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INTRODUCTION |
After insulin binds to its receptor, it activates an intrinsic
tyrosine kinase activity, which mediates the tyrosine phosphorylation of a variety of endogenous substrates including insulin receptor substrates (IRS)1 1-4 (1,
2). Binding of these tyrosine phosphorylated substrates to the Src
homology (SH) domain-2 of the regulatory subunit of the heterodimeric
p85/p110 phosphatidylinositol (PI) 3-kinase leads to a 3-5-fold
stimulation in its enzymatic activity and an increase in the PI
3,4-bisphosphate and 3,4,5-trisphosphate in the cell (3, 4). As a
result of the increase in these lipids, there is a pronounced
stimulation in the enzymatic activity of the Ser/Thr kinases called
Akt, and a more modest increase in the enzymatic activity of particular
isoforms of protein kinase C, which ultimately lead to numerous
biological responses (5, 6).
The IRS 1-4 all contain an amino-terminal pleckstrin homology (PH)
domain next to a phosphotyrosine binding (PTB) domain (1). The PH
domain is homologous to a region in pleckstrin and has been found in
over 120 proteins including serine/threonine kinases, tyrosine kinases,
phospholipases, GTPase-activating proteins, GTPases, and cytoskeletal
proteins (7). PH domains are often involved in the attachment of
proteins to membranes, either by directly binding to phospholipids
and/or by protein-protein interactions with, for example, the 
subunits of the heterotrimeric G proteins (8). In the case of the PH
domain of IRS-1, both phospholipid binding as well as protein binding
have been reported (9-11).
In contrast, the PTB domain of IRS-1 has been documented to bind to
phosphotyrosines in the motif found in the juxtamembrane region of the
insulin receptor (i.e. NPXpY) (12, 13). Although the binding specificities of these two regions is quite distinct, they
share a similar overall structure, with each containing a
-sandwich
formed by two nearly orthogonal antiparallel
-sheets of 4 and 3 strands, respectively. In recent studies, the crystal structure of the
region of IRS-1 containing both the PH and PTB domains has been
determined (14). These data indicate that the two binding domains may
act cooperatively to localize IRS-1 at the membrane in association with
the receptor and thereby allow multiple tyrosine phosphorylations of
IRS-1.
A variety of experimental approaches have also been utilized to test
the role of the PH and PTB domains of the IRS proteins in their
interactions with the IR and the subsequent tyrosine phosphorylations.
Mutant IRS molecules in which either the PH or PTB domain have been
deleted or replaced with homologous structures of other proteins have
been expressed in mammalian cells (15-18). These studies have
documented a primary role for the PH domain in the subsequent ability
of IRS-1 to be tyrosine phosphorylated in intact cells after insulin
stimulation. In either in vitro studies or yeast two-hybrid
systems, a primary role of the IRS-1 PTB domain has been documented in
receptor interactions (15, 19). In the case of IRS-2, a more central
region of the molecule has also been identified as playing a role in
receptor interactions (20, 21).
Although numerous studies have documented the roles of the PH and PTB
domains of the IRS proteins in the interactions with the IR and the
subsequent tyrosine phosphorylation of the IRS proteins, no studies
have tested the roles of these two domains on subsequent signals
induced by the IRS·PI 3-kinase complex. Because mutations in the PH
and PTB domains interfere with the interaction of the IRS with the IR
and its subsequent tyrosine phosphorylation, it is impossible to
determine the effect of these mutations on the downstream signals
emanating from the IRS·PI 3-kinase complex. To overcome this
obstacle, we now describe a constitutively active form of IRS-1. In
this chimeric molecule, the inter-SH2 domain of the p85 regulatory
subunit of PI 3-kinase is attached to various portions of the IRS-1
molecule. The inter-SH2 domain of p85 has previously been documented to
bind to the 110-kDa catalytic unit of the PI 3-kinase (22). Thus the
resultant chimeric IRS-1 molecules now constitutively bind to the PI
3-kinase. By using these chimeric molecules, we have tested the role of
different regions of the IRS-1 molecule in eliciting a subsequent
biological response, the stimulation of the Ser/Thr kinase Akt. We
demonstrate that the PH domain alone is sufficient to target the
constitutively active PI 3-kinase to induce the activation of Akt and
that this requires the lipid binding properties of the PH domain.
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EXPERIMENTAL PROCEDURES |
Reagents--
Cell culture medium DME-H21 and oligonucleotides
were from Life Technologies, Inc. Glutathione-agarose beads and
monoclonal anti-GST antibodies were from Sigma, protein A-Sepharose was
from Repligen (Cambridge, MA), 12CA5 monoclonal anti-HA antibodies were
from Roche Molecular Biochemicals (Mannheim, Germany), rabbit polyclonal anti-Akt antibodies directed against the PH domain of Akt1
were produced as described (23). [
-32P]ATP (3000 Ci/mmol) and PI 4,5-bisphosphate, [2-3H]inositol
were from PerkinElmer Life Sciences, nitrocellulose (Protran) membrane
was from Schleicher and Schuell (Keene, NH), phosphatidylinositol was
from Avanti (Alabaster, