From the Department of Molecular Pharmacology,
Stanford University, Stanford, California 94305-5174, ¶ Howard
Hughes Medical Institute, University of Pennsylvania School of
Medicine, Philadelphia, Pennsylvania 19104,
Thermo Finnigan, San
Jose, California 95134, and ** Hopkinton Division of
BioSource International, Hopkinston, Massachusetts 01748
Received for publication, October 22, 2002, and in revised form, December 2, 2002
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ABSTRACT |
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Akt (also called protein kinase B) is one of
the major downstream targets of the phosphatidylinositol 3-kinase
pathway. This protein kinase has been implicated in insulin signaling,
stimulation of cellular growth, and inhibition of apoptosis as well as
transformation of cells. Although a number of cellular proteins have
been identified as putative targets of the enzyme, additional
substrates may play a role in the varied responses elicited by this
enzyme. We have used a combination of 14-3-3 binding and recognition by
an antibody to the phosphorylation consensus of the enzyme to identify
and isolate one of the major substrates of Akt, which is also a 14-3-3 binding protein. This 40-kDa protein, designated PRAS40, is a proline-rich Akt substrate. Demonstration that it is a substrate of Akt
was accomplished by showing that 1) PRAS40 was phosphorylated in
vitro by purified Akt on the same site that was phosphorylated in
insulin-treated cells; 2) activation of an inducible Akt was alone
sufficient to stimulate the phosphorylation of PRAS40; and 3) cells
lacking Akt1 and Akt2 exhibit a diminished ability to phosphorylate
this protein. Thus, PRAS40 is a novel substrate of Akt, the
phosphorylation of which leads to the binding of this protein to
14-3-3.
The phosphatidylinositol
(PI)1 3-kinase pathway is
activated by many extracellular growth factors including insulin (1, 2). A number of approaches have implicated this pathway in the
subsequent responses elicited by this hormone. One of the major
downstream targets of the lipid products generated by this enzyme (PI
3,4,5-P and PI 3,4-P) is the Ser/Thr kinase called Akt or
protein kinase B. Which of the particular responses elicited by the PI
3-kinase are actually caused by Akt has been the subject of numerous
studies, and these works have implicated Akt in many of the responses
including stimulation of glucose uptake and cell growth as well
as inhibition of apoptosis (3-5). The Akt enzyme phosphorylates
proteins on Ser or Thr residues in the motif
RXRXX(S/T) (6). A number of substrates of Akt
that contain this motif have been identified. These substrates include
glycogen synthase kinase (GSK)-3, several mammalian homologs of the
Caenorhabditis elegans DAF-16 transcription factor, the
anti-apoptotic protein BAD, phosphodiesterase 3B, a Rab
GTPase-activating protein, ATP-citrate lyase, and most recently,
tuberous sclerosis complex-2 (1, 2, 7-10).
The phosphorylation of a substrate by Akt will in several cases
(i.e. BAD, the DAF-16 homologs, and tuberous sclerosis
complex-2) result in the subsequent binding of the substrate to another
protein called 14-3-3 (11-13). 14-3-3 is one of a family of seven
related proteins that in some cases (i.e. BAD, the DAF-16
homologs) can induce a change in the subcellular localization of a
protein, whereas in other cases this protein can activate or inhibit
the intrinsic enzymatic activity of a protein (i.e.
