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J Biol Chem, Vol. 275, Issue 12, 8271-8274, March 24, 2000
§ and
From the ¶ Department of Pharmacology, University of
California at San Diego, La Jolla, California 92093-0640 and
the Signal Transduction Group, Boston Biomedical Research
Institute, Boston, Massachusetts 02114
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
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The function of Akt (protein kinase B) is
regulated by phosphorylation on two sites conserved within the AGC
kinase family: the activation loop (Thr-308) in the kinase core and a
hydrophobic phosphorylation site on the carboxyl terminus (Ser-473).
Thr-308 is phosphorylated by the phosphoinositide-dependent
kinase-1, (PDK-1), whereas the mechanism of phosphorylation of the
hydrophobic site, tentatively referred to as the PDK-2 site, is
unknown. Here we report that phosphorylation of the hydrophobic motif
requires catalytically competent Akt. First we show that a
kinase-inactive construct of Akt fails to incorporate phosphate at
Ser-473 following IGF-1
stimulation in vivo but does incorporate phosphate at
Thr-308 and a second carboxyl-terminal site, Thr-450; this ligand
triggers the phosphorylation of both sites in wild-type enzyme. Neither does a catalytically inactive construct in which phosphorylation at the
activation loop is blocked, T308A, become phosphorylated on the
hydrophobic site in response to stimulation. Second, we show that Akt
autophosphorylates on the hydrophobic site in vitro: phosphorylation of the activation loop by PDK-1 triggers the
phosphorylation of the hydrophobic site in kinase-active, but not
thermally inactivated, Akt Phosphorylation exquisitely regulates a plethora of cellular
functions, including the function of the protein kinases themselves. Many members of this superfamily are controlled by two phosphorylation switches, one on a segment near the entrance to the active site and a
second on the carboxyl terminus. Phosphorylation at one or both
positions appears to be required to correctly align residues to create
a catalytically competent conformation. For kinases such as protein
kinase A and the conventional protein kinase Cs (PKC),1 phosphorylation of
these positions is constitutive and part of the processing of the
enzyme (1). In contrast, phosphorylation of kinases such as p70 S6
kinase is tightly regulated by specific stimuli (2). The
carboxyl-terminal switch contains two motifs: a turn motif, typically
rich in Pro, which, in the case of protein kinase A, is positioned at
the apex of a tight turn (Ser-338) (3), and a hydrophobic motif
(FXXS/TXF) first identified in protein kinase C
and p70 S6 kinase (4, 5).
The serine/threonine kinase, Akt (protein kinase B) transduces signals
that result in phosphoinositide-3-kinase activation, (6-13). The
membrane-localized enzyme becomes rapidly phosphorylated at two
positions, the activation loop (Thr-308) and the hydrophobic phosphorylation motif (Ser-473). The activation loop reaction is
catalyzed by the recently discovered PDK-1 (14-16). The
phosphorylation of the hydrophobic motif is tightly coupled to that of
the activation loop; however, the mechanism of this phosphorylation is
unknown. The mitogen sensitivity of this site, like that of the
activation loop, has led to the proposal that it, too, is
phosphorylated by an agonist-sensitive kinase, tentatively named PDK-2
(14). Nonetheless, such a kinase has remained refractory to molecular or biochemical identification (17). In this regard, protein kinase C
This contribution addresses the mechanism of phosphorylation of
the carboxyl-terminal sites of Akt. Specifically, we ask which sites
depend on the intrinsic catalytic activity of the enzyme in order to
dissect the contribution of autophosphorylation versus heterologous phosphorylation in the regulation of Akt.
Dipalmitoylphosphatidylinositol tris-3,4,5-phosphate
(PtdIns-3,4,5-P3) was obtained from Matreya, palmitoyl
oleoyl phosphatidylcholine (PC), and bovine brain
L- cDNA Constructs--
The murine Akt T308A and K179M
mutants were a kind gift of Philip Tsichlis (Thomas Jefferson
University). The Akt T450A mutant was made by polymerase chain
reaction-based site-directed mutagenesis using the Pfu Quickchange
methodology (Stratagene).
Cell Culture--
Human embryonic kidney HEK 293E cells and
HepG2 cells expressing the Phosphatase Treatment of Akt--
Purified Akt (80 nM) was incubated with the catalytic subunit of protein
phosphatase 1, in the presence of lipid vesicles, as described in the
legend to Fig. 1. Incubation mixtures included PC vesicles (100 µM total lipid) containing no other lipid, 10 mol % PtdIns-3,4,5-P3, or 10 mol % PtdIns-3,4,5-P3
and 45 mol % PS. Microcystin (4 µM) was included in some
experiments. Protein kinase C Mammalian Cell Transfections--
HEK 293E and HepG2 cells were
transiently transfected either by the calcium-phosphate procedure (24)
or by the LipofectAMINE procedure (Life Technologies, Inc.) as
described previously (25). Cells were serum-starved for 24 h in
Dulbecco's modified Eagle's medium with no added serum. Cells were
stimulated with either IGF-1 (20 ng ml In Vitro Phosphorylation of Akt--
A highly purified insect
cell-expressed recombinant Akt preparation was used to detect
phosphorylation of relevant sites by PDK-1 in vitro.
Recombinant human PDK-1 was expressed as a fusion protein in insect
cells. For in vitro assays, 0.1 µg of each protein was
mixed in a nonradioactive kinase assay reaction (using 80 µM [ATP]) either in the presence or absence of
PS:PC:PtdIns-3,4,5-P3 vesicles (see above).
Carboxyl Terminus of Akt Contains Two Conserved Phosphorylation
Sites--
The carboxyl terminus of Akt is phosphorylated at two
positions: Thr-450 is constitutively phosphorylated (18, 27) and Ser-473 is phosphorylated in a mitogen-sensitive manner (18). Alignment
of the carboxyl-terminal residues of Akt isozymes with those of protein
kinase C
First, we tested whether unstimulated Akt is phosphorylated on
the turn motif. Homogeneously pure Akt from the baculoviral expression
system was used for this study; mass spectrometric analysis revealed
there was no phosphate on either the activation loop or the hydrophobic
site.2 Pure Akt was treated
with the catalytic subunit of protein phosphatase 1 (PP1), a
phosphatase that dephosphorylates the turn motif of protein kinase C
(4). The silver stain in Fig. 1B shows that untreated Akt
migrated as a major band with an apparent molecular mass of 64 KDa,
with a minor (<10%) faster migrating species (apparent molecular mass
of 62 kDa). Treatment with PP1 resulted in disappearance of the upper
band and accumulation of the faster migrating species. Samples from
this experiment were also probed with an antibody that recognizes the
phosphorylated, but not unphosphorylated, turn motif of protein kinase
C (see "Materials and Methods"). Fig. 1B, lower
panel, shows that this antibody cross-reacted with untreated Akt
(lane 1). However, it did not react with the faster migrating species resulting from phosphatase treatment. These data
reveal that unstimulated Akt is almost quantitatively phosphorylated on
the turn motif. For comparison, lanes 6 and
7 show the migration of purified, baculoviral-expressed
protein kinase C Characterization of Kinase-inactive Constructs of Akt--
To
address whether autophosphorylation or phosphorylation by an upstream
kinase regulates the two carboxyl-terminal positions of Akt, we first
examined the agonist-dependent protein kinase activities of
wild-type and mutant Akt alleles in vivo. HA-tagged constructs of Akt were expressed in HepG2 cells expressing the wild-type Thr-450 Is Phosphorylated in Kinase-inactive Constructs of
Akt--
Previous studies have indicated that the turn motif in Akt,
Thr-450, is constitutively phosphorylated in serum-starved cells and
that phosphorylation of this site does not contribute to the activity
of the enzyme (18, 27). The mechanism of regulation of this site has
not been examined to date. The Western blots in Fig. 2, A
and B, show the expression of wild-type Akt, the activation
loop mutant T308A, the turn motif mutant T450A, and the kinase-inactive
mutant K179M in HEK 293E cells detected using anti-HA antibodies
(upper panels). Wild-type Akt migrated as a doublet
(lanes 2 and 3). The upper band comigrated with
Akt that is phosphorylated on Thr-450 and was labeled with the
phospho-turn motif antibody (Fig. 3,
Phosphorylation of Ser-473 Requires the Intrinsic Catalytic
Activity of Akt--
We next explored whether phosphorylation of the
hydrophobic site depended on the intrinsic catalytic activity of Akt.
The Western blot in Fig. 3 shows immunoprecipitated Akt proteins from control and IGF-1-stimulated HEK 293 cells probed with an antibody that
specifically labels phosphorylated Thr-473 (
One possibility is that Thr-473 phosphorylation requires
phosphorylation of Thr-308. Thus, we addressed the phosphorylation state of the activation loop in the kinase-inactive constructs using an
antibody that specifically labels phosphorylated Thr-308 ( Phosphorylation at the Activation Loop Triggers Autophosphorylation
of Ser-473--
We next tested whether the phosphorylation of Thr-308
by PDK-1 in vitro triggered the autophosphorylation of pure
Akt. Homogeneously pure Akt and PDK-1 were incubated with
Mg2+/ATP, in the presence of
PS:PC:PtdIns-3,4,5-P3
vesicles. The electrophoretic mobility and phosphorylation of specific residues was assessed by
immunoblotting with either anti-HA, anti-P308, or anti-P473 antibodies.
Two forms of Akt were used for this experiment: untreated Akt or Akt
that had been thermally inactivated by sonication. Fig. 4A,
middle panel, shows that both forms of Akt were
phosphorylated on Thr-308, with maximal phosphorylation catalyzed
within 5 min. Thus, the untreated and thermally inactivated forms of
Akt were equally good substrates for PDK-1, indicating that the
sonication had not grossly perturbed the structure of the kinase.
Analysis with the P473 antibody revealed incubation with
Mg2+/ATP resulted in phosphorylation of Ser-473. The
kinetics of phosphorylation mirrored those of Thr-308, with maximal
phosphorylation observed within the first 5 min of the reaction. Note
that a mobility shift to a slower migrating species (single
arrow) lagged behind the phosphorylation of Thr-308/Ser-473; this
shift likely represents additional autophosphorylations that follow the
two priming events. A more detailed analysis of the initial
phosphorylation (Fig. 4C) revealed that phosphorylation of
Ser-473 closely followed that of Thr-308, with Thr-308 being the first
phosphorylation event (compare 20-s time points). Fig. 4B,
left panels, shows that the phosphorylation of Ser-473 was
abolished in the thermally inactivated sample. This reveals that the
Ser-473 phosphorylation requires the activity of Akt and cannot result
from contaminating kinases or from phosphorylation by PDK-1, as
suggested recently (28).
Conclusions--
The hydrophobic site has generally been accepted
to be regulated by a heterologous kinase, referred to as PDK-2 (14) or Ser-473 kinase (9). Interestingly, a yeast two-hybrid screen led to the
identification of the carboxyl terminus of the protein kinase C-related
kinase, PRK2, as a module interacting with PDK-1 (28). In
vitro studies revealed that a peptide based on this sequence, PIF
(PDK-1 interacting fragment), promoted the incorporation of phosphate
onto Ser-473 of Akt in the presence of PDK-1. This led to the proposal
that PDK-2 is actually PDK-1 which undergoes a dramatic, and
unprecedented, switch in substrate specificity through its
interaction with PIF. Because this study did not examine whether
kinase-inactive constructs of Akt are phosphorylated by PIF-bound
PDK-1, it did not address whether autophosphorylation was
promoted by binding of PDK-1 to PIF. One possibility is that PDK-1
binding to Akt sterically or conformationally blocks the hydrophobic
site, thus preventing autophosphorylation. Autophosphorylation may be
triggered by release of PDK-1 from Akt, an event that may be promoted
by PIF.
Taken together with previous studies, our data support the
following, alternative model for the regulation of Akt. In unstimulated cells, Akt is present in the cytosol in a conformation that blocks access of PDK-1 to the activation loop. Specifically, the PH domain masks Thr-308. Mitogen stimulation results in the membrane recruitment of Akt, unmasking of the activation loop, and allowing the
phosphorylation of Thr-308 by PDK-1. The phosphorylation by PDK-1
renders Akt catalytically competent and causes the autophosphorylation
of Ser-473 of the hydrophobic phosphorylation motif. This mechanism of
regulation is similar to that recently elucidated for the regulation of
the protein kinase C family of isozymes. Whether a hydrophobic site
kinase, tentatively referred to as PDK-2, exists for other kinases such
as p70 S6 kinase, or whether these kinases also autophosphorylate at
the hydrophobic site, remains to be determined.
. Thus, Akt is regulated by
autophosphorylation at the Ser-473 hydrophobic site.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
II has recently been shown to be regulated by autophosphorylation, and not by a heterologous kinase, at its hydrophobic phosphorylation motif (19, 20). In addition to the mitogen-sensitive sites, 32P labeling studies have revealed that Akt is
constitutively phosphorylated at Thr-450 in vivo (18). This
position corresponds to the turn motif in the protein kinase Cs as it
has the consensus motif T/SPXD (21).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
-phosphatidylserine (PS) were obtained from Avanti
Polar Lipids. Microcystin and peroxidase-conjugated goat anti-rabbit
antibodies were from Calbiochem. Protein kinase C II (rat) was
purified from the baculovirus expression system as described (22).
Baculoviral expressed, homogeneously pure mouse Akt was a generous
gift from Dr. Frank Sicheri, Samuel Lunenefeld Research Institute. An
antibody that recognizes the phosphorylated Thr-641 in the turn motif
of protein kinase C II (23) was provided by David Sweatt (Baylor
College of Medicine). Antibodies, which recognize phosphorylated
Thr-308 or phosphorylated Ser-473 were obtained from New England
Biolabs. In one experiment, an antibody that recognizes the
phosphorylated hydrophobic motif of Akt and all the protein kinase C
isozymes was used (New England Biolabs, PAN-PKC antibody). The
catalytic subunit of protein phosphatase 1 was a generous gift from Dr.
Anna DePaoli-Roach (Indiana University). All other chemicals were
reagent-grade.
subunit of the wild-type
platelet-derived growth factor-receptor (-PDGF-R, a kind gift of
Andrius Kazlauskas, Harvard Medical School) were maintained in
Dulbecco's modified Eagle's medium (Life Technologies, Inc.)
containing 10% fetal bovine serum.
II (45 nM) was treated
similarly except that lipid was presented in the form of sonicated
dispersions of phosphatidylserine (140 µM) and
diacylglycerol (4 µM) and 200 nM
CaCl2 was present. Reactions were quenched by addition of
SDS-PAGE sample buffer and samples analyzed by SDS-PAGE (7.5%
polyacrylamide) followed by silver staining or Western blot analysis.
1, Life
Technologies, Inc.) or with PDGF-BB (50 ng ml1, Life
Technologies, Inc.), typically for 10 min. Immunoprecipitated Akt
proteins were assayed for protein kinase activity using histone H2B as
substrate, as described by Franke et al. (26).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
II revealed the Thr-450 corresponds to the turn motif
described for the protein kinase Cs (Fig.
1A). The mechanism of
phosphorylation of both positions is autophosphorylation for protein
kinase C (19). Given the high sequence similarity between these two
kinases, we explored whether such a mechanism accounted for the
phosphorylation of the corresponding sites in Akt.
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Fig. 1.
A, alignment of activation loop and
carboxyl-terminal sequences of Akt and related kinases showing
conservation (shaded areas) of phosphorylation sites on the
activation loop, turn motif, and hydrophobic motif. Phosphorylated
residues are in bold with the numbering for Akt indicated
on top. Shown are the sequences for murine Akt (29), human Akt (30), human Akt 
(31), rat protein kinase C II (32), rat S6
kinase (33), and murine protein kinase A (34). B,
dephosphorylation of unstimulated Akt results in a specific shift in
electrophoretic mobility and loss of immunoreactivity with phospho-turn
motif antibody. Silver-stained gel (silver; upper panel) and
corresponding Western blot probed with an antibody that specifically
recognizes the phosphorylated turn motif in protein kinase C II
(P-turn motif; lower panel) showing Akt (0.15 µg) before
(lane 1) and after treatment with the catalytic subunit of
PP1 (4 µg ml1) for 5 (lane 2), 11 (lane 3), 30 (lane 4), or 60 (lane 5)
min at 22 °C, in the presence of phosphatidylcholine vesicles (100 µM total lipid) containing 10 mol % PtdIns-3,4,5-P3; protein kinase C II (0.12 µg) was
treated without (lane 6) or with (lane 7) the
catalytic subunit of PP1 (2 µg ml1) for 60 min,
22 °C, in the presence of 200 µM Ca2+ and
sonicated dispersions of phosphatidylserine (140 µM) and
diacylglycerol (4 µM). Akt was also treated without
(lane 8) or with (lanes 9 and 10) PP1
(2 µg ml1), and in the presence of microcystin (4 µM, lane 10), for 60 min at 22 °C.
