From the Department of Molecular Pharmacology,
Stanford University School of Medicine, Stanford, California 94305, the § Howard Hughes Medical Institute, University of
Pennsylvania School of Medicine, Clinical Research Building,
Philadelphia, Pennsylvania 19104-6148, and the ¶ Department of
Pharmacology, University of Virginia School of Medicine,
Charlottesville, Virginia 22908
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ABSTRACT |
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Akt is a serine/threonine kinase that requires a functional phosphatidylinositol 3-kinase to be stimulated by insulin and other growth factors. When directed to membranes by the addition of a src myristoylation sequence, Akt becomes constitutively active. In the present study, a conditionally active version of Akt was constructed by fusing the Akt containing the myristoylation sequence to the hormone binding domain of a mutant murine estrogen receptor that selectively binds 4-hydroxytamoxifen. The chimeric protein was expressed in NIH3T3 cells and was shown to be stimulated by hormone treatment 17-fold after only a 20-min treatment. This hormone treatment also stimulated an approximate 3-fold increase in the phosphorylation of the chimeric protein and a shift in its migration on SDS gels. Activation of this conditionally active Akt resulted in the rapid stimulation of the 70-kDa S6 kinase. This conditionally active Akt was also found to rapidly stimulate in these cells the phosphorylation of properties of PHAS-I, a key protein in the regulation of protein synthesis. The conditionally active Akt, when expressed in 3T3-L1 adipocytes, was also stimulated, although its rate and extent of activation was less then in the NIH3T3 cells. Its stimulation was shown to be capable of inducing glucose uptake into adipocytes by stimulating translocation of the insulin-responsive glucose transporter GLUT4 to the plasma membrane.
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INTRODUCTION |
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Akt is a serine/threonine kinase that contains a pleckstrin homology (PH)1 domain at its amino terminus (1, 2). The PH domain is a protein module found in many signal transduction proteins that can mediate either protein-lipid or protein-protein interactions (1, 2). The kinase activity of Akt is stimulated by a number of different growth factors, including insulin and platelet-derived growth factor (3-5). Several studies have shown that Akt stimulation requires prior activation of phosphatidylinositol 3-kinase (PI 3-kinase) (3-5), possibly due to a direct interaction of the Akt PH domain with these lipids products (6) or, alternatively, due to the phosphorylation of Akt by a distinct Ser/Thr kinase, which is activated by PI 3-phosphates (7).
Further interest in Akt has been stimulated by the finding that this enzyme can induce a variety of biological responses. In particular, Akt has been proposed to positively regulate the 70-kDa S6 kinase (4) and negatively regulate the GSK-3 kinase (8). Moreover, Akt has been shown to be capable of stimulating the differentiation of 3T3-L1 cells into adipocytes (9, 10) and to inhibit apoptosis of neuronal cells, as well as fibroblasts (11, 12). Finally, Akt has been shown to stimulate lipogenesis (9) and to induce glucose uptake into adipocytes by stimulating GLUT4 translocation to the plasma membrane (9, 13).
To determine whether a particular biological response can be mediated via Akt, the above studies made use of a constitutively active form of Akt in which the enzyme is targeted to membranes via the addition of either the src myristoylation signal or a myristoylated gag sequence (4, 8-13). Such studies have the problem that the Akt kinase activity is unregulated, and thus the kinase is active as soon as it is expressed in cells. In contrast, the responses one is attempting to mimic, such as stimulation of glucose uptake, are stimulated by insulin within minutes. In addition, such studies require one to utilize different populations of cells to compare a particular response; for example, cells expressing the constitutively active Akt must be compared with control cells.
In this report, we describe and characterize a conditionally active
form of the Akt molecule. This conditionally active form of Akt was
created by fusing the hormone binding domain (HBD) of a mutant murine
estrogen receptor (14) to a variant of Akt that lacks its PH domain but
contains a src myristoylation signal at its amino terminus
(myrAkt 4-129) (15). The Akt construct without the HBD has
unregulated constitutive kinase activity (15). This mutant HBD of the
estrogen receptor, which has also been used to make a conditionally
active myc (14), does not bind 17
-estradiol but is responsive to
4-hydroxytamoxifen (14). In the present work, we show that the kinase
activity of the Akt-estrogen receptor fusion protein (myrAkt
4-129-ER) is dependent on 4-hydroxytamoxifen (HT). This
conditionally active form of Akt was used to demonstrate that acute
activation of Akt was sufficient to stimulate the phosphorylation of
PHAS-I (phosphorylated heat- and
acid-stable protein) and to induce its
dissociation from eIF4E, a key step in the regulation of protein
synthesis.