Raf) (14, 15). The binding of 14-3-3 to a protein also has been
proposed to regulate either the proteolysis and/or the phosphorylation
state of the bound protein. Most important for the purposes of the
present discussion has been the finding that the 14-3-3 proteins bind to phosphoserine- and phosphothreonine-containing motifs in a sequence-specific manner (15-17). The particular motif recognized by
these proteins includes the consensus phosphorylation site of Akt, a
phosphorylated Ser/Thr with an Arg residue present at position In the present work we have used a combination of the
14-3-3 protein and anti-pAkt substrate antibodies (18) to screen and isolate substrates of Akt. The major 14-3-3 binding protein observed in
cells after insulin treatment was a 40-kDa molecule. This protein (designated PRAS40) was purified, sequenced, and identified as a
proline-rich molecule without any major homology to other proteins in
the data base and also lacking any recognizable domains. We have
demonstrated that this protein is a substrate of Akt by showing that it
can be phosphorylated in vitro with purified Akt, that the
activation of an inducible Akt (called mer-Akt) (19) was alone
sufficient to induce PRAS40 phosphorylation, and that the phosphorylation of this protein was decreased in cells lacking Akt1 and
Akt2 (3, 4).
Materials and Cell Lines--
LY294002 and rapamycin were
from Calbiochem. The 14-3-3-GST expression constructs were a
gift of Dr. Andrey Shaw, and the fusion protein was purified as
described (17). Cloning enzymes and competent DH5 Purification and Identification of PRAS40--
Confluent 150-mm
dishes of H4IIE cells were treated without or with 100 nM
insulin for 10 min and then lysed with lysis buffer (50 mM
HEPES, 150 mM NaCl, 1% Triton X-100, 1 mM
phenylmethylsulfonyl fluoride, 1 mM sodium ortho-vanadate,
5 mM PRAS40 Cloning and Generation of Expression Constructs--
An
EST containing the entire coding region of the human cDNA
(GenBankTM accession number BC007416, IMAGE clone
number 2988648) was purchased (Incyte Genomics) and amplified via PCR
with the addition of a BamHI site (5') and an
EcoRI site (3') and subcloned into the pcDNA3.1(+)
vector (Invitrogen) containing either an amino-terminal Myc tag or a
carboxyl-terminal HA tag for mammalian cell expression. For bacterial
expression as a GST fusion protein, the full-length construct was also
subcloned into pGEX-KG vector (a modification of pGEX-2T, obtained from
R. Scheller, Amersham Biosciences). Site-directed mutagenesis of
Thr-246 was performed using a QuikChange kit (Stratagene). The cDNA
encoding the Myc-tagged PRAS40 (wt and mutant) was also subcloned into
pWZL-blasticidin S retroviral vectors (provided by Dr. Nolan, Stanford University).
Expression and Analyses of PRAS40--
PRAS40 (wt or mutant) was
expressed by either transient transfection in human embryonic kidney
cells (293t) or by stable expression after retroviral infection of
NIH3T3-MER cells. In some experiments the PRAS40 was co-transfected
with epitope-tagged constitutively active Akt as indicated. Cells were
lysed as described above, and either the total lysates were analyzed or
the PRAS40 was precipitated with either an antibody to the epitope tag
or an antibody to the PRAS40 protein. The pGEX-PRAS40 construct was
expressed in Escherichia coli BL21(RIL) (Stratagene) and
purified and injected into rabbits (Lampire) for antibody production.
An antibody that recognizes PRAS40 when it is phosphorylated on Thr-246
(anti-pPRAS40) was obtained from BIOSOURCE
(Camarillo, CA). This rabbit polyclonal antibody was affinity-purified
using both negative and positive affinity purification methods to
optimize specificity for the targeted phosphoepitope.
Northern Blot--
The full-length PRAS40 cDNA was isolated
and gel-purified, and 100 ng of DNA were labeled by the random primer
labeling system (Invitrogen) according to the manufacturer's protocol.
A human tissue mRNA blot (HB-2010; Origene Technologies, Inc.) was
probed with the labeled PRAS40 according to the manufacturer's
protocol. The labeled bands were detected by autoradiography.
In Vitro Phosphorylation of PRAS40--
An epitope-tagged
constitutively active Akt (myr-Akt) was precipitated from transfected
cells with anti-HA antibody 12CA5 adsorbed to protein A-agarose beads.
The precipitated kinase was incubated with 4 µg of purified
PRAS40-GST fusion protein or GST alone for 30 min at 30 °C in the
presence of 10 mM MgCl2 and either 5 µM ATP (containing 2 µCi of
[ 14-3-3 Far Western Blot Analyses--
Total cell lysates of
control or insulin-treated H4IIE cells or PRAS40 precipitates from PC-3
cells were separated by SDS-PAGE and transferred to nitrocellulose
membranes. After blocking in 5% nonfat dry milk in Tris-buffered
saline buffer, membranes were incubated with purified 14-3-3-GST
(10 µg/ml). Bound 14-3-3-GST was detected using either an anti-GST
(Covance) or an anti-14-3-3 antibody (Santa Cruz) and the appropriate
secondary antibody.
Identification and Isolation of a 40-kDa Protein That Binds 14-3-3, the Phosphorylation of Which Is Induced by Insulin--
A rat
hepatoma, H4IIE, was treated with insulin and lysed, and the lysates
were either probed directly or after purification on a 14-3-3 affinity
column with an antibody directed against the phosphorylation motif of
Akt substrates (18). In total lysates, several proteins of various
molecular masses were detected with this antibody with the major band
at 25-kDa (Fig. 1a), presumably ribosomal protein S6 (18). However, after elution from the 14-3-3 affinity column, the major band detected was a 40-kDa molecule (Fig.
1a). This protein was not bound by a mutant 14-3-3 (Arg-56 and -60 changed to Ala) that lacks the ability to bind phosphorylated peptides (17). To look for other proteins that could bind to 14-3-3 in
the total lysates but that may not have been recovered in the 14-3-3 affinity column, total lysates from insulin-treated cells were probed
with the 14-3-3 in a far Western. Again, the major insulin-stimulated
14-3-3 binding protein observed in the total lysates was a 40-kDa
protein (Fig. 1b).
To determine which signal cascade was responsible for the
insulin-stimulated increase in 14-3-3 binding to the 40-kDa protein, H4IIE cells were treated with various inhibitors before insulin stimulation. Wortmannin, an inhibitor of the PI 3-kinase/Akt pathway, was found to largely block the insulin-stimulated increase in phosphorylated 40-kDa protein in the 14-3-3 eluates. In contrast, rapamycin, an inhibitor of the downstream kinase mammalian target of
rapamycin, had only a slight effect on the amount of phosphorylated 40-kDa protein detected (Fig. 1c). In contrast, an inhibitor
of the mitogen-activated protein kinase pathway (PD 098059) had no effect on the phosphorylation of the 40-kDa protein (data not shown).
To further characterize this 40-kDa protein, two-dimensional gel
electrophoresis was used. The isoelectric point (pI) of the protein was
estimated by this procedure to be ~4.5 (Fig. 1d). This point was considerably different from any previously
identified substrate of Akt in this size range (i.e.
glycogen synthase kinase-3
The above results suggested that the 40-kDa protein may be
a novel substrate of Akt and a major 14-3-3 binding protein. We therefore isolated sufficient amounts of this protein for tryptic digestion and identification by mass spectrometry. Five peptides from
the isolated protein were found to match a predicted mouse protein
gi:12834425 of unknown function and a predicted
Mr of 27,500 and pI of 4.6. A human homolog
(gi:14150199) of this protein was also present in the EST data base.
The deduced sequences of these proteins contained a consensus Akt
phosphorylation site (Thr-246) but no other recognizable motif (Fig.
2). It was, however, highly proline-rich,
with 15% of its amino acids being proline (versus 5% for a
typical protein), and these proline-rich regions are potential SH3
and/or WW domain binding partners (21). The protein has therefore been
named PRAS40, for proline-rich Akt substrate of 40 kDa.
Verification That the Identified cDNA Encodes for the 40-kDa
Substrate of Akt and That Thr-246 Is the Major Phosphorylation
Site--
PRAS40 was first expressed as a GST fusion protein in
E. coli. This fusion protein was found to be phosphorylated
by Akt in vitro, either by inclusion of radioactive ATP in
the kinase reaction and autoradiography (Fig.