II before (lane 6) and after treatment
with PP1 (lane 7). The shift in electrophoretic mobility
following PP1 treatment arises from loss of phosphate at the turn motif
and the hydrophobic motif; a smaller shift, similar to that observed
for Akt above, results from loss of phosphate at the hydrophobic motif
only (4). The increased electrophoretic mobility and loss of
immunoreactivity with the phospho-specific antibody did not result from
proteolysis: the presence of microcystin in the incubation medium
(lane 10) prevented both the shift and loss of
immunoreactivity revealing that phosphatase activity was required for
these effects.
PDGF-R or in HEK 293E cells (18, 26). HEK 293E cells
(Fig. 2A) or HepG2 cells (Fig.
2B) were stimulated for 10 min with IGF-1 or PDGF-BB,
respectively, and Akt activity from HA immunoprecipitates was assayed
using histone H2B as substrate. Fig. 2A, lower
panel, shows that wild-type Akt was activated 12.5-fold by
treatment of HEK 293 cells with IGF-1. Importantly, the activity of
T450A was similarly (10.7-fold) stimulated by IGF-1. Identical results
were obtained with PDGF-stimulation of HepG2 cells: PDGF induced an
8.3-fold increase in wild-type Akt activity and a 7.5-fold increase in
the activity of the T450A mutant (Fig. 2B, lower
panel). Thus, the effect of the T450A mutation in Akt was not
restricted to one cell type alone. In contrast, neither the K179M
(lanes 8 and 9) or the T308A (lanes 4 and 5) constructs had any detectable activity in the absence
or presence of mitogens. These data reveal that negative charge at
position 450 does not influence either the basal or stimulated activity
of Akt.
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Fig. 2.
Phosphorylation of Thr-450 in Akt does not
depend on the intrinsic catalytic activity of Akt. A,
HEK 293 cells transfected with the indicated Akt plasmids and left
unstimulated ( 
) or stimulated (+) with 20 ng/ml IGF-1 for 10 min.
B, HepG2 cells expressing the wild-type PDGF-R were
transfected with the indicated Akt plasmids and left unstimulated ()
or stimulated (+) with PDGF-BB (50 ng/ml) for 10 min. The top
panels of A and B show an immunoblot of Akt
from 5% of total cell lysates detected with anti-HA
(-HA) antibody. The bottom panels show an
immune-complex kinase assay of Akt using histone H2B as substrate. Fold
stimulation of Akt was quantitated on a Molecular Imager. The data are
representative of four independent experiments.
-P450), whereas the lower band comigrated with completely
dephosphorylated Akt (Fig. 1B and data not shown). Importantly, the T450A mutant (lanes 6 and
7) comigrated with the faster migrating wild-type species
consistent with lack of negative charge at position 450 (see Fig.
1B). Similar to wild-type Akt, the kinase-inactive mutant
K179M (lanes 8 and 9) migrated as a doublet,
consistent with partial phosphorylation of Thr-450. Similarly, the
activation loop mutant, T308A (lanes 4 and 5)
also comigrated with wild-type enzyme, although it was enriched in upper band. This mutant was labeled with the anti-phospho-turn antibody
(Fig. 3, -P450). These data reveal that Thr-450 is
phosphorylated by a reaction that depends neither on the catalytic
competence of Akt nor on the phosphorylation state of Thr-308. These
results indicate that both wild-type Akt as well as the T450A mutant
retain full catalytic competency, whereas both T308A and K179M are
completely inactive.
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Fig. 3.
Phosphorylation of Thr-473 in vivo
requires the intrinsic catalytic activity of Akt. HEK 293 cells were
transfected with the indicated Akt plasmid DNA's, serum-starved, and
stimulated for 10 min with IGF-1 (20 ng/ml). The top panel
shows 5% of total cell lysate probed with the anti-HA
( -HA) antibody. Phosphorylation of Akt was detected by
immunoprecipitation with HA antibody, followed by immunoblotting with
the anti-Thr-308(P) (-P308), anti-Ser-473(P)
(-P473), and anti-phospho-turn (-P450)
antibodies. The immunoblots are representative of three independent
experiments.