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EXPERIMENTAL PROCEDURES |
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Constructs--
To make an estradiol-dependent human
Akt protein kinase, the hormone binding domain of a mutant murine
estrogen receptor (ER) (a gift from Dr. Martin McMahon) that no longer
binds 17-estradiol but is activated by the synthetic steroid
4-hydroxytamoxifen (14) was first subcloned into the pWZLneo retroviral
vector (16), which carries a neomycin resistance gene, using the
EcoRI and SalI restriction sites at the 5'- and
3'-ends, respectively. In addition, the polymerase chain reaction was
used to modify myrAkt
4-129 and A2myrAkt
4-129 (15) by
replacing the stop codon after the hemagglutinin (HA) epitope tag with
an in-frame EcoRI site. The fragment obtained by the
polymerase chain reaction was confirmed by sequencing. The Akt
constructs were then fused to the estrogen receptor in pWZLneo using
the 5' BamHI and 3' EcoRI restriction sites,
resulting in the formation of myrAkt
4-129-ER and A2myrAkt
4-129-ER.
Retroviral Infection--
NIH3T3 fibroblasts and 3T3-L1
preadipocytes were infected with either myrAkt 4-129-ER or A2myrAkt
4-129-ER as described previously (15).
Akt, p70 S6, PI 3-Kinase Assays, PHAS-I Shift, and eIF4E Dissociation-- The Akt immunoprecipitations and immunoblotting were performed as described previously (9), except that a single 100-mm plate was used for each treatment, and each plate was lysed in 400 µl of lysis buffer. In most experiments, a peptide (sequence GRPRTSSFAEG) assay was utilized to measure Akt kinase activity as described (8), except that a 40% SDS-polyacryamide gel was utilized to separate the incorporated label from free ATP. This assay was linear for up to 90 min, and all the values determined were within the linear range for Akt levels. To detect phosphorylation of Ser473, immunoblotting was performed with the phospho-specific Akt antibody from New England BioLabs.
The expressed p70 S6 kinase was immunoprecipitated using a monoclonal antibody directed against the myc epitope tag (Babco) that was preadsorbed to protein G-Sepharose. The nonspecific background was measured by incubating lysates with normal mouse immunoglobulin that was also preadsorbed to protein G-Sepharose. The kinase activity was measured as described previously (15). 3× Laemmli sample buffer (45 µl) was also added to the remaining beads. The bound protein was eluted by incubating for 4 min at 100 °C, and these samples were electrophoresed on 10% SDS-polyacrylamide gels. The gels were transferred and immunoblotted using the anti-myc antibody to detect p70 S6 kinase. The PI 3-kinase assay using pure phosphatidylinositol (Sigma) as a substrate was performed as described previously (17), except that cells were serum-starved for 16 h before being treated and lysed. The PHAS-I shift and eIF4E dissociation were measured by PHAS-I immunoblotting either whole cell extracts and m7GTP-Sepharose bound material, respectively, as described previously (18).3T3-L1 Preadipocytes and Adipocytes--
3T3-L1 preadipocytes
infected with either myrAkt 4-129-ER or A2myrAkt
4-129-ER were
cultured, selected, and differentiated as described previously (9).
These cell lines were used to measure glucose uptake and to isolate
crude membrane fractions for detection of GLUT1 and GLUT4 expression as
described previously (9). Translocation of GLUT4 to the plasma membrane
was performed and quantitated as described previously (19, 20).
In Vivo Labeling of myrAkt 4-129-ER--
Medium was replaced
with Krebs-Ringer bicarbonate buffer containing 10 mM
glucose and [32P]orthophosphate (500 µCi/plate). After
3 h at 37C/5%CO2, NIH3T3 cells expressing myrAkt
4-129-ER were treated without or with 1 µM HT for
additional 40 min, placed on ice, washed with ice-cold HBS, and lysed
with 500 µl of lysis buffer (50 mM Hepes, pH 7.4, 1%
Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, 300 nM
okadaic acid, 1 mM NaF, 10 mM
-glycerol
phosphate, 10 µg/ml aprotinin). Lysates were immunoprecipitated with
monoclonal anti-HA (12CA5) bound to Protein-A agarose beads. Beads were
washed, and bound proteins were eluted and analyzed by
SDS-polyacrylamide gel electrophoresis and transferred to
nitrocellulose. To confirm which band was Akt, the membrane was
immunoblotted with anti-HA antibodies. The autoradiographs were
scanned, imported into Adobe Photoshop, and quantitated after
subtracting a background region of the gel.
