Inhibition of Phosphatidylinositol 3-Kinase Activity by
Adenovirus-mediated Gene Transfer and Its Effect on Insulin
Action*
Prem M.
Sharma
,
Katsuya
Egawa
,
Yi
Huang
,
Jody L.
Martin
,
Ivana
Huvar§,
Gerry R.
Boss§, and
Jerrold M.
Olefsky
¶
**
From the
Department of Medicine, Division of
Endocrinology and Metabolism, the
Whittier Diabetes Institute,
and the § Department of Medicine, University of California,
San Diego, La Jolla, California 92093, the ¶ Veterans
Administration Research Service, San Diego, California 92161
 |
ABSTRACT |
Phosphatidylinositol 3-kinase (PI
3-K) is implicated in cellular events including glucose transport,
glycogen synthesis, and protein synthesis. It is activated in
insulin-stimulated cells by binding of the Src homology 2 (SH2) domains
in its 85-kDa regulatory subunit to insulin receptor substrate-1
(IRS-1), and, others. We have previously shown that IRS-1-associated PI
3-kinase activity is not essential for insulin-stimulated glucose
transport in 3T3-L1 adipocytes, and that alternate pathways exist in
these cells. We now show that adenovirus-mediated overexpression of the
p85N-SH2 domain in these cells behaves in a dominant-negative manner,
interfering with complex formation between endogenous PI 3-K and its
SH2 binding targets. This not only inhibited insulin-stimulated
IRS-1-associated PI 3-kinase activity, but also completely blocked
anti-phosphotyrosine-associated PI 3-kinase activity, which would
include the non-IRS-1-associated activity. This resulted in inhibition
of insulin-stimulated glucose transport, glycogen synthase activity and
DNA synthesis. Further, Ser/Thr phosphorylation of downstream molecules
Akt and p70 S6 kinase was inhibited. However, co-expression of a
membrane-targeted p110C with the p85N-SH2
protein rescued glucose transport, supporting our argument that the
p85N-SH2 protein specifically blocks insulin-mediated PI 3-kinase
activity, and, that the signaling pathways downstream of PI 3-kinase
are intact. Unexpectedly, GTP-bound Ras was elevated in the basal
state. Since p85 is known to interact with GTPase-activating protein in
3T3-L1 adipocytes, the overexpressed p85N-SH2 peptide could titrate out
cellular GTPase-activating protein by direct association, such that it
is unavailable to hydrolyze GTP-bound Ras. However, insulin-induced
mitogen-activated protein kinase phosphorylation was inhibited. Thus,
PI 3-kinase may be required for this action at a step independent of
and downstream of Ras. We conclude that, in 3T3-L1 adipocytes,
non-IRS-1-associated PI 3-kinase activity is crucial for insulin's
metabolic signaling, and that overexpressed p85N-SH2 protein inhibits a
variety of insulin's ultimate biological effects.
 |
INTRODUCTION |
Insulin binding to its cell surface receptors initiates diverse
metabolic and mitogenic signals by activation of a complex signaling
cascade of protein tyrosine and serine/threonine kinases, as well as
lipid kinases (1, 2). Phosphatidylinositol
(PI)1 3-kinase (PI 3-kinase),
a dual protein and lipid kinase is a heterodimeric enzyme composed of a
110-kDa catalytic subunit (p110) associated with an 85-kDa regulatory
subunit (p85). Two isoforms of the catalytic subunit (p110
and
p110
) and several isoforms of the regulatory subunit (p55
,
p55PIK, p85
, and p85
) have been cloned so far. The regulatory
subunit contains several well known functional domains: one Src
homology 3 (SH3) domain, homology to the breakpoint cluster region
(bcr) gene, two proline-rich motifs, and two Src homology region 2 (SH2) domains (3). The p85
isoform is insulin-responsive and
is predominantly expressed in 3T3-L1 adipocytes (4).
The insulin receptor is a tyrosine kinase, which, when activated by
insulin, phosphorylates cellular substrates such as IRS-1, IRS-2,
IRS-3, IRS-4 and the protein Shc, etc. (5-8). IRS-1 is the most well
characterized among members of the IRS family. Following insulin
stimulation, the phosphorylated YXXM motifs in IRS-1 binds to the SH2 domains of p85 stimulating the lipid kinase activity of the
p110 subunit. Through the same SH2 mechanism, PI 3-kinase can associate
with other proteins such as IRS-2-4, and the insulin receptor itself
(9, 10). PI 3-kinase phosphorylates phosphatidylinositol (PI),
phosphatidylinositol-4-monophosphate, and
phosphatidylinositol-4,5-bisphosphate on the D-3 position of the
inositol ring producing phosphatidylinositol 3-monophosphate, phosphatidylinositol 3,4-bisphosphate, and
phosphatidylinositol 3,4,5-triphosphate, respectively. Insulin induces
the production of D3-phosphorylated phosphoinositides through
stimulation of PI 3-kinase activity in various tissues and cultured
cells.
Insulin is a pleiotropic hormone that initiates a variety of biologic
effects through a complex set of signaling cascades. As a general view,
however, two main branches of the insulin signaling pathway can be
activated in most cell types: one is controlled predominantly by PI
3-kinase (1, 11) and the other by the small GTP-binding protein
p21ras (1). The major route by which insulin stimulates PI
3-kinase activity is through tyrosine phosphorylation of IRS-1, as
described above. Insulin stimulates Ras activity through a dual
mechanism involving Shc, and to a lesser extent, IRS-1, which
subsequently binds to the Grb2·SOS complex, activating p21ras
(12). Stimulated Ras then activates a cascade of protein
serine/threonine kinases which includes Raf, MEK, and the
mitogen-activated protein (MAP) kinases, ERK1 and ERK2 (reviewed in
Refs. 1, 5, and 6). Activation of both of these pathways appears to be
necessary for the mitogenic effects of insulin (12, 14, 15), whereas the metabolic effects of insulin are primarily activated by PI 3-kinase-dependent steps (16, 17). Thus, it has been shown that activated PI 3-kinase is both necessary and sufficient for insulin-stimulated GLUT4 translocation and glucose uptake (18-23), and
downstream targets of PI 3-kinase such as AKT and p70 S6 kinase may
play important roles in the ultimate metabolic actions of this hormone
(24, 25).
By no means are the PI 3-kinase and Ras/MAP kinase pathways completely
separate. For example, Yamauchi et al. (26) have demonstrated that PI 3-kinase may lie upstream of Ras and Raf in
mediating mitogenic effects of insulin. On the other hand, experiments
using wortmannin to inhibit PI 3-kinase activity have shown that the
effect of insulin on p21ras·GTP loading in CHO cells is
independent of PI 3-kinase (27). However, reciprocal relationships
between PI 3-kinase and p21ras have also been demonstrated
(28). Thus, one report has shown that activated Ras can stimulate PI
3-kinase activity, whereas other investigators have shown that the
biologic effects of constitutively activated PI 3-kinase are
Ras-dependent (29). Thus, although the exact relationship
between PI 3-kinase and p21ras is unclear, abundant data exist
to indicate that they are interconnected in some way (30).
In the current studies, we have extensively evaluated the role of PI
3-kinase activity in a diverse array of insulin actions. Our approach
was based upon the notion that cellular overexpression of the N-SH2
domain of the p85 subunit would disrupt complex formation between
endogenous PI 3-kinase and its SH2 domain binding targets, such as
IRS-1. This, in turn, would prevent insulin activation of PI 3-kinase
enzymatic activity, allowing us to elucidate the biologic actions of
this enzyme in insulin target cells such as 3T3-L1 adipocytes. Since
gene transfer into 3T3-L1 adipocytes can be problematic, we elected to
use recombinant adenovirus to achieve these goals. We (31), and others
(32), have recently demonstrated that adenovirus mediated gene delivery
allows high efficiency gene transduction and protein expression in
terminally differentiated 3T3-L1 adipocytes. Thus, we prepared a
recombinant adenovirus encoding the N-SH2 domain of the p85
subunit
of PI 3-kinase in order to overexpress this protein domain within
3T3-L1 adipocytes. Using this approach, we have investigated the acute effects of interrupting p85 association with insulin-stimulated target
proteins on the ultimate biologic effects of insulin.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Porcine insulin was kindly provided by Lilly.
Mouse monoclonal anti-phosphotyrosine (PY-20), mouse monoclonal
anti-ERK1, and the anti-Shc-SH2 antibodies were purchased from
Transduction Laboratories. Anti-IRS-1 and anti-human S6 kinase (p70S6K)
were from Upstate Biotechnology Inc. (UBI). Phosphospecific MAPK and
p70S6K antibodies were from New England Biolabs, Inc. The goat
polyclonal AKT (C-20) antibody of human origin, anti-Ras antibody
(Y13-259), and horseradish peroxidase-linked anti-rabbit, -mouse, and
-goat antibodies were purchased from Santa Cruz Biotechnology.
Dulbecco's modified Eagle's medium, fetal bovine serum, restriction
enzymes, and the random primer DNA labeling kit were purchased from
Life Technologies, Inc. . All radioisotopes were obtained from NEN Life
Science Products. Hoechst dye 33342 was obtained from Molecular Probes
(Eugene, OR). XAR-5 film was obtained from Eastman Kodak. All other
reagents and chemicals were purchased from Sigma.
Cell Culture--
3T3-L1 cells were grown and maintained in DMEM
high glucose containing 50 units/ml penicillin, 50 µg/ml
streptomycin, and 10% fetal bovine serum in a 10% CO2
environment. The cells were allowed to grow 2 days postconfluence, then
differentiated by addition of the same medium containing
isobutylmethylxanthine (500 µM), dexamethasone (25 µM), and insulin (4 µg/ml) for 3 days and the medium
containing insulin for 3 additional days. The medium was then changed
every 3 days until the cells were fully differentiated, typically by 10 days. Prior to experimentation, the adipocytes were trypsinized and
reseeded in the appropriate culture dishes. Rat 1 fibroblasts that had
been stably transfected and that overexpress wild type human insulin
receptor (HIRcB cells) were cultured as described previously (14). The
Ad-E1A-transformed human embryonic kidney cell line 293 was cultured in
Dulbecco's modified Eagle's medium containing 10% fetal bovine
serum.
Plasmid Construction--
The N-SH2 domain of P85
subunit of
bovine PI 3-Kinase were amplified by polymerase chain reaction (PCR)
using Pfu polymerase (Boehringer Mannheim, France) with the
following primers (5' to 3'), using full-length P85
subunit
as the template cDNA.
ctagtctagagccgccaccatgGTATGAATAACAATATGTCCTTA (primer 1, sense),
gccgaagcttCA*ATTCAGATCCTCTTCTGAGATGAGTTTTTGTTCATCTTCTTTGACAACTTGATCCT (primer 2, antisense), XbaI and HindIII;
the 5' and the 3' cloning sites are underlined. The PCR products
digested with XbaI and HindIII were cloned into
pACCMVpLpA (vector containing ampicillin selection marker), digested
with XbaI and HindIII. Kozak sequences including
a methionine initiation codon (indicated in lowercase, bold letters)
were inserted upstream of the 5' end of the sense (primer 1) PCR
primer. The antisense PCR primer 2 (written above in 5' to 3'
orientation) contained the Myc tag sequences (uppercase, bold),
downstream of the homologous sequences for the N-SH2 domain, followed
by a stop codon (*). The inserted fragment was in-frame with the
initiation codon. The plasmids pAC-p85N-SH2 (395 bp, including the
methionine and Myc) obtained by cloning the cDNA fragments was
amplified with the primer set 1 and 2. This plasmid encodes the
p85N-SH2 domain specific protein (encoding nucleotides 962-1320) of
the bovine p85
protein. The nucleotide sequence of the cloned
plasmid was verified by DNA sequence analysis using the 5' and the 3'
end specific primers around the cloning sites (XbaI and
HindIII) of the plasmid, pACCMVpLpA.
Preparation of Recombinant Adenovirus--
The recombinant
adenovirus containing the cDNA encoding the p85N-SH2 domain of
bovine p85
protein were isolated by homologous recombination using
two plasmids, pACCMVpLpA (33) and pJM17 (34). The recombinant plasmids
pAC-p85N-SH2 and pJM17 were purified and co-transfected into 293 cells.
Since 293 cells were originally derived from adenovirus transformation,
the missing E1 gene function of pJM17 is provided in trans.
The resulting recombinant virus containing the p85N-SH2 domain of
bovine p85
is denoted as Ad5-p85N-SH2, which is
replication-defective (at least in cells lacking the E1 region of
adenovirus), but fully infectious.
Detection of Recombinant Ad5-p85N-SH2 or Wild-type by
PCR--
DNA templates for PCR were extracted from the supernatant of
the culture medium (350 µl) of the 293 cells that were infected with
each plaque isolates/viruses at multiplicity of infection (m.o.i.) of
50 plaque-forming units (pfu)/ml. A multiplex PCR was performed on
one-tenth of virus DNA using E1A and E2B region specific primers (35)
and analyzed for the presence of recombinant or wild type adenoviruses.
The presence of p85N-SH2 domain specific cDNA inserts of bovine p85
in the recombinant virus was confirmed by PCR analysis of viral DNA
with p85N-SH2 cDNA specific primers. One clone of each of the
recombinant viruses were amplified further in 293 cells. Purification
by cesium chloride centrifugation resulted in higher titer stocks of
the recombinant viruses, typically 1-2 × 1010 pfu/ml
as determined by limiting dilution. All recombinant adenoviruses were
stored at
70 °C in phosphate-buffered saline (PBS) supplemented with 10% glycerol.
Cell Treatment--
HIRcB cells were transduced at a
multiplicity of infection of 1-20 pfu/cell for 1 h with stocks of
either a control recombinant adenovirus (Ad5-Ctrl/Ad5-CMV) containing
the cytomegalovirus promoter, pUC 18 polylinker, and a fragment of the
SV40 genome or the recombinant adenoviruses Ad5-p85N-SH2. 3T3-L1
adipocytes required either 10-fold more virus or longer infection time
(14-16 h) than HIRcB cells to achieve >90% infection in a transient
system. Transduced cells were incubated for 48-72 h at 37 °C in 5%
CO2 and DMEM with 2% heat-inactivated serum in order to
minimize cell division and to avoid a hormonal effect on gene
expression and/or on the activities of overexpressed proteins, followed
by incubation in the starvation media required for the assay. The
experiments were performed 60-96 h after the viral incubation and were
repeated three to five times. The efficiency of adenovirus mediated
gene transfer was approximately 90% as measured by histocytochemical
staining for
-galactosidase (
-gal) of the cells infected with a
recombinant adenovirus Ad5-lacZ, containing the bacterial
-galactosidase gene (data not shown). The survival of the
differentiated 3T3-L1 adipocytes was unaffected by incubation of cells
with the different adenovirus constructs since the total cell protein
remained the same in infected or uninfected cells. However, the HIRcB
cells demonstrated about 10% decrease in total cellular protein,
thereby indicating inhibition of cell cycle progression.
RNA Extraction and Northern Blot Analysis--
Total RNA was
isolated from the control (uninfected) and Ad5-p85N-SH2-infected cells
as described elsewhere (36). Total RNA (15 µg) was denatured and
electrophoresed on a 1% agarose, 6.6% formaldehyde gel and blotted
onto a Hybond-N+ membrane. A 360-bp cDNA fragment of
bovine p85N-SH2 domains was labeled with [
-32P]dCTP by
random priming. Northern blot was hybridized with the labeled probe and
washed as described previously (36). Ethidium bromide staining of the
electrophoresed RNA was used to visualize the loading pattern. The
autoradiograph was obtained after a 6-h exposure and analyzed with a
desktop scanner (Hewlett Packard Scan Jet II P), utilizing a scan
analysis program.
DNA Synthesis--
Cells were infected with the indicated
adenoviruses at m.o.i. of 2.0 and 10 for 1 h at room temperature,
serum-starved for 36 h, and stimulated with insulin (100 ng/ml) in
the presence of BrdUrd for 16 h at 37 °C. They were then fixed
and processed for immunofluorescence by sequential incubation with rat
anti-BrdUrd antibody and rhodamine-conjugated anti-donkey antibody.
Cells were counter stained with Hoechst stain to visualize nuclei.
BrdUrd-incorporated cells were identified by the nuclear red rhodamine
staining. An average of 200 cells per coverslip were counted. Results
are expressed as the percentages of stained cells exhibiting nuclear
fluorescence.
Phosphatidylinositol 3'-Kinase Activity--
In vitro
phosphorylation of phosphatidylinositol was carried out in immune
complexes as described previously (37). Cells were infected with
Ad5-p85N-SH2 or Ad5-CMV (control), or Ad5-lacZ (control) at the
indicated m.o.i. and time and grown in medium containing
heat-inactivated serum (2%) for 48 h. Serum-starved (16 h) cells
were incubated in the absence (basal) or presence of insulin (100 ng/ml) for 10 min, washed once with ice-cold PBS, lysed, and subjected
to immunoprecipitation (400-500 µg of total protein) with antibodies
to IRS-1 or to phosphotyrosine residues (2-4 µg), overnight at
4 °C. Immune complexes were precipitated from the supernatant with
protein A-Sepharose (Sigma) and washed as described (37). The washed
immune complexes were incubated with phosphatidylinositol (Avanti) and
[
-32P]ATP (3000 Ci/mmol) for 10 min at room
temperature. Reactions were stopped with 20 µl of 8 N HCl
and 160 µl of CHCl3:methanol (1:1) and centrifuged, and
the lower organic phase was removed and applied to a silica gel
thin-layer chromatography (TLC) plate (Merck) that had been coated with
1% potassium oxalate. TLC plates were developed in
CHCl3:CH3OH:H2O:NH4OH
(60:47:11.3:2), dried, and visualized, and quantitated on a Molecular
Dynamics PhosphorImager.
2-Deoxyglucose Transport--
The procedure for glucose
transport was a modification of the methods described by Klip et
al. (38). Differentiated 3T3-L1 adipocytes were infected with
Ad5-p85N-SH2 or Ad5-LacZ/Ad5-CMV (controls) at the indicated m.o.i. for
12 to 6 h at 37 °C and grown in medium containing
heat-inactivated serum (2%) for 48 h. Serum and glucose deprived
cells were incubated in
-MEM in the absence (basal) or presence of
100 ng/ml insulin for 1 h at 37 °C. In another set of
experiments, cells were incubated with 1, 10, and 100 ng/ml insulin for
1 h at 37 °C. Glucose uptake was determined in duplicate at
each point after the addition of 10 µl of substrate
([2-3H]deoxyglucose/L-[14C]glucose,
0.1 µCi, final concentration 0.01 mmol/liter) to provide a
concentration at which cell membrane transport is rate-limiting. The
value for L-glucose was subtracted to correct each sample for the contributions of diffusion and trapping. Data are
representative of three different observations and each value was
corrected for protein content.
Western Blotting--
Differentiated 3T3-L1 adipocytes
uninfected or infected with Ad5-p85N-SH2 or Ad5-CMV/Ad5-lacZ (negative
control) were starved for 24 h in serum-free Cellgro media. The
cells were stimulated with insulin (100 ng/ml or as indicated in the
figure legend) for 5-30 min at 37 °C and lysed in a solubilizing
buffer containing 30 mM Tris, 150 mM EDTA, 1%
Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM
Na3VO4, pH 7.4, for 20 min at 4 °C. The cell
lysates were centrifuged to remove insoluble materials. For Western
blot analysis, whole cell lysates (60-80 µg of protein/lane) from
the indicated cells were denatured by boiling in Laemmli sample buffer containing 100 mM dithiothreitol and resolved by SDS-PAGE.
Gels were transferred to Immobilon-P by electroblotting in Towbin
buffer containing 0.02% SDS and 20% methanol and immunoblotted with
the indicated antibody. Blots were then incubated with horseradish peroxidase-linked second antibody followed by chemiluminescence detection, according to the manufacturer's instructions (Pierce).
Glycogen Synthase Activity--
Glycogen synthase activity was
determined as described previously (39), with some modifications.
Confluent 3T3-L1 adipocytes (six-well culture dishes) were infected
with the indicated adenovirus as described above for 2-deoxyglucose
(2-DOG) assay, starved for 3 h in glucose and serum free DMEM
containing 0.1% bovine serum albumin and 2 mM sodium
pyruvate. Following this, the cells were either preincubated with 30 µM MEK inhibitor and stimulated with 200 ng/ml insulin
for additional 30 min in DMEM containing 0.1% bovine serum albumin, 5 mM glucose, 2 mM sodium pyruvate. The cells
were rinsed thrice with ice-cold PBS and then lysed in 150 µl of
homogenization buffer (50 mM Tris-HCl, 10 mM
EDTA, 100 mM KF (pH 7.4)) using a sonicator. Homogenates
were centrifuged at 4,000 rpm for 10 min at 4 °C. Thirty µl of
homogenate (equivalent to 10 µg of protein) was added to 60 µl of
preheated glycogen synthase assay buffer (10% oyster glycogen, 10 µM uridine 5'-diphosphoglucose, 50 mM
Tris-HCl, 25 mM Na2SO4, 50 mM NaF, 27 mM EDTA, 0.3 µCi of UDP-[14C]glucose per ml (pH 7.6) with and without 10 mM D-glucose 6-phosphate). The mixture was
incubated at 30 °C for 30 min. Seventy-five microliters of each
reaction mixture was applied to Whatman no. 4 filter paper. The filters
were air-dried and washed three times for 1 h each in 66%
ethanol. The final wash was with 100% acetone. Filters were dried and
placed in scintillation fluid for counting. Glycogen synthase
activation was calculated by subtracting glucose
6-phosphate-independent activity from glucose
6-phosphate-dependent activity.
Ras GTP/GDP Assay--
Differentiated 3T3-L1 adipocytes were
infected with Ad5-p85N-SH2, or Ad5-CT at 50 m.o.i. for 16 h
at 37 °C and grown in medium containing heat-inactivated serum (2%)
for 48 h. Following 24 h of serum starvation, the cells were
stimulated with or without insulin (100 ng/ml) for 10 min, washed with
PBS, scraped, and frozen at
70 °C immediately. Frozen cell pellets
were extracted in ice-cold RIPA buffer (50 mM Hepes, pH
7.4, 10 mM MgCl2, 150 mM NaCl, 1%
Nonidet P-40, 0.5 mM phenylmethylsulfonyl fluoride, and 10 µg/ml each of aprotinin, leupeptin, and pepstatin) by shaking for 5 min at 4 °C. The resulting cell extracts were centrifuged at
10,000 × g for 2 min. The supernatants were divided in
half and either 3 µg of the anti-Ras antibody Y13-259 (Santa Cruz
Biotechnology) or 3 µg of rat IgG (Cappel) were added. To both
samples, goat anti-rat IgG and protein G-agarose were added as well as
NaCl, sodium dodecyl sulfate (SDS), and deoxycholate to final
concentrations of 500 mM, 0.05%, and 0.5%, respectively.
The samples were shaken gently for 1 h at 4 °C and then the
immunoprecipitates were washed four times in RIPA buffer containing 500 mM NaCl, 0.05% SDS, and 0.5% deoxycholate and two times
in 20 mM TrisPO4, pH 7.4. The washed
immunoprecipitates were resuspended in 30 µl of 5 mM
TrisPO4, pH 7.4, 2 mM dithiothreitol, 2 mM EDTA (TED buffer), heated to 100 °C for 3 min, cooled
on ice, and centrifuged at 10,000 × g for 2 min. The
immunoprecipitates were washed with an additional 15 µl of TED
buffer, which was combined with the first 30 µl of TED buffer and GTP
and GDP were measured as described below.
GTP was converted to ATP using NDP kinase and ADP with the ATP measured
in the luciferase/luciferin system according to the following
reactions.
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PPi is pyrophosphate. This assay is sensitive to 1 fmol of GTP and was performed essentially as described previously (40). GDP was converted to GTP using pyruvate kinase and phosphoenolpyruvate with the GTP measured as described above:
Because the final product is again emitted light, this assay is
also sensitive to 1 fmol. The reaction mixture was incubated for 30 min
at 30 °C and contained in a final volume of 15 µl of 50 mM glycine, pH 7.8, 10 mM dithiothreitol, 8 mM MgSO4, 50 µM phosphoenolpyruvate, 3 milliunits of pyruvate kinase, and 5 µl of
sample or GDP standard. It should be noted that this assay measures the
sum of GTP + GDP; thus, the amount of GTP in the sample must be
subtracted from the amount of GTP + GDP to yield the amount of GDP. DNA
was measured by a standard fluorescence method using the fluorescent
dye bisbenzimidazole, and protein was measured by the Bradford method.
The amounts of GDP and GTP in the samples are determined from standard
curves prepared with each set of samples and the data are expressed as
femtomoles of GTP or GDP per microgram DNA or milligram protein in the
cell lysate (40).
 |
RESULTS |
Detection of Recombinant Ad5-p85N-SH2 or Wild-type Virus by
Multiplex PCR Analysis--
Multiplex PCR amplification of the viral
DNA was performed using E1A and E2B specific primers as described
previously (31). The E1A DNA (indicative of wild-type virus) was not
detected from any plaque-purified recombinant Ad5-p85N-SH2 (Fig.
1A, top panel, lanes 2-6), but was specifically detected when DNA from the
wild-type infected cell cultures was used as a template (Fig.
1A, lane 7). These results indicate that the
recombinants were free of wild-type virus contamination. To further
confirm the presence of recombinant cDNAs in E2B PCR positive
clones, the Ad5-p85N-SH2 virus DNAs were PCR amplified with specific
primers derived from the Ad5 polylinker (5' and 3' of the cloning site)
and with cDNA specific primers named 1 and 2, respectively (see
"Experimental Procedures"). A 386-bp cDNA fragment was detected
from the p85N-SH2 domain clones (Fig. 1B, bottom
panel, lanes 2-6), indicating the presence of recombinant DNA. No PCR product was detected in either the control or
the wild-type infected cell culture DNA (Fig. 1B,
lanes 1 and 7, respectively).

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Fig. 1.
Detection of recombinant p85N-SH2 or
wild-type virus in cell culture medium by PCR amplification of the
viral DNA. DNA templates for PCR were extracted from the
supernatant of the culture medium (350 µl) of the 293 cells that were
infected with each plaque isolates/viruses (clones 1-5) at an m.o.i.
of 50 pfu/ml. A (top panel), a multiplex PCR was
performed on a tenth of virus DNA using E1A and E2B region specific
primers. The E1A DNA (indicative of wild-type virus) was not detected
from the five plaque-purified recombinant Ad5 p85N-SH2 (lanes
2-6) but was specifically detected when the DNA template from the
wild-type infected cell culture was used as a template (lane
7). These results indicate that the recombinants were free of
wild-type virus contamination. B (bottom panel),
the recombinant Ad5 p85N-SH2 virus DNA (clones 1-5) were PCR-amplified
with cDNA specific primers. A 386-bp fragment was detected in all
the clones containing the recombinant Ad5 p85N-SH2 (lanes
2-6) but was not detected in the wild-type infected cell culture
DNA (lane 7).
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Northern and Western Blot Analysis of Overexpressed p85N-SH2
mRNAs and Proteins--
Northern blot analysis was used to confirm
the expression of p85N-SH2 mRNAs in 293 cells (cells used for
isolation of the recombinant adenoviruses) and HIRcB cells following
infection at an m.o.i. of 1 and 10 pfu/cell of Ad5-p85N-SH2 (clones 2 and 4) adenovirus. The filter was hybridized with a 386-bp cDNA
fragment of p85N-SH2 as a probe. Densitometric scanning of the blot
revealed that treatment of the HIRcB cells (Fig.
2A, lanes 2 and
3) and 293 (lanes 3 and 4) cells with
Ad5-p85N-SH2 resulted in a 20-100-fold increase in mRNA levels of
the p85N-SH2 domain of PI 3-kinase relative to the uninfected cells
(lane 1).

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Fig. 2.
A, Northern blot analysis of
overexpression of p85N-SH2 mRNA in 293 and HIRcB cells. Total RNA
(15 µg) isolated from control (uninfected) and Ad5 p85N-SH2-infected
293 and HIRcB cells was electrophoresed on a 1% agarose, 6.6%
formaldehyde gel and blotted onto a Hybond-N+ membrane. The
filter was hybridized with a 386-bp fragment of p85N-SH2 cDNA as a
probe. The autoradiograph was obtained after a 6-h exposure.
Ctrl, control. B, expression of p85N-SH2 protein
in 3T3-L1 adipocytes. Cells were infected with the p85N-SH2 expressing
adenovirus at 10 and 50 m.o.i. for 12-16 h. After 3 days, 60 µg
of protein were analyzed for expression by Western blotting using a
monoclonal anti-Myc antibody. Immunoreactivity corresponding to the Myc
epitope tag at the N terminus of the recombinant p85N-SH2 is indicated
by arrow on the right side. The results shown are
representative of several independent experiments on cells from
separate infections.
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The differentiated 3T3-L1 adipocytes were infected with the same clones
of the p85N-SH2 expressing adenovirus at the indicated m.o.i. for
16 h and protein expression was examined 48 h later by
Western blotting. Fig. 2B shows the results of an anti-Myc blot. Specific bands appeared at 18-20 kDa for the Myc-tagged p85N-SH2
protein in a viral dose-dependent manner.
Studies of HIRcB Fibroblasts: IRS-1-associated PI 3-Kinase
Activity--
It is well established that tyrosine phosphorylated
IRS-1 associates with and activates PI 3-kinase (1, 5, 6). Therefore, we measured IRS-1-associated PI 3-kinase activity in
p85N-SH2-overexpressing HIRcB cells. In theory, the p85-derived N-SH2
domain should competitively inhibit interactions between the SH2 domain
of endogenous p85 and its protein partners. To assess this, whole cell
lysates from insulin-treated HIRcB fibroblasts overexpressing p85N-SH2
proteins were subjected to immunoprecipitation with anti IRS-1 antibody and PI 3-kinase activity was determined in the precipitates. In control
cells, insulin stimulation resulted in a 25-30-fold increase in
IRS-1-associated PI 3-kinase (Fig. 3) and
this effect was unchanged in Ad5-lacZ- or Ad5-CMV-infected cells (data
not shown). Overexpression of increasing amounts of the p85N-SH2 domain
led to a dose-dependent inhibition of IRS-1-associated PI
3-kinase activity (Fig. 3). These results are consistent with the
hypothesis that the overexpressed p85N-SH2 protein behaves in a
dominant negative manner, interfering with the interactions of
endogenous p85 with phosphorylated IRS-1.

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Fig. 3.
Effects of Ad5p85N-SH2 overexpression on
insulin-stimulated PI3-kinase activity in HIRcB cells. Cells were
infected with Ad5p85N-SH2 for 1 h at room temperature and grown in
medium containing heat-inactivated serum (2%) for 48 h.
Serum-starved (16 h) cells were incubated in the absence (basal) or
presence of insulin (100 ng/ml) for 10 min, lysed, and subjected to
immunoprecipitation with antibodies to IRS-1. The washed
immunoprecipitates were assayed for PI 3-kinase activity with
phosphatidylinositol as substrate, and the labeled PI 3-phosphate
product (PI-3P) was resolved by thin-layer chromatography
and visualized by autoradiography. This experiment was repeated four
times with appropriate controls.
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Effect of p85N-SH2 Domain on Insulin-stimulated DNA
Synthesis--
To evaluate a more distal biological effect,
insulin-stimulated cell cycle progression was measured by monitoring
BrdUrd incorporation into newly synthesized DNA. Cells were plated on
coverslips and infected with Ad5-p85N-SH2, and Ad5-
-gal at m.o.i. of
2.0 and 10 pfu/cell, serum-starved for 36 h and then stimulated
with insulin. As seen in Fig. 4, 45%
of the uninfected cells underwent DNA synthesis in response to insulin
and serum, respectively, and this response was not altered by
Ad5-
-gal infection. In contrast, overexpression of the p85N-SH2
domain completely inhibited insulin-stimulated DNA synthesis, showing
that p85N-SH2 behaves in a dominant/negative fashion with respect to
insulin-stimulated mitogenesis.