3a) or by reaction with the
anti-pAkt substrate antibody used to screen the total cell lysates
(Fig. 3b). These results indicate that PRAS40 is readily
phosphorylated in vitro and that this phosphorylation leads
to the recognition of PRAS40 by the anti-pAkt substrate antibody.
The PRAS40-GST protein was also used to generate a polyclonal antibody
to the protein. This antibody was capable of immunoprecipitating from
lysates of insulin-treated H4IIE cells a protein that ran in an
identical position on SDS-PAGE as the 14-3-3 bound material and the
phosphorylation of which was stimulated by insulin (Fig. 4a). In addition, an identical
anti-pAkt substrate-reactive band was immunoprecipitated with an
antibody generated to the carboxyl-terminal 19 amino acids of the
deduced sequence of PRAS40 (Fig. 4a). The antibody to PRAS40
also immunoprecipitated a similar band, the phosphorylation of which
was stimulated by insulin in 3T3-L1 adipocytes (Fig.
4b).
An HA-epitope-tagged version of PRAS40 was also expressed
transiently in human embryonic kidney cells (293 cells). Lysates of
cells expressing the PRAS40 cDNA showed a phosphorylated 40-kDa band by blotting with the anti-pAkt substrate antibody, the levels of
which were greatly increased in comparison with cells transfected with
plasmid alone (Fig. 5). The phosphorylation
of this band was further increased when the cells were also transfected
with a constitutively active version of Akt (called myr-Akt) (22) (Fig.
5). Wortmannin, but not rapamycin, almost completely inhibited the
phosphorylation of the expressed PRAS40 (Fig. 5), as was previously observed for the endogenous protein. Wortmannin did not block the
phosphorylation of the expressed PRAS40 when the constitutively active
Akt was also co-expressed. These results are consistent with PRAS40
being a direct substrate of Akt.
To determine whether the predicted Akt consensus phosphorylation site
(Thr-246) was in fact the site recognized by the anti-pAkt substrate
antibody, a mutant form of the HA-tagged PRAS40 (T246A) was expressed
in which this site was changed to Ala. The anti-pAkt substrate did not
recognize the mutant PRAS40, even when the constitutively active Akt
was co-expressed with this protein (Fig. 5). Expression of the mutant
PRAS40 was verified by the presence of an HA-reactive band in these
transfected cells. These results indicate that Thr-246 is the sole
phosphorylation site in PRAS40 that is recognized by this antibody.
Detection of PRAS40 mRNA in Various Tissues--
Northern blot
analyses of a number of tissues demonstrated the presence of two PRAS40
mRNA species, one at 2.6 and the other at 1.8 kilobases (Fig.
6). An examination of the EST data base indicated the presence of two alternatively spliced forms of the human
mRNA that were of sizes consistent with the two mRNA species observed (gi:16359151 and gi:14150198); however, both ESTs contained the same predicted coding region. Of the tissues tested, the highest levels of both mRNAs were observed in liver and heart. However, a
PRAS40 band was readily detected in all the tissues tested. Western
blot analyses of various rat tissues confirmed the presence of PRAS40
in all of the tissues examined (data not shown).
