDK1
induced by EGF. Rat2 cells were grown on coverslips in 6-well
plates and transfected with pEGFP-PDK1. Following serum starvation for
16 h, cells were left untreated ( ) or treated with forskolin
(F) or wortmannin (wort) for 15 min and further
stimulated with EGF for 5 min as indicated. Fixed cells were examined
under a laser-scanning confocal microscope as described under
"Experimental Procedures." The results shown are representative of
three independent experiments.
View larger version (42K):
[in a new window]
Fig. 8.
Co-expressed myristoylated PDK1 blocks the
inhibition of myrAkt activity by cAMP. COS cells transfected with
pECE-HA-myrAkt alone or pBJ5-FLAG-myrPDK1 and pECE-HA-myrAkt were
pretreated with forskolin (F) for 20 min prior to cell
lysis. Cell lysates were immunoprecipitated with anti-HA monoclonal
antibody, and the immune-complexes were subjected to phosphotransferase
assays for Akt. Phosphorylated substrates were visualized by
autoradiography (second panel) and quantified with
phosphorimager analyses (first panel). Anti-HA for myrAkt
and anti-FLAG for myrPDK1 immunoblot analyses were completed from the
same cell lysates (third and fourth panels). The
values in the first panel represent the mean of three
independent cell preparations ± S.D.
-HA-p110, and treated
them with EGF and either forskolin or wortmannin. EGF treatment
strongly stimulated the PI3K activity (5.3-fold over unstimulated
control) (Fig. 9A, compare lanes 1 and 4). Consistently, both forskolin and
wortmannin inhibited the PI3K lipid kinase activities to below the
unstimulated control level (Fig. 9A, lanes 2,
3, 5, and 6). To further confirm the effect of cAMP on PI3K, we examined the effect of forskolin on the
production of PtdIns-3,4,5-P3 in vivo. Quiescent
COS cells were labeled with 32PO4 and treated
with forskolin for 15 min prior to EGF stimulation. In Fig.
9B, the levels of phosphatidylinositol monophosphates and
PtdIns-3,4,5-P3 were decreased by cAMP and wortmannin.
Therefore, we conclude that cAMP inhibits PI3K lipid kinase activity
in vivo.
View larger version (33K):
[in a new window]
Fig. 9.
Inhibition of PI3K activity and
PtdIns-3,4,5-P3 production in vivo by
cAMP. A, COS cells transfected with the HA-tagged p110 subunit of
PI3K in pSR were serum-starved for 16 h. Cells were pretreated
with forskolin (F) or wortmannin (wort) for 15 min and stimulated with EGF for 5 min. Cell lysates were
immunoprecipitated with anti-HA monoclonal antibody, and the
immune-complexes were subjected to PI3K lipid kinase assays as
described under "Experimental Procedures." Phosphorylated lipids
were separated by TLC, and the 3'-OH phosphorylated phosphoinositides
were detected by autoradiography (middle panel) and
quantified with phosphorimager analyses (top panel).
Immunoblot analyses showed that similar protein amounts of p110 were
used in the assays (bottom panel). The values in the
top panel represent the mean of three independent cell
preparations ± S.D. B, quiescent COS cells were
labeled with 32PO4 as described under
"Experimental Procedures." Cells were pretreated with forskolin
(F) or wortmannin (wort) for 15 min and treated
with EGF for 30 s, and then lipids were extracted and separated by
TLC with phosphoinositide standards. Phosphorylated lipids were
visualized by autoradiography (middle panel) and quantified
with phosphorimager analyses (top panel). The values in the
top panel represent the mean of three independent cell
preparations ± S.D.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Drs. D. R. Alessi, J. Blenis, J. Downward, and R. A. Roth for providing reagents.
![]() |
FOOTNOTES |
---|
* This work was supported by Korea Research Foundation Grant KRF-2000-015-DS0034.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.
Supported by the Korea Advanced Institute of Science and
Technology Brain Korea 21 Program. To whom correspondence should be
addressed. Tel: 82-42-869-2620; Fax: 82-42-869-2610; E-mail: jchung@mail.kaist.ac.kr.
Published, JBC Papers in Press, January 26, 2001, DOI 10.1074/jbc.M001492200
2 J. Chung and S. Kim, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PI3K, phosphatidylinositol 3-kinase; EGF, epidermal growth factor; PDK, phosphoinositide-dependent kinase; cAMP, cyclic AMP; Br, bromo; PKA, cAMP-dependent protein kinase; myrAkt, myristoylated Akt; p70S6K, p70 S6 kinase; PtdIns-3, 4,5-P3, phosphatidylinositol 3,4,5-trisphosphate; PtdIns-3, 4-P2, phosphatidylinositol 3,4-bisphosphate; HA, hemagglutinin; Jak/STAT, Janus kinase/signal transducers and activators of transcription.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Rameh, L. E.,
and Cantley, L. C.