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Fig. 4.
Inhibition of DNA synthesis by overexpression
of p85N-SH2 protein following infection with Ad5p85N-SH2 into HIRcB
cells. Cells were infected with the indicated adenoviruses at
increasing m.o.i. for 1 h at RT, serum-starved for 36 h, and
stimulated with insulin (100 ng/ml) in the presence of BrdUrd for
16 h at 37 °C. They were then fixed and processed for
immunofluorescence by sequential incubation with rat anti-BrdUrd
antibody and rhodamine-conjugated anti-donkey antibody. Cells were
counterstained with Hoechst stain to visualize nuclei.
BrdUrd-incorporated cells were identified by the nuclear red rhodamine
staining. An average of 200 cells per coverslip were counted. Results
are expressed as the percentages ± S.E. of stained cells
exhibiting nuclear fluorescence from three different experiments.
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Studies in 3T3-L1 Adipocytes--
Effects of Overexpression of
p85N-SH2 Domain on Insulin-induced PI 3-kinase Activation. 3T3-L1
adipocytes provide an excellent in vitro model system to
study the metabolic actions of insulin. As seen in Fig. 2, these cells
can be readily infected with adenoviral vectors, allowing us to examine
the effect of p85N-SH2 domain expression in these cells. No differences
in the extent of insulin-induced tyrosine phosphorylation of the
insulin receptor or IRS-1 were detected in cell lysates from uninfected
3T3-L1 adipocytes or those expressing p85N-SH2 domain (data not shown).
However, insulin-stimulated PI 3-kinase activity in anti-IRS-1
immunoprecipitates was markedly attenuated in 3T3-L1 adipocytes
expressing p85N-SH2 domain compared with control or noninfected cells
(Fig. 5A). Thus, in control cells, insulin led to a 10-15-fold increase in IRS-1-associated PI
3-kinase activity, which was markedly inhibited by expression of the
p85N-SH2 domain in a dose-dependent manner. At 50 m.o.i., the insulin-induced PI 3-kinase activity was almost completely abolished. In contrast, infection of the 3T3-L1 adipocytes with Ad5-CMV
was without effect. Pretreatment of cells with 100 nM wortmannin for 30 min prior to insulin stimulation resulted in ~80%
reduction in IRS-1-associated PI 3-kinase activity (data not
shown).

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Fig. 5.
A, effects of Ad5p85N-SH2 overexpression
on insulin-stimulated PI3-kinase activity in 3T3-L1 adipocytes. Cells
were infected with Ad5p85N-SH2 for 16 h at 37 °C and grown in
medium containing heat-inactivated serum (2%) for 48 h.
Serum-starved (16 h) cells were incubated in the absence (basal) or
presence of insulin (100 ng/ml) for 10 min, lysed, and subjected to
immunoprecipitation with antibodies to IRS-1. The washed
immunoprecipitates were assayed for PI 3-kinase activity with
phosphatidylinositol as substrate, and the labeled PI 3-phosphate
product (PI-3P) was resolved by thin-layer chromatography
and visualized by autoradiography. This experiment was repeated four
times with appropriate controls. Ctrl, control.
B, cell lysates from 3T3-L1 adipocytes infected with control
Ad5 or the p85N-SH2 expressing adenovirus, stimulated with or without
insulin were subjected to immunoprecipitation with anti-phosphotyrosine
antibody. The washed immunoprecipitates were assayed for PI 3-kinase
activity as described above. Ctrl, control.
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We also assessed PI3-K activity associated with phosphotyrosine
residues in anti-phosphotyrosine immunoprecipitates from control and
p85N-SH2-infected 3T3-L1 adipocytes in the presence and absence of
insulin. In control cells, insulin stimulation resulted in ~15-fold
increase in phosphotyrosine-associated PI3-K activity, which was
completely inhibited by expression of the p85N-SH2 domain (Fig.
5B).
p21ras·GTP Loading--
Since the relationship
between PI 3-kinase and Ras is a matter of debate, we examined the
effect of PI 3-kinase inhibition on the insulin-induced stimulation of
p21ras in 3T3-L1 adipocytes. Stimulation of 3T3-L1 adipocytes,
infected with a control adenovirus (Ad5-CMV) with 100 ng/ml insulin for 10 min resulted in a 2-3-fold stimulation of p21ras activation
(Fig. 6). Surprisingly, unstimulated
cells overexpressing the p85N-SH2 protein showed a 2-3-fold higher,
basal p21ras·GTP activity, which is similar to the
insulin-induced state. Stimulation of these cells with insulin for 10 min did not have any further effect on p21ras·GTP
loading.

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Fig. 6.
Effects of overexpression of the p85N-SH2
protein on Ras activation in 3T3-L1 adipocytes. Cells were
infected with Ad5-Ctrl and Ad5-p85N-SH2 expressing adenoviruses at an
m.o.i. of 50/cell in medium containing heat-inactivated serum (2%) for
12-16 h. Following infection, cells were serum-starved (16 h) and were
incubated in the absence (basal) or presence of insulin (100 ng/ml) for
5 min, lysed. Cell lysated were assayed for GTP- and GDP-bound Ras as
described under "Experimental Procedures." Data are expressed as
femtomoles of guanine nucleotide per mg of cellular DNA and are the
mean ± S.E. of three independent experiments. Ctrl,
control.
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Insulin-stimulated Activation of MAPK--
To investigate whether
PI 3-kinase is required for the induction of MAPK activity, or
alternatively, whether it plays an indirect role in regulating MAPK
activation through its effect on Ras-GTP levels, we determined the
activation state of MAPK in uninfected, control, or p85N-SH2-infected
3T3-L1 adipocytes in the presence and absence of insulin. Cell lysates
were subjected to Western blot analysis with phosphospecific MAPK
antibodies. This antibody detects the tyrosine-phosphorylated (position
204) activated form of p44 and p42 (ERK1 and ERK2) MAPK. In uninfected
cells, insulin stimulated phosphorylation of MAPK by about 20-fold
(Fig. 7A, lane 2).
Overexpression of the p85N-SH2 domain led to a
dose-dependent inhibition of both p44 and p42 MAPK
phosphorylation and activation (Fig. 7A, lanes
3-6). Expression of MAPK was assessed by Western blotting with a
polyclonal MAPK antiserum that recognizes both nonphosphorylated and
phosphorylated forms and protein levels were not altered by infection
with either the p85N-SH2 domain or Ctrl adenoviruses (Fig.
7B, lanes 1-6).

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Fig. 7.
Effects of overexpression of the p85N-SH2
domain on insulin-stimulated MAPK phosphorylation and expression in
3T3-L1 adipocytes. Cells were infected with Ad5-p85N-SH2
expressing adenovirus at an m.o.i. of 10 and 50/cell in medium
containing heat-inactivated serum (2%) for 12-16 h. Following
infection, cells were serum-starved (16 h) and were incubated in the
absence (basal) or presence of insulin (100 ng/ml) for 5 min, lysed,
and, were subjected to SDS-PAGE and immunoblotted with phosphospecific
MAPK antibody. Insulin stimulated phosphorylation of both p44 and p42
MAPKs as indicated (A). Phosphorylation of both p44 and p42
MAPKs were inhibited by overexpression of p85N-SH2 protein in a
dose-dependent manner. This experiment was repeated twice.
To detect the levels of expression of p44 and p42 MAPKs, the above
filter was stripped and reblotted with anti-ERK1 antibody. There was no
significant difference in the levels of expression between uninfected
and infected 3T3-L1 adipocytes (B).
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Insulin-stimulated Activation of p70S6K and AKT--
It has been
shown that p70S6K is downstream of PI 3-kinase, and activation of this
enzyme was determined by Western blot analysis of whole cell lysates
with a phosphospecific p70S6K antibody that detects p70S6K only when
phosphorylated at Thr-421/Ser-424 and does not cross-react with other
phosphorylated protein kinases. As shown in Fig.
8A (upper panel,
lane 2), stimulation of 3T3-L1 adipocytes with insulin
resulted in activation of p70 S6 kinase, which was inhibited by
pretreatment with rapamycin (Fig. 8A, upper panel, lane 1). The p85N-SH2-overexpressing cells
demonstrate a 60-90% inhibition of insulin-induced p70S6K activation
at 10 and 50 m.o.i, respectively (Fig. 8A, upper
panel, lanes 5 and 7). Similar results were
obtained when p70S6K activation was assessed by retarded migration on
SDS-PAGE using anti p70S6K antibody. As shown in Fig. 8A
(lower panel, lane 2), stimulation of cells with
insulin resulted in activation of p70S6K, which was blocked by p85N-SH2
expression (Fig. 8A, lower panel, lanes 5 and
7).

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Fig. 8.
A, effects of overexpression of the
Ad5-p85N-SH2 protein on insulin-stimulated p70S6K activation in 3T3-L1
adipocytes. Cells were infected with Ad5-p85N-SH2 expressing adenovirus
at 10 and 50 m.o.i. for 12-16 h. Serum-starved (16 h) cells were
incubated in the absence (basal) or presence of insulin (100 ng/ml) for
30 min, lysed, and subjected to SDS-PAGE and immunoblotted with
phospho-specific p70 S6 kinase antibody (upper panel). The
above filter was stripped and reblotted with p70 S6 kinase antibody.
Insulin stimulation of serine phosphorylation of p70 S6 kinase is
detected by a retarded migration of the enzyme (lower panel,
lane 2). Preincubation with 100 nM rapamycin for
30 min inhibited activation of p70 S6 kinase (lower panel, lane
1). Similarly, overexpression of PTB and SAIN proteins inhibited
p70 S6 kinase activation in a dose-dependent manner
(lower panel, lanes 4-7). This experiment was repeated
twice. B, effects of overexpression of Ad5-p85N-SH2 protein
on insulin-stimulated AKT activation in 3T3-L1 adipocytes. Whole cell
lysates (60 µg) were prepared as described in A, were
subjected to SDS-PAGE and immunoblotted with AKT antibody. Insulin
stimulation of serine phosphorylation of AKT is detected by a retarded
migration of the enzyme (lane 2). Preincubation with 100 nM wortmannin for 30 min inhibited activation of of AKT
(lane 1). Overexpression of the p85N-SH2 protein
(lanes 4-7) inhibited insulin-induced AKT activation
(lanes 5 and 7). This experiment was repeated
three times. Ctrl, control.
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AKT (PKB) is also activated by insulin in a PI
3-kinase-dependent manner, and we assessed the degree of
AKT activation in cell lysates, derived from insulin-stimulated 3T3-L1
cells as determined by retarded migration on SDS-PAGE (Fig.
8B, lane 2). Insulin treatment stimulated an AKT
mobility shift, which was inhibited by wortmannin or p85N-SH2
expression (Fig. 8B, lanes 1, 5, and
7, respectively). These findings suggest that
IRS-1-associated PI 3-kinase activity is necessary for insulin
stimulation of p70S6K and for AKT activation.
Insulin-stimulated Glucose Transport--
Cells were infected with
Ad5-p85N-SH2, or Ad5-CMV and 2-DOG uptake was measured 48 h later.
As seen in Fig. 9A, insulin
led to an 8-fold increase in 2-DOG uptake, and this was not affected by
Ad5-CMV infection. p85N-SH2 expression at 20 and 50 m.o.i. markedly inhibited insulin-stimulated 2-DOG uptake. Expression of the
p85N-SH2 also led to a slight decline in the basal 2-DOG. Viral
mediated expression of the unrelated Shc SH2 domain had no effect on
basal or insulin-stimulated glucose uptake (data not shown). To further
evaluate this finding, additional experiment was performed in which
insulin dose-response studies were carried out in control,
Ad5-p85N-SH2-, and Ad5-CMV-infected cells. As shown in Fig.
9B, insulin-mediated glucose transport was inhibited by
p85N-SH2, while control adenovirus was without effect.

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Fig. 9.
A, inhibition of 2-deoxyglucose
transport in 3T3L1 adipocytes infected with increasing m.o.i. of
adenovirus expressing p85N-SH2. Differentiated 3T3-L1 adipocytes were
infected with Ad5-p85N-SH2 or Ad5-ctrl at the indicated m.o.i. for
16 h at 37 °C and grown in medium containing heat-inactivated
serum (2%) for 48 h. Serum- and glucose-deprived cells were
incubated in -MEM in the absence (basal) or presence of 100 ng/ml
insulin for 1 h at 37 °C. Cells were then washed with
glucose-free media, and 2-deoxyglucose uptake was measured. Each
measurement was performed in duplicate. Infection of differentiated
3T3-L1 adipocytes with Ad5-p85N-SH2 resulted in a viral
dose-dependent inhibition of insulin-stimulated percent
incorporation of 2-deoxyglucose transport. Control Ad5 virus had no
effect on glucose transport. Data are representative of four different
observations ± S.E., and each value was corrected for protein.
B, inhibition of 2-deoxyglucose transport in 3T3L1
adipocytes as a function of insulin concentration. Differentiated
3T3-L1 adipocytes were infected with 50 m.o.i. of Ad5-p85N-SH2 for
16 h at 37 °C and grown in medium containing heat-inactivated
serum (2%) for 48 h. Serum- and glucose-deprived cells were
incubated in -MEM in the absence (basal) or presence of 1, 10, and
100 ng/ml insulin for 1 h at 37 °C. Cells were then washed in
glucose-free media, and 2-deoxyglucose uptake was measured. Each
measurement was performed in duplicate. Insulin stimulated percent
incorporation of 2-deoxyglucose in 3T3-L1 adipocytes in a
dose-dependent manner, which was inhibited by
overexpression of the p85N-SH2 protein. Control Ad5 virus had no effect
on glucose transport. Data are representative of four different
observations. C, effect of co-expression of p110C and p85N-SH2 expressing adenoviruses
on 2-deoxyglucose transport in 3T3L1 adipocytes. Differentiated 3T3-L1
adipocytes were infected with 20 and 60 m.o.i. of Ad5-p85N-SH2 and
Ad5-p110C (a membrane-targeted, p110 subunit of
PI 3-kinase), respectively, either individually or in combination, for
16 h at 37 °C and grown in medium containing heat-inactivated
serum (2%) for 48 h. Serum- and glucose-deprived cells were
incubated in -MEM in the absence (basal) or presence of 100 ng/ml
insulin for 1 h at 37 °C. Cells were then washed in
glucose-free media, and 2-deoxyglucose uptake was measured. Each
measurement was performed in duplicate. Data are representative of
three different observations ± S.E.
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Co-expression of p110CAAX Rescues 2-DOG Uptake
Inhibited by the p85N-SH2 Protein--
The effect of coexpression of
p85N-SH2 domain and a membrane-targeted, p110 subunit of PI 3-kinase
(p110C) on 2-deoxyglucose uptake in 3T3-L1
adipocytes is shown in Fig. 9C. Insulin stimulated glucose
transport about 8-fold in uninfected cells (Fig. 9C).
Overexpression of a membrane-targeted (by C-terminal prenylation), p110
subunit of PI 3-kinase
(p110C)2
stimulated glucose transport to the same extent as insulin, and addition of insulin had no further effect. The p85N-SH2 blocks insulin
induced 2DOG uptake; however, co-expression of
p110C with p85N-SH2 rescued 2-DOG uptake by
~90% without any added insulin.
Glycogen Synthase Activity--
Fig.
10 shows glycogen synthase activity in
differentiated 3T3-L1 adipocytes. Glucose 6-phosphate-independent
activity is expressed as a percentage of total glycogen synthase
activity. In both nontransfected (not shown) and Ad5-CMV
(control)-infected cells, insulin stimulated glycogen synthase activity
~3-fold. Preincubation of cells with 30 µM MEK
inhibitor (PD 98059) had no effect on insulin induced glycogen synthase
activity. The p85N-SH2 inhibited glycogen synthase activity by about
50%. These data suggest that activation of PI 3-kinase is necessary
for activation of glycogen synthase, whereas MAPK does not appear to be
involved.

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Fig. 10.
Effect of overexpression of p85N-SH2 protein
on glycogen synthase activity in 3T3-L1 adipocytes. Differentiated
3T3-L1 adipocytes were infected with 50 m.o.i. of Ad5-Ctrl and
Ad5-p85N-SH2 for 16 h at 37 °C and grown in medium containing
heat-inactivated serum (2%) for 72 h. The cells were glucose- and
serum-starved in DMEM containing 0.1% bovine serum albumin, 2 mM sodium pyruvate for 2.5 h, then incubated with or
without 50 µM MEK inhibitor for 30 min, following which
cells were stimulated with 200 ng/ml insulin for 30 min in 5 mM glucose-containing medium. After which the cells were
scraped, sonicated, and centrifuged. The ability of the supernatant to
stimulate incorporation of UDP-glucose into glycogen was determined in
the presence and absence of glucose 6-phosphate (G-6-P).
Results are expressed as mean ± S.E. of percentage of glycogen
synthase index (% GSI) from three independent experiments.
Ctrl, control.
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DISCUSSION |
Insulin stimulation leads to extensive tyrosine phosphorylation of
IRS-1. Phosphorylated IRS-1 binds to the SH2 domains of the p85 subunit
of PI 3-kinase and activates its p110 subunit, and this is a major
mechanism by which insulin stimulates this enzyme. Several lines of
evidence suggest that PI 3-kinase is involved in mediating mitogenic
effects of various growth factors (2, 5, 42) and is a necessary
molecule for insulin stimulation of glucose transport (18-23). Despite
a great deal of information on both the structure and function of PI
3-kinase, the precise mechanism of its involvement in mediating insulin
signaling is not fully understood.
In the current study, we have extensively evaluated the role of PI
3-kinase activity in a diverse array of insulin actions. Our approach
is based on the hypothesis that cellular overexpression of the N-SH2
domain of the p85 subunit would competitively inhibit complex formation
between endogenous PI 3-kinase and its SH2 domain binding targets such
as IRS-1, -2, -3, -4, the insulin receptor, and others. This in turn,
will prevent insulin activation of PI 3-kinase enzymatic activity,
allowing us to elucidate the biologic actions of this enzyme in insulin
target cells such as 3T3-L1 adipocytes. Indeed our data show not only a
marked inhibition of insulin-stimulated IRS-1-associated PI 3-kinase
activity, but we find a complete blockade of insulin-stimulated
anti-phosphotyrosine-associated PI 3-kinase activity, which would
include the non-IRS-1-associated activity. Predictably, and in
agreement with previous findings, inhibition of PI 3-kinase activity
resulted in abrogation of insulin's effects on glucose uptake and DNA
synthesis (12-17). Other major findings of these experiments are that
inhibition of PI 3-kinase activity resulted in inhibition of glycogen
synthase activity and stimulation of MAPK activation by insulin. In
contrast, overexpression of the p85N-SH2 protein led to a 2-fold
increase in basal, insulin-independent GTP-bound levels of
p21ras. However, insulin treatment did not further alter the
levels of GTP-bound p21ras in these cells. Inhibition of the PI
3-kinase activity, also led to significant impairment of other
downstream signaling effects of insulin such as Akt and p70 S6 kinase
phosphorylation. These results lead to several predictions and
conclusions.
The activation of MAPK by insulin depends on activation of Ras (1, 13).
Tyrosine-phosphorylated Shc (12), and to a lesser extent IRS-1 (1, 5,
6), binds to the adaptor protein, Grb-2, which is preassociated with
the guanine nucleotide exchange factor, Sos, that promotes the
formation of the active GTP-bound state of Ras. Formation of GTP-bound
Ras leads to activation of the protein serine/threonine kinase cascade
including Raf-1 kinase, MEK and MAPK (1, 13). Recent studies using
specific inhibitors of PI 3-kinase or constitutively active and
dominant negative mutants of the enzyme, have yielded confusing, and
potentially conflicting results suggesting that PI 3-kinase can be
either downstream or upstream of Ras (26-30). In the present study,
using 3T3-L1 adipocytes, we have demonstrated that inhibition of PI 3-kinase decreased insulin-induced activation of MAPK, although the
levels of GTP-bound Ras were increased ~2-fold in the basal state.
Thus, PI 3-kinase may be required for the activation of MAPK at a step
independent of and downstream of Ras.
The importance of PI 3-kinase for activation of ERKs 1 and 2 has been
controversial. Expression of activated forms of p110
has been
reported to stimulate the MAPK pathway in one case (29), but not in
others (32, 43-45). A recent study found that overexpression of the
p110
isoform resulted in activation of ERKs 1 and 2, whereas p110
was without effect (45). Our findings indicating the involvement of PI
3-kinase in the activation of MAPK seem to be consistent with a recent
report showing that treatment of 3T3-L1 adipocytes, either with
wortmannin or LY294002, inhibited insulin-induced activation of Raf-1
and MAPKs with no effect on the formation of GTP-bound Ras (46).
However, wortmannin did not affect epidermal growth factor-induced
activation of Raf and MAPK in the same system, indicating that
differential mechanisms to activate Raf-1 and MAPKs by insulin and
epidermal growth factor exist in 3T3-L1 adipocytes. Very similar
observations have been reported using rat adipocytes (47). However, the
involvement of PI 3-kinase in MAPK activation appears to be cell
type-specific, because wortmannin did not inhibit insulin-induced
activation of MAPK in CHO cells in the above study (46).
As mentioned earlier, the relationship between PI 3-kinase and
Ras is a matter of debate. Some observations indicate that PI 3-kinase
could be upstream, downstream, or independent of Ras; these
observations are perhaps related to cell-type differences (reviewed in
Ref. 48). Our data reinforce the idea that PI 3-kinase is upstream and
can activate the Ras pathway. However, our unexpected findings were the
influence of overexpression of the p85N-SH2 domain of PI 3-kinase to
stimulate formation of p21ras·GTP in 3T3-L1 adipocytes. PI
3-kinase and Ras form a complex, suggesting an intimate relation
between the function of these molecules (28), and it can be argued that
a loss of PI 3-kinase activity could disrupt basal Ras-p110 binding,
which may be required for PI 3-kinase signaling.
It is known that insulin stimulates p21ras·GTP loading by
increasing the guanine nucleotide exchange activity of Sos (49, 50). Return of p21ras into its inactive conformation is facilitated
by GAP that hydrolyzes GTP to GDP. Therefore, overexpression of the
p85N-SH2 domain of PI 3-kinase can stimulate formation of
p21ras·GTP either by stimulating the guanine exchange
activity of Sos or by diminishing the activity of GAP. Stimulation of
Sos activity is unlikely because Langolis et al. (51) have
shown that immunodepletion of PI 3-kinase from lysates of
insulin-stimulated cells does not alter the activity level of the
guanine nucleotide exchange factor Sos. Therefore, the most likely
explanation for the increase in basal GTP-bound Ras in the p85N-SH2
expressing cells is that the p85N-SH2 depletes the pool of GAP in these
cells. Indeed, in 3T3-L1 adipocytes, the p85 subunit of PI 3-kinase is
already in excess compared with the p110 subunit, and it has been shown
to interact with GAP (presumably via p62, a GAP-associated protein)
(52). Overexpression of the p85N-SH2 peptide could titrate out cellular GAP by direct association, such that GAP is not available to hydrolyze p21ras·GTP. The elevated p21ras·GTP levels do not
appear to affect MAPK activity, since we find that the p85N-SH2 protein
inhibits insulin-induced MAPK phosphorylation in 3T3-L1 adipocytes.
Therefore, the inhibitory effect of PI 3-kinase on the MAPK cascade
most likely occurs at a level independent of Ras. Therefore, in these
cells, insulin is able to stimulate MAPK via a Ras-independent pathway.
These results are in agreement with the observations of Carel et
al. (53), who also found that insulin activates MAPK by a
Ras-independent pathway in 3T3-L1 adipocytes and by a
Ras-dependent pathway in 3T3-L1 fibroblasts.
In an earlier report, we utilized an adenovirus system to express the
PTB domain of IRS-1 in 3T3-L1 adipocytes. This led to a marked decrease
in IRS-1-associated PI 3-kinase activity, but no inhibition of
insulin-stimulated glucose transport (31). The current study further
extends our earlier proposed hypothesis that complete blockade of PI
3-kinase activity (i.e. IRS-1 and the non-IRS-1-associated
components) would be necessary to inhibit insulin-induced glucose
transport. Indeed, we find that this is the case from these results. As
shown in Fig. 5B, the dominant negative p85 completely
blocks PI 3-kinase activity in the phosphotyrosine immunoprecipitates.