Verification That PRAS40 Is a 14-3-3-binding
Protein--
To determine whether the expressed PRAS40 could bind
14-3-3, transiently expressed protein was immunoprecipitated from 293 cells via the use of antibodies to the added epitope tag. The precipitates were analyzed by immunoblotting for 14-3-3. Insulin was
found to stimulate the amount of 14-3-3 present in the precipitates (Fig. 7a). To determine whether
the endogenous PRAS40 was also capable of directly binding 14-3-3, we
used the prostate cancer cell line called PC-3 that lacks the inositol
1,4,5-triphosphate lipid phosphatase PTEN and thus has constitutively
active Akt (23). Precipitates of PRAS40 from these cells could directly bind 14-3-3 in a far Western (Fig. 7b). To determine whether
this was also regulated by PRAS40 phosphorylation, we treated these cells overnight with a reversible PI 3-kinase (LY294002) (24) and then
either lysed the cells or washed out the inhibitor and incubated the
cells for an additional 2 h. As seen in the anti-pAkt blot of
these lysates, the overnight incubation with LY294002 resulted in a
more than 95% decrease in phosphorylated Akt that was almost
completely reversed after a subsequent 2-h incubation without inhibitor
(Fig. 7b). The 14-3-3 did not bind to PRAS40 from cells
treated with LY294002 but did bind PRAS40 after removal of the
inhibitor and the 2 h incubation (Fig. 7b). This
correlated with the phosphorylation of the PRAS40 (Fig. 7b).
These results indicate that 14-3-3 can bind directly to PRAS40 and that
this binding is regulated by PRAS40 phosphorylation.
Demonstration That PRAS40 Is an in Vivo Target of Akt--
Because
insulin as well as LY294002 washout will activate various Ser/Thr
kinases downstream of inositol 1,4,5-triphosphate in addition to Akt
(1, 2), we sought a method to test whether activation of Akt alone was
sufficient to stimulate phosphorylation of PRAS40. To this end, we
expressed PRAS40 in mammalian cells that contain a conditionally active
Akt. This expressed variant Akt can be activated by treatment of the
cells with 4-hydroxytamoxifen, a treatment that should not activate any
other downstream targets of PI 3-kinase (19). Treatment of these cells
with 4-hydroxytamoxifen stimulated the phosphorylation of wt PRAS40 but
not the mutant PRAS40 in which Thr-246 was mutated (Fig.
8). These results demonstrate that activation
of Akt alone is sufficient to induce phosphorylation of PRAS40.
To determine whether endogenous Akt is responsible for the
phosphorylation of PRAS40 in nontransfected cells, we have used MEFs
from mice lacking either Akt1 or Akt2, or both Akt1 and Akt2 (3, 4).
Phosphorylation of endogenous PRAS40 was greatly stimulated by PDGF in
the MEFs from the wt mice and those lacking Akt2 (Fig.
9a). MEFs lacking Akt1 showed some
decrease in the PDGF-stimulated phosphorylation of PRAS40
(approximately a 30% decrease), whereas those lacking both Akt1 and
Akt2 showed the largest decrease (70%) in PDGF-stimulated
phosphorylation (Fig. 9a). These values were consistent with
the decreases observed in the PDGF-stimulated phosphorylation of GSK-3
(Fig. 9b), another substrate of Akt (25), as well as the
amount of PDGF-stimulated phosphorylated Akt present in the four cell
lines (Fig. 9b). These results implicate endogenous Akt in
the PDGF-stimulated phosphorylation of PRAS40.
The present studies have identified a new substrate of
Akt. Demonstration that PRAS40 is a substrate of Akt was
accomplished by showing that it could be phosphorylated in
vitro by Akt at the same site where it is phosphorylated in
vivo after activation by insulin. Moreover, we showed that
activation of an inducible Akt was alone sufficient to stimulate its
phosphorylation. Finally, we showed that cells lacking Akt1 and Akt2
exhibit a diminished ability to phosphorylate this protein. This
protein appears to be the major 14-3-3 binding protein which is
responsive to insulin. It is also quite similar in size to a recently
reported 14-3-3 binding protein (whose sequence is unknown) that is
responsive to growth factors and nutrients in PC-12 cells (26).
Although the function of this protein is not known, the presence of
several proline-rich regions may allow it to interact with various SH3 or WW domain-containing proteins, thereby modifying the function of
these molecules.