(1999)
J. Biol. Chem.
274,
8347-8350 |
2. | Vanhaesebroeck, B., Leevers, S. J., Panayotou, G., and Waterfield, M. D. (1997) Trends Biochem. Sci. 22, 267-272[CrossRef][Medline] [Order article via Infotrieve] |
3. | Anderson, K. E., Coadwell, J., Stephens, L. R., and Hawkins, P. T. (1998) Curr. Biol. 8, 684-691[Medline] [Order article via Infotrieve] |
4. | Alessi, D. R., James, S. R., Downes, C. P., Holmes, A. B., Gaffney, P. R., Reese, C. B., and Cohen, P. (1997) Curr. Biol. 7, 261-269[Medline] [Order article via Infotrieve] |
5. |
Stephens, L.,
Anderson, K.,
Stokoe, D.,
Erdjument-Bromage, H.,
Painter, G. F.,
Holmes, A. B.,
Gaffney, P. R.,
Reese, C. B.,
McCormick, F.,
Tempst, P.,
Coadwell, J.,
and Hawkins, P. T.
(1998)
Science
279,
710-714 |
6. | Ferguson, K. M., Lemmon, M. A., Sigler, P. B., and Schlessinger, J. (1995) Nature Struct. Biol. 2, 715-718[Medline] [Order article via Infotrieve] |
7. |
Datta, S. R.,
Brunet, A.,
and Greenberg, M. E.
(1999)
Genes Dev.
13,
2905-2927 |
8. | Yao, R. J., and Cooper, G. M. (1995) Science 267, 2003-2006[Medline] [Order article via Infotrieve] |
9. | Kulik, G., Klippel, A., and Weber, M. J. (1997) Mol. Cell. Biol. 17, 1595-1606[Abstract] |
10. |
Del Peso, L.,
Gonzalez-Garcia, M.,
Page, C.,
Herrera, R.,
and Nunez, G.
(1997)
Science
278,
687-689 |
11. | 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] |
12. |
Cardone, M. H.,
Roy, N.,
Stennicke, H. R.,
Salvesen, G. S.,
Franke, T. F.,
Stanbridge, E.,
Frisch, S.,
and Reed, J. C.
(1998)
Science
282,
1318-1321 |
13. | Brunet, A., Bonni, A., Zigmond, M. J., Lin, M. Z., Juo, P., Hu, L. S., Anderson, M. J., Arden, K. C., Blenis, J., and Greenberg, M. E. (1999) Cell 96, 857-868[Medline] [Order article via Infotrieve] |
14. | Cross, D. A. E., Alessi, D. R., Cohen, P., Andjelkovich, M., and Hemmings, B. A. (1995) Nature 378, 785-789[CrossRef][Medline] [Order article via Infotrieve] |
15. | Frevert, E. U., and Kahn, B. B. (1997) Mol. Cell. Biol. 17, 190-198[Abstract] |
16. | Cheatham, B., Vlahos, C. J., Cheatham, L., Wang, L., Blenis, J., and Kahn, C. R. (1994) Mol. Cell. Biol. 14, 4902-4911[Abstract] |
17. | Thomas, G., and Hall, M. N. (1997) Curr. Opin. Cell Biol. 9, 782-787[CrossRef][Medline] [Order article via Infotrieve] |
18. |
Scott, P. H.,
Brunn, G. J.,
Kohn, A. D.,
Roth, R. A.,
and Lawrence, J. C., Jr.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
7772-7777 |
19. | Burgering, B. M., and Coffer, P. J. (1995) Nature 376, 599-602[CrossRef][Medline] [Order article via Infotrieve] |
20. |
Kohn, A. D.,
Summers, S. A.,
Birnbaum, M. J.,
and Roth, R. A.
(1996)
J. Biol. Chem.
271,
31372-31378 |
21. | Koh, H., Jee, K., Lee, B., Kim, J., Kim, D., Yun, Y. H., Kim, J. W., Choi, H. S., and Chung, J. (1999) Oncogene 18, 5115-5119[CrossRef][Medline] [Order article via Infotrieve] |
22. |
Ohlmeyer, J. T.,
and Kalderon, D.
(1997)
Genes Dev.
11,
2250-2258 |
23. |
Wang, B.,
and Kuspa, A.
(1997)
Science
277,
251-254 |
24. |
Thomason, P. A.,
Traynor, D.,
Cavet, G.,
Chang, W.,
Harwood, A. J.,
and Kay, R. R.
(1998)
EMBO J.
17,
2838-2845 |
25. | Grieco, D., Porcellini, A., Avvedimento, E. V., and Gottesman, M. E. (1996) Science 271, 1718-1723[Abstract] |
26. |
DeBernadi, M. A.,
and Brooker, G.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
4577-4582 |
27. |
Rogue, P. J.,
Humbert, J. P.,
Meyer, A.,
Freyermuth, S.,
Krady, M. M.,
and Malviya, A. N.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
9178-9183 |
28. |
David, M.,
Petricoin, E., 3rd,
and Larner, A. C.