The number of insulin receptor substrates is expanding rapidly, with
the addition of IRS-2, -3, and -4 to the earlier insulin-substrates:
IRS-1 and Shc. Among these, IRS-2 and IRS-3 (pp60) are tyrosine
phosphorylated by insulin and bind to PI 3-kinase (7, 54). It is
possible that insulin stimulates glucose transport by at least two
parallel pathways, which may be interacting or redundant. The current
data support the notion that inhibition of glucose transport results
from blockade of a yet to be identified, non-IRS-1-associated PI
3-kinase activity. Consistent with this, studies by Kaburagi et
al. (16) on fat cells derived from IRS-1 knockout mice, and in
which IRS-2 is negligible, show only a 50% reduction in maximal
insulin-stimulated glucose transport, indicating the existence of an
IRS-1/2-independent, but PI 3-kinase-dependent pathway for
transport stimulation (16). In addition, studies by Krook et
al. (17) and Isakoff et al. (55) are also consistent
with the notion that IRS-1 phosphorylation, and its associated PI
3-kinase activation, are not sufficient to initiate metabolic
signaling, suggesting that additional insulin-derived metabolic inputs
must exist.
We also examined additional targets of insulin action which are thought
to be downstream of PI 3-kinase, to further explore the effects of PI
3-kinase inhibition. Akt, a serine/threonine kinase, is a downstream
target of PI 3-kinase and is activated by a dual mechanism involving
the binding of phosphatidylinositol 3,4-bisphosphate to its PH domain,
as well as serine/threonine phosphorylation by one or more Akt kinases,
which may themselves be stimulated by the lipid products of PI 3-kinase
(56). Expression of a constitutively active Akt in rat adipocytes (57)
and 3T3-L1 adipocytes (58) increases glucose uptake and GLUT4
translocation. Consistent with these findings, our data show that Akt
activation is dependent on PI 3-kinase activity, since we find that
pretreatment of 3T3-L1 adipocytes with wortmannin, a PI 3-kinase
inhibitor, as well as overexpression of the dominant inhibitory
p85N-SH2 protein, leads to an inhibition of insulin-induced
serine/threonine kinase activity of Akt. In addition, insulin-induced
glucose transport and glycogen synthase were inhibited when 3T3-L1
adipocytes were pretreated with wortmannin, or when the p85N-SH2
protein was overexpressed. Similarly, insulin-induced phosphorylation
of another downstream target of PI 3-kinase, p70 S6 kinase, was
inhibited by Rapamycin as well as by the dominant inhibitory p85N-SH2
protein. These results suggest a role for Akt as a PI
3-kinase-dependent upstream activator of glucose transport
and/or a role for p70 S6 kinase as a PI 3-kinase-dependent
upstream activator of glycogen synthase activity.
Our data on expression of the membrane-targeted
p110C indicate that membrane localization of PI
3-kinase is critical for biologic functions, since this form of the
p110 subunit leads to stimulation of 2-deoxyglucose transport similar
to the insulin induced levels in 3T3-L1 adipocytes. Along similar
lines, it has been shown in COS-7 cells, that targeting p110 to the
membrane, either by N-terminal myristoylation or by C-terminal
farnesylation, can stimulate p70 S6 kinase and Akt activity in the
absence of insulin. Our results on co-expression of the
p110C with the N-SH2 domain strongly support the
idea that the p85N-SH2 protein specifically blocks insulin mediated PI
3-kinase activity, by competitively interfering with the targeting of
p85 to its natural protein partners. Thus, although the functional
activity of downstream signaling molecules, such as p70 S6 kinase, Akt, and GLUT4, are functionally blocked by expression of the p85N-SH2, the
fact that the biologic activity can be rescued by co-expression of the
N-SH2 domain with p110C demonstrates that the
signaling pathways downstream of p85 targeting are intact and that the
dominant/negative effects of the p85N-SH2 domain do not reflect
nonspecific actions at other cellular loci.
Our studies also show that overexpression of the p85N-SH2 domain
markedly inhibits insulin-stimulated glycogen synthase activity. This
leads to the conclusion that PI 3-kinase stimulation is necessary for
insulin activation of glycogen synthase. The molecular mechanisms by
which glycogen synthase is regulated by insulin remains one of the
crucial issues in insulin action. Insulin stimulates glycogen synthesis
via a coordinated response involving activation of protein phosphatase
1, by phosphorylation of its G subunit and by a phosphorylation-induced inactivation of glycogen synthase kinase 3 (GSK3) (59, 60). It has been
suggested that pp1 might be downstream of the MAPK pathway, but a
number of reports have indicated that this is not the case (61). In
addition, our own results show that the MEK inhibitor (PD098059) did
not lead to a decrease in insulin stimulation of glycogen synthesis.
Thus, it is unlikely that any effect of the p85N-SH2 protein on the
MAPK pathway is mediating the inhibitory effect on glycogen synthesis.
It has been reported that GSK3 is a downstream target of Akt, which, in
turn, is dependent on PI 3-kinase activity (62). Thus, one potential
interpretation of our results is that inhibition of PI 3-kinase,
through the dominant/negative p85N-SH2 domain, prevents
insulin-stimulated GSK3 phosphorylation, and that this leads to
inhibition of glycogen synthesis. However, it has recently been shown
that GSK3 expression is either very low, or not expressed, in 3T3-L1
adipocytes (63-65), making this possibility less likely. Thus,
although the exact mechanism is unknown, our results clearly show that
insulin-stimulated glycogen synthesis is dependent on proper targeting
of PI 3-kinase, and this is consistent with other reports showing that
insulin-stimulated glycogen synthesis can be inhibited by wortmannin or
LY294002 (21, 66), both of which are relatively specific inhibitors of
PI 3-kinase activity.
In conclusion, we have shown that overexpression of the p85N-SH2 domain
in 3T3-L1 adipocytes inhibits a variety of insulin's ultimate biologic
effects, and that this domain will also block insulin-stimulated DNA
synthesis in fibroblasts. These results are consistent with our earlier
findings, which suggested that a complete blockade of PI 3-kinase
targeting to all of its cellular substrates (IRS-1 as well as non-IRS-1
targets) would be necessary for inhibition of AKT activation and
glucose transport stimulation. We also find that in 3T3-L1 adipocytes,
PI 3-kinase can regulate p21ras·GTP levels and that the
dominant/negative effects of the p85 NSH2 domain on the MAP kinase
pathway are exerted distal to the stimulatory effect of
p21ras·GTP on the MAPK pathway.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Research Grant R01 DK 36651 (to J. M. O.) and a grant from
the Uehara Memorial Foundation (to K. E.).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 Medicine
(0673), University of California, San Diego, 9500 Gilman Dr., La Jolla,
CA 92093-0673. Tel.: 619-534-6651; Fax: 619-534-6653.
1
The abbreviations used are: PI,
phosphatidylinositol; GAP, GTPase-activating protein; GLUT4,
insulin-responsive glucose transporter; IRS, insulin receptor
substrate; MAP, microtubule-associated protein; MAPK,
microtubule-associated protein kinase; p85, 85-kDa regulatory subunit
of PI 3-kinase; BrdUrd, bromodeoxyuridine; m.o.i., multiplicity of
infection; bp, base pair(s);
-gal,
-galactosidase; 2-DOG, 2-deoxyglucose; SH, Src homology; PBS, phosphate-buffered saline; PAGE,
polyacrylamide gel electrophoresis;
-MEM,
-minimal essential medium; PCR, polymerase chain reaction; DMEM, Dulbecco's modified Eagle's medium; ERK, extracellular signal-regulated kinase; MEK, MAP
kinase/ERK kinase; GSK3, glycogen synthase kinase 3.
2
K. Egawa, P. M. Sharma, N. Nakashima, Y. Huang,
E. Huvar, G. Boss, J. Downward, and J. M. Olefsky, manuscript in
preparation.
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