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ABSTRACT
INTRODUCTION
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RESULTS AND DISCUSSION
CONCLUSIONS
REFERENCES
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EXPERIMENTAL PROCEDURES
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ABSTRACT
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cells were from
Invitrogen. Plasmid purification kits were from Qiagen (Valencia, CA).
Pfu Turbo and the QuikChange XL mutagenesis kit were from Stratagene
(La Jolla, CA). Enhanced chemiluminescence detection reagents were from
Pierce. Porcine insulin was from Roche Molecular Biochemicals, goat
anti-mouse and anti-rabbit phosphatase-conjugated antibodies were from
Promega, anti-mouse and anti-rabbit antibodies coupled to horseradish
peroxidase were from Amersham Biosciences, monoclonal anti-FLAG,
anti-FLAG-agarose, and glutathione-agarose beads and other chemicals
were from Sigma. Anti-pAkt and the anti-pAkt substrate antibodies were
from Cell Signaling (Beverly, MA). H4IIE cells were grown as described
previously (20). MEF cells lacking Akt1 and Akt2 and control MEFs were prepared from E13.5 day embryos of the appropriate mice (3, 4) by
mincing and trypsinization and genotyped by reverse
transcription PCR, and expression of the Akt isozymes was
confirmed by Western blotting. Isoelectrofocusing strips, pH 3-10,
were Immobilon (13 cm) from Amersham Biosciences. A peptide containing
the carboxyl-terminal 19 residues of PRAS40 (DLPRPRLNTSDFQKLKRKY) was
synthesized, coupled to keyhole limpet hemocyanin, and used to
generate a polyclonal rabbit antibody to PRAS40 by BioCarta (Carlsbad, CA).
-glycerol phosphate, 10 mM NaF, 100 nM okadaic acid, 10 µg/ml aprotinin, and 10 µg/ml leupeptin). Lysates were centrifuged at 10,000 × g for
15 min at 4 °C, and the supernatants were incubated with 14-3-3-GST
bound to glutathione-agarose beads at 4 °C. The beads were washed
three times with HEPES-buffered saline, pH 7.6, and the bound proteins eluted by the addition of 1 ml of 0.5% Empigen BB. Eluted proteins were concentrated with the Page Perfect kit (Geno Technology Inc.) and
then analyzed or purified on either SDS-PAGE gels or via
two-dimensional gel electrophoresis. To find the position of the PRAS40
band, gels were transferred to nitrocellulose and blotted with
anti-pAkt substrate antibody. The PRAS40 bands were digested
with trypsin, and the tryptic peptides were sequenced by microcapillary
liquid chromatography mass spectrometry/mass spectrometry on an
ion trap mass spectrometer at both Thermo Finnigan and the Harvard
Microchemistry Facility.
-32P]-ATP) or 2.5 mM ATP. The samples
were then analyzed by SDS-PAGE and either autoradiography or
immunoblotting with anti-pAkt substrate antibody, respectively.
RESULTS AND DISCUSSION
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ABSTRACT
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REFERENCES
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Fig. 1.
Identification of a 40-kDa phosphoprotein as
an insulin-responsive 14-3-3-binding protein. a, binding to
14-3-3 columns. H4IIE cells were treated without or with 100 nM insulin for 10 min as indicated. Cells were lysed, and
either the total cell lysates or the material that bound and was eluted
from a 14-3-3 affinity column was analyzed by SDS-PAGE and
immunoblotting with an anti-pAkt substrate antibody. As a control,
lysates were also adsorbed and eluted from an affinity column composed
of an inactive mutant 14-3-3 (R56,60A). b, far
Western blots with 14-3-3. Total lysates as described above were also
analyzed by SDS-PAGE, and the transferred blots were probed with either
the wt 14-3-3 or the mutant 14-3-3 (R56,60A), and the bound
14-3-3 was detected as described under "Experimental Procedures."