(1996)
J. Biol. Chem.
271,
4585-4588 |
29. | Liu, M., and Simon, M. I. (1996) Nature 382, 83-87[CrossRef][Medline] [Order article via Infotrieve] |
30. | Wu, J., Dent, P., Jelinek, T., Wolfman, A., Weber, M. J., and Sturgill, T. W. (1993) Science 262, 1065-1069[Medline] [Order article via Infotrieve] |
31. | Cook, S. J., and McCormick, F. (1993) Science 262, 1069-1072[Medline] [Order article via Infotrieve] |
32. | Burgering, B. M., Pronk, G. J., Van Weesen, P., Chardin, P., and Bos, J. L. (1993) EMBO J. 12, 4211-4220[Abstract] |
33. | Graves, L. M., Bornfeldt, K. E., Raines, E. W., Potts, B. C., Macdonald, S. G., Ross, R., and Krebs, E. G. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 10300-10304[Abstract] |
34. | Monfar, M., Lemon, K. P., Grammer, T. C., Cheatham, L., Chung, J., Vlahos, C. J., and Blenis, J. (1995) Mol. Cell. Biol. 15, 326-337[Abstract] |
35. |
Du, K.,
and Montminy, M.
(1998)
J. Biol. Chem.
273,
32377-32379 |
36. | Traynor-Kaplan, A. E., Harris, A. L., Thompson, B. L., Taylor, P., and Sklar, L. A. (1988) Nature 334, 353-356[CrossRef][Medline] [Order article via Infotrieve] |
37. |
Tamura, M.,
Gu, J.,
Danen, E. H.,
Takino, T.,
Miyamoto, S.,
and Yamada, K. M.
(1999)
J. Biol. Chem.
274,
20693-20703 |
38. | Franke, T. F., Yang, S. I., Chan, T. O., Datta, K., Kazlauskas, A., Morrison, D. K., Kaplan, D. R., and Tsichlis, P. N. (1995) Cell 81, 727-736[Medline] [Order article via Infotrieve] |
39. |
Andjelkovic, M.,
Jakubowicz, T.,
Cron, P.,
Ming, X.,
Han, J.,
and Hemmings, B. A.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
5699-5704 |
40. |
Banfic, H.,
Downes, C. P.,
and Rittenhouse, S. E.
(1998)
J. Biol. Chem.
273,
11630-11637 |
41. | Andjelkovic, M., Alessi, D. R., Meier, R., Fernandez, A., Lamb, N. J. C., Frech, M., Cron, P., Lucocq, J. M., and Hemmings, B. A. (1997) J. Biol. Chem. 273, 31515-31524[CrossRef] |
42. | Alessi, D. R., Andjelkovic, M., Caudwell, B., Cron, P., Morrice, N., Cohen, P., and Hemmings, B. A. (1996) EMBO J. 15, 6541-6551[Abstract] |
43. |
Stokoe, D.,
Stephens, L. R.,
Copeland, T.,
Gaffney, P. R.,
Reese, C. B.,
Painter, G. F.,
Holmes, A. B.,
McCormick, F.,
and Hawkins, P. T.
(1997)
Science
277,
567-570 |
44. | Filippa, N., Sable, C. L., Filloux, C., Hemmings, B., and Van Obberghen, E. Mol. Cell Biol. 19, 4989-5000 |
45. |
Fang, X., Yu, S. X.,
Lu, Y.,
Bast, R. C., Jr.,
Woodgett, J. R.,
and Mills, G. B.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
11960-11965 |
46. |
Li, M.,
Wang, X.,
Meintzer, M. K.,
Laessig, T.,
Birnbaum, M. J.,
and Heidenreich, K. A.
(2000)
Mol. Cell. Biol.
20,
9356-9363 |
47. |
Moule, S. K.,
Welsh, G. I.,
Edgell, N. J.,
Foulstone, E. J.,
Proud, C. G.,
and Denton, R. M.
(1997)
J. Biol. Chem.
272,
7713-7719 |
48. | Ruchaud, S., Seite, P., Foulkes, N. S., Sassone-Corsi, P., and Lanotte, M. (1997) Oncogene 15, 827-836[CrossRef][Medline] [Order article via Infotrieve] |
49. | Weissinger, E. M., Eissner, G., Grammer, C., Fackler, S., Haefner, B., Yoon, L. S., Lu, K. S., Bazarov, A., Sedivy, J. M., Mischak, H., and Kolch, W. (1997) Mol. Cell. Biol. 17, 3229-3241[Abstract] |
50. | Srivastava, R. K., Srivastava, A. R., Cho-Chung, Y. S., and Longo, D. L. (1999) Oncogene 18, 1755-1763[CrossRef][Medline] [Order article via Infotrieve] |