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RESULTS |
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Production of a Conditionally Active Akt Kinase-- Several strategies exist to make a conditionally active Akt kinase, the activity of which can be regulated. Tetracycline-regulatable systems have been described in which tetracycline can be added to suppress or induce expression of a given construct (21). In addition, one could use drug-induced dimerization systems exploiting synthetic bivalent membrane permeable compounds that bind to drug binding domains fused to intracellular signaling molecules (22). Finally, conditionally active forms of the transcription factor Myc and the protein kinases Raf1 and Abl have been made by fusing them with the HBD of steroid receptors, particularly the estrogen receptor (23-25). Although we were unable to create a conditionally active form of Akt using the tetracycline-regulatable or drug-induced dimerization systems (data not shown), we were able to generate such an enzyme by constructing a HBD fusion protein.
We have previously described a constitutively active form of Akt (myrAkt
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Studies on the Mechanism of Activation of the Conditionally Active
Akt--
As shown above, although the levels of the myrAkt
4-129-ER protein did not change after HT treatment of cells, the
protein did shift to a higher molecular weight on SDS gels (Fig.
2B), consistent with an increase in phosphorylation. To
directly test this, metabolically labeled cells expressing myrAkt
4-129-ER were treated for 40 min with either HT or ethanol and
lysed, and the lysates were immunoprecipitated with anti-HA antibodies.
An approximate 3-fold increase (range, 1.8-3.4; n = 3)
in labeling of the myrAkt
4-129-ER was observed with HT treatment
(Fig. 2D).
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The Conditionally Active Akt Rapidly Stimulates p70 S6 Kinase-- Prior studies have shown that constitutively active forms of Akt stimulate the enzymatic activity of p70 S6 kinase (4, 15). In these reports, the constitutively active Akt was unregulated, so its kinase activity was chronically elevated, raising the possibility that p70 S6 kinase was stimulated not because of a phosphorylation cascade initiated by Akt but rather because of other cellular events arising secondary to long-term expression of a constitutively active kinase. The conditionally active Akt allowed us to resolve this question.
NIH3T3 cells already expressing either myrAkt
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The Conditionally Active Akt Stimulates Glucose Uptake and GLUT4
Translocation in 3T3-L1 Adipocytes--
The unregulated constitutively
active Akt has previously been shown to stimulate glucose uptake in
3T3-L1 adipocytes and induce the translocation of GLUT4 to the plasma
membrane (9, 13). To test whether the conditionally active Akt could
also stimulate this response, we infected 3T3-L1 preadipocytes with
either myrAkt 4-129-ER or A2myrAkt
4-129-ER. The kinase
activity of the Akt fusion proteins was measured in immunoprecipitates
prepared from differentiated adipocytes that had been treated with
either HT or ethanol, the control vehicle, for different periods of
time by utilizing a peptide as exogenous substrate (8). This assay showed no activation of Akt in the adipocytes after a 1-h stimulation, but after 6 h, there was a 20-fold stimulation (Fig.
2C), and the activity remained elevated after 16 h. In
contrast, the A2myrAkt
4-129-ER construct was not significantly
stimulated at any of the times tested, and the myrAkt
4-129-ER was
not activated in the absence of HT (Fig. 2C). The slower
rate of activation of myrAkt
4-129-ER with HT in the adipocytes
could be due to the lower level of expression of these constructs in
the adipocytes in comparison to the NIH3T3 cells or for some other
unknown reason.
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The Conditionally Active Akt Rapidly Stimulates PHAS-I Phosphorylation in NIH3T3 Cells-- In addition to regulating glucose uptake, insulin stimulates protein synthesis in a variety of cell types (31). The constitutively active Akt, myrAkt, was found to activate protein synthesis to a level comparable to insulin stimulation.2 One mechanism whereby insulin has been found to regulate protein synthesis is via the phosphorylation of the eIF4E-binding protein, PHAS-I (31). The nonphosphorylated form of PHAS-I inhibits protein synthesis by tightly binding to the mRNA cap-binding protein, eIF4E. When PHAS-I is phosphorylated on the appropriate sites, it releases eIF4E, which is then free to participate in translation initiation (31). The phosphorylation of PHAS-I has been shown to be controlled by a rapamycin-sensitive pathway involving the mammalian target of rapamycin, mTOR (31). There is also evidence that PI 3-kinase is an upstream element in this pathway, but the role of Akt in the control of PHAS-I phosphorylation is not known.
Experiments were therefore performed to investigate the effect of the conditionally active Akt on the phosphorylation of PHAS-I and its association with eIF4E. NIH3T3 cells expressing myrAkt D4-129-ER (MER-3T3) or control cells were treated with 1 µM HT and lysed, and the lysates were either analyzed by SDS-polyacrylamide gel electrophoresis and immunoblotting for PHAS-I or first incubated with m7GTP-Sepharose to determine the amount of PHAS-I complexed with eIF4E. Phosphorylation of PHAS-I has been shown to markedly decrease its electrophoretic mobility in SDS gels (18). By gel shift analysis, HT treatment of MER-Akt-expressing cells was found to rapidly stimulate PHAS-I phosphorylation to an extent comparable to insulin (Fig. 6A). In contrast, HT was without effect in the control cells (Fig. 6A), whereas insulin stimulated the shift of PHAS-I in both cell types. In addition, tamoxifen stimulated a dissociation of PHAS-I from eIF4E in the MER-Akt cells but not in the control cells, as detected by a decrease in the amount of the PHAS-I/eIF4E complex bound to the m7GTP-Sepharose (Fig. 6).
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DISCUSSION |
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The results presented here demonstrate that fusion of the hormone
binding domain of the estrogen receptor to an activated form of Akt
renders the kinase activity of this protein dependent on the addition
of exogenous HT. Using HT, the kinase activity can be rapidly turned on
in several cell types, including NIH3T3 cells (Fig. 2A), a
mouse hepatoma cell line, and primary
myoblasts.3 The induction of
Akt kinase activity correlates with the phosphorylation of this
protein, including at a key regulatory site, Ser473 (Fig.
2, B, D, and E), consistent with the hypothesis
that phosphorylation regulates the enzymatic activity of this chimera
as it does the the wild type enzyme (32). The rapid activation of the
chimera with HT treatment is consistent with the previously described model, in which hormone binding stimulates the release of the 90-kDa
heat shock protein from the HBD, thereby allowing access of other
proteins (e.g. a kinase) to the chimeric molecule (33). An
alternative model would be that the HBD could induce oligomerization of
the chimera, and this oligomerization could be sufficient to result in
transphosphorylation and activation of Akt. This did not appear to be
the case because A2myrAkt 4-129-ER, which only lacks the glycine
that is myristoylated, was not activated. Thus, the Akt chimera must be
present in a membrane compartment to be activated. Presumably, this is
due to the presence of a kinase in the membrane that is responsible for
phosphorylating Akt. This kinase could be the recently described Akt
kinase kinase, which is stimulated by PI 3,4,5-trisphosphate and PI
3,4-bisphosphate (7). Alternatively, a distinct membrane kinase could
also be involved. In this regard, it is intriguing that wortmannin and LY294002, inhibitors of PI 3-kinase, could block the activation of
myrAkt
4-129-ER by HT (Fig. 3 and data not shown). The latter has
recently been shown to also cause a deactivation of the constitutively active Akt (34). This inhibition could be caused by these drugs directly inhibiting the kinase that is responsible for phosphorylating Akt (possibly the PI 3-kinase itself) or, more likely, by the ability
of these drugs to lower the basal levels of PI 3-phosphates in the
cells and thereby decrease the basal activity of a kinase sensitive to
these compounds. The slower rate of activation of myrAkt
4-129-ER
in 3T3-L1 adipocytes in comparison to the NIH3T3 cells could be due to
a lower level of the membrane kinase responsible for phosphorylating
and activating Akt and/or a lower basal level of the necessary PI
3-phosphates. Alternatively, it may be due to the lower levels of
expression of the Akt chimera in these cells or for some other
reason.
The generation of a conditionally active Akt allowed us to investigate
downstream targets of this enzyme. Prior studies have shown that
expression of different constitutively active forms of Akt induce
various subsequent biological responses, including stimulation of the
p70 S6-kinase and glucose uptake (4, 8-13). In the present study, we
could show activation of the p70 S6-kinase activity in NIH3T3 cells
expressing myrAkt 4-129-ER after only a 10-min stimulation with HT
(Fig. 4), a time very close to that required for induction of Akt
enzymatic activity in these cells (Fig. 2A). These results
indicate that the activation of the 70-kDa S6 kinase by Akt is an
immediate consequence of stimulation of Akt kinase activity, rather
than an event secondary to long-term expression of a constitutively
active kinase.
In 3T3-L1 adipocytes, the activation of myrAkt 4-129-ER was
considerably slower than in NIH3T3 cells (Fig. 2C), and the
maximal amount of activity was less. A detectable increase in glucose uptake was observed after 4- and 6-h incubations with HT. After 16 h, glucose uptake was stimulated approximately 1/4 as well as a
maximal dose of insulin. The extent of stimulation of glucose uptake
after HT treatment corresponded with the extent of GLUT4 translocation
and the relative amounts of Akt stimulated by HT in comparison to
insulin. That is, the amount of Akt kinase activity and glucose uptake
observed with a 16-h HT stimulation of cells expressing myrAkt
4-129-ER was approximately 1/4 the maximal amount of glucose
uptake and endogenous Akt kinase activity observed in insulin-treated
cells. It is possible that the longer period of time required to
maximally stimulate Akt activity and glucose uptake in these cells
allowed other processes to occur in addition to Akt activation, raising
the question of whether Akt activation alone is sufficient for
stimulation of glucose uptake. For example, it was possible that Akt
activation stimulates the release of a secreted molecule that feeds
back to activate glucose uptake. However, the supernatants of these
cells were incapable of stimulating glucose uptake in 3T3-L1
adipocytes,3 indicating that this increase in glucose
uptake was at least not due to the release of a secreted factor. Recent
additional evidence of a role for Akt in mediating the insulin-induced
increase in glucose uptake has come from studies showing that
expression of an inactive Akt can hinder the ability of insulin to
stimulate GLUT4 translocation in adipocytes (35).
In the present study, we also utilized the conditionally active Akt to determine whether this enzyme can modulate the phosphorylation of PHAS-I, a key regulator of protein synthesis, as well as its association with eIF4E (31). A constitutively active Akt, myrAkt, can increase protein synthesis in 3T3-L1 adipocytes to the same extent as insulin2 and stimulate an increase in leptin production in these cells (36). One mechanism by which insulin and other growth factors regulate protein synthesis is a stimulation in phosphorylation of PHAS-I and its subsequent release from eIF4E (31). Prior studies have demonstrated that insulin stimulates a phosphorylation of PHAS-I and its subsequent release from eIF4E, thereby increasing the amount of eIF4E available to participate in translation of capped mRNAs (31). In the present study, we showed that activation of the conditionally active Akt caused a shift of the PHAS-I protein within 10 min of stimulation (Fig. 6). This phosphorylation was associated with a release of PHAS-I from eIF4E. Thus, acute activation of Akt was sufficient to mimic the ability of insulin to stimulate PHAS-I phosphorylation and release of eIF4E, indicating that Akt is upstream of the kinase(s) responsible for phosphorylation of PHAS-I.
In summary, the present study provides further evidence that Akt can directly regulate activation of the 70-kDa S6 kinase and glucose uptake. In addition, we provide the first evidence that activation of Akt can regulate the phosphorylation of PHAS-I, as well as its association with eIF4E, providing a potential mechanism whereby insulin may regulate protein synthesis.
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ACKNOWLEDGEMENTS |
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We thank Dr. Martin McMahon for the HB domain construct, Dr. Garry Nolan for the Phoenix retroviral packaging cell line and the retroviral vectors, and Drs. Gerry Crabtree and John Blenis for the p70 S6 kinase clones.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants DK 34926 and DK 39615, a grant from the Cox Institute, Medical Scientist Training Program Grant 5T32 GM07365 (to A. D. K.), and a Feodor Lynen fellowship of the Alexander von Humboldt Stiftung (to A. B.).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: Dept. of Molecular
Pharmacology, Stanford Medical Center, Stanford, CA 94305. Tel.:
650-723-5933; Fax: 650-725-2952; E-mail: roth{at}cmgm.stanford.edu.
1 The abbreviations used are: PH, pleckstrin homology; PI, phosphatidylinositol; HT, hydroxytamoxifen; ER, estrogen receptor; HBD, hormone binding domain; HA, hemagglutinin.
2 A. D. Kohn and R. A. Roth, unpublished observations.
3 Andreas Barthel and R. A. Roth, unpublished observations.
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REFERENCES |
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