c, the role of PI 3-kinase in the insulin-stimulated
phosphorylation of the 40-kDa protein. H4IIE cells were treated with
insulin in either the absence or presence of 1 µM
wortmannin or 1 µM rapamycin. The cells were lysed, the
lysates were absorbed with 14-3-3, and the bound proteins were analyzed
with the anti-pAkt substrate antibody. d, two-dimensional
gel analysis of the 40-kDa protein. H4IIE cells were treated without or
with insulin and lysed, and the 14-3-3-bound proteins were analyzed by
two-dimensional gel electrophoresis and transferred to nitrocellulose,
and the blots were probed with the anti-pAkt substrate antibody.
has a pI of 8.9). A similar
14-3-3-binding protein was also observed in 3T3-L1 adipocytes as well
as in various transformed cells like the prostate cancer cell line PC-3
(data not shown).
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Fig. 2.
Schematic of the deduced sequence of the
40-kDa protein (PRAS40). Scaled-up preparations of the 40-kDa
14-3-3 bound rat protein were electrophoresed on SDS-PAGE and digested
with trypsin, and the resulting peptides were analyzed by mass
spectrometry. Five peptides were identified that matched the deduced
sequence of a mouse protein in the data base (gi:12834425). This
protein was highly homologous to that of a human protein (gi:14150199).
The single predicted Akt phosphorylation site (Thr-246) and the
proline-rich regions are indicated in a schematic of the PRAS40.
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Fig. 3.
In vitro phosphorylation of PRAS40
by Akt. Four µg of purified PRAS40-GST fusion protein or GST
alone were incubated with a constitutively active Akt
immunoprecipitated from transfected cells. After a 30-min incubation at
30 °C in the presence of 10 mM MgCl2 and 5 µM ATP (containing 2 µCi of
[ -32P]ATP), the reaction mixtures were analyzed by
SDS-PAGE followed by transfer to nitrocellulose and autoradiography.
a, the reaction was also performed in the presence of either
0 or 2.5 mM ATP, and the products were analyzed by
immunoblotting with the anti-pAkt substrate antibody
(b).
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Fig. 4.
Antibodies to PRAS40 precipitate a 40-kDa
insulin-stimulated phosphoprotein. a, H4IIE cells were
treated with or without 100 nM insulin for 10 min at
37 °C as indicated. Total cell lysates, 14-3-3 bound proteins, or
precipitates with control antibodies (IgG) or anti-PRAS40
antibodies were analyzed by SDS-PAGE and immunoblotting with anti-pAkt
substrate antibody. Antibodies to PRAS40 were generated against either
the PRAS40-GST fusion protein ( PRASgst) or the
carboxyl-terminal 19 amino acids of the deduced PRAS40 sequence
(
PRASpep). b, PRAS40 in 3T3-L1 adipocytes.
Cells were treated or not with 100 nM insulin for 10 min at
37 °C as indicated and lysed, and either the total cell lysates or
anti-PRAS40 precipitates were analyzed by SDS-PAGE and immunoblotting
with anti-pAkt substrate antibody or anti-PRAS40.
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Fig. 5.
In vivo phosphorylation of the
expressed PRAS40. Human embryonic kidney (293) cells were
transfected with either empty pCDNA vector (V), an
HA-tagged version of PRAS40wt-pCDNA (PRAS40 wt), or
PRAS40/T246A-pCDNA (PRAS/T246A). As indicated, the
plasmid encoding constitutively active Akt (myr-Akt) was
included in some transfections. After 48 h, cells were treated
with either vehicle, 1 µM wortmannin, or 1 µM rapamycin for 40 min at 37 °C and then lysed as
described under "Experimental Procedures." Lysates were analyzed by
SDS-PAGE and immunoblotting with anti-pAkt substrate antibody
(a) or anti-HA 12CA5 (b) antibody.
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Fig. 6.
Northern blot of PRAS40. A human tissue
mRNA blot (Origene Technologies, Inc., HB-2010) was probed with the
labeled PRAS40 according to the manufacturer's protocol. The labeled
bands were detected by autoradiography.
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Fig. 7.
PRAS40 interacts with 14-3-3. a, in vivo interaction with expressed PRAS40. 293 cells were transfected with either empty vector (V) or
plasmid encoding a Myc-tagged PRAS40. After 46 h, the cells were
serum-starved for 2 h and then treated with or without 100 nM insulin for 10 min at 37 °C, lysed, and the lysates
were incubated with the anti-Myc antibody 9E10 prebound to protein
G-agarose. The bound proteins were analyzed by Western blot analysis
using either anti-Myc or anti-14-3-3 antibodies. b, direct
14-3-3 binding to PRAS40 from PC-3 cells. PC-3 cells were either
untreated ( ), treated overnight with LY294002 (+), or treated with
LY294002 overnight, washed, and allowed to recover for 2 h (+/
).
The cells were lysed, and the total cell lysates were either analyzed
directly for phosphorylated Akt or pPRAS40 or adsorbed with either
control antibodies (IgG) or anti-PRAS40 antibodies as
indicated. The precipitates were analyzed by SDS-PAGE and blotting with
either 14-3-3 or anti-PRAS40 antibodies as indicated.
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Fig. 8.
Activation of Akt alone is sufficient to
stimulate PRAS40 phosphorylation. A Myc-tagged PRAS40 wt as well
as the mutant PRAS40/T246A were expressed in NIH3T3 cells containing a
conditionally active Akt (mER-Akt). The cells were treated with 2.5 µM 4-hydroxytamoxifen for 20 min at 37 °C as
indicated, lysed, and precipitated with an anti-Myc tag antibody.
Precipitated proteins were analyzed by SDS-PAGE and Western blotting
with either anti-pPRAS40 antibody (a) or anti-Myc 9E10
antibody (b).
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Fig. 9.
Phosphorylation of PRAS40 is dependent on the
presence of Akt. Lysates of control or PDGF-treated (50 nM for 20 min at 37 °C) MEFs were either analyzed
directly or precipitated with either control antibody (lane
1) or anti-PRAS40 antibody (lanes 2-4), and the
precipitated proteins were analyzed by SDS-PAGE and blotting with
either anti-pAkt substrate antibody or anti-PRAS40 antibody, as
indicated. Phosphorylation of GSK-3 and Akt was examined in the same
experiment by immunoblotting total cell lysates with the respective
antibodies. The MEFs studied were either from mice with all three Akt
isoforms (WT), mice lacking Akt1 (1KO), mice
lacking Akt2 (2KO), or mice lacking both Akt1 and Akt2
(1,2KO).
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RESULTS AND DISCUSSION
CONCLUSIONS
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Dr. Andrey Shaw (Washington University) for the generous gift of 14-3-3 GST plasmid, Dr. Jesika Faridi for the cells expressing myr-Akt, Dr. Garry Nolan for the pWZL retroviral construct, and Dr. Chandra Kumar (Schering-Plough Institute) for a gift of baculovirus-produced Akt.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants DK34976 (to R. A. R.) and DK56886 (to M. J. 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.
§ Both authors contributed equally to this work.
To whom correspondence should be addressed. Tel.: 650-723-5933;
Fax: 650-723-2253; E-mail: rroth@stanford.edu.
Published, JBC Papers in Press, January 10, 2003, DOI 10.1074/jbc.M210837200
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ABBREVIATIONS |
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The abbreviations used are: PI, phosphatidylinositol; PRAS40, proline-rich Akt substrate of 40 kDa; GST, glutathione S-transferase; wt, wild type; PDGF, platelet-derived growth factor; PKB, protein kinase B; EST, expressed sequence tag; MEF, mouse embryonic fibroblasts; HA, hemagglutinin; GSK, glycogen synthase kinase; myr-Akt, myristoylated Akt; mer, myristoylated estrogen receptor.
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