-P473). This antibody strongly labeled wild-type Akt from stimulated (lane 3), but not unstimulated (lane 2), cells. Similarly, it
labeled the T450A construct in stimulated (lane 7), but not
unstimulated (lane 6), cells. For both wild-type and the
T450A construct, IGF-1 stimulation resulted in a 13-15-fold increase
in immunoreactivity. In contrast, IGF-1 treatment had no detectable
effect on the immunoreactivity of the two catalytically inactive
constructs, T308A (lanes 4 and 5) or K179M
(lanes 8 and 9). Based on the sensitivity of the
detection method, any phosphorylation of Ser-473 would be less than 5%
that observed for wild-type enzyme. Thus, phosphorylation of Ser-473 requires the catalytic competence of Akt.
-P308). Fig. 3, lower panel, shows that IGF-1
caused a marked increase in the phosphorylation of the activation loop
of wild-type enzyme (lane 3), T450A (lane 7), but
not T308A (lane 5), which has an alanine at the
phosphoacceptor position. Importantly, the K179M mutant was
phosphorylated on the activation loop to comparable levels as wild-type
enzyme following IGF-1 stimulation. Thus, despite having a phosphate on
the activation loop, the K179M mutant was unable to incorporate
phosphate at position Ser-473. Identical results were obtained upon
expression of kinase-inactive mutants in PDGF-stimulated HepG2 cells
(data not shown). These data reveal that phosphorylation of the
hydrophobic site requires the intrinsic catalytic competence of Akt.
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Fig. 4.
Phosphorylation of Ser-473 in
vitro requires prior phosphorylation of the activation loop
and requires the intrinsic catalytic activity of Akt. A,
Western blot showing native Akt (left panels) or Akt
thermally inactivated by sonication (right panels) incubated
with Mg2+/ATP in the presence of
PS:PC:PtdIns-3,4,5-P3 vesicles and PDK-1 for the indicated
times. The immunoblot was probed with antibody to the HA tag
( -HA), the phosphorylated activation loop
(-P308), or the phosphorylated hydrophobic site
(-P473; New England Biolabs PAN antibody that recognizes
the hydrophobic phosphorylation motif in all the protein kinase Cs as
well as Akt). , no ATP added. B, Western blot showing Akt
incubated with Mg2+/ATP in the presence of
PS:PC:PtdIns-3,4,5-P3 vesicles and PDK-1 for the indicated
times. The immunoblots were probed with the antibodies described in the
legend to A. The immunoblots are representative of two
independent experiments performed in triplicate.
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ACKNOWLEDGEMENTS |
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We thank Frank Sicheri and Kenji Miyata for providing the purified Akt, Anna DePaoli Roach for the gift of the catalytic subunit of protein phosphatase, David Sweatt for providing the antibody against phosphorylated Thr-641, Michael Comb (New England Biolabs) for the phospho-hydrophobic site antibody, and Carmen Baca (University of California at San Diego) and Wendy Chaplis (Boston Biomedical Research Institute) for excellent technical assistance.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants GM 43154 (to A. C. N.) and CA 75134 (to A. T.).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 can be addressed. Present address: Dept. of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Ave., RN-237, Boston, MA 02215. Tel.: 617-667-8535; Fax: 617-667-3616; E-mail: atoker@caregroup.harvard.edu.
To whom correspondence can also be addressed: Dept. of
Pharmacology, UCSD, La Jolla, CA 92093-0640 (Tel.: 858-534-4527; Fax: 858-534-6020; E-mail: anewton@ucsd.edu).
2 F. Sicheri, personal communication.
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ABBREVIATIONS |
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The abbreviations used are: PKC, protein kinase C; IGF-1, insulin-like growth factor-1; PDGF, platelet-derived growth factor; PAGE, polyacrylamide gel electrophoresis; PDK-1, phosphoinositide-dependent kinase 1; PIF, PDK-1 interacting fragment; PC, phosphatidylcholine; PS, phosphatidylserine; PtdIns-3, 4,5-P3, phosphatidylinositol 3,4,5-tris-phosphate; PP1, protein phos- phatase 1; HA, hemagglutinin.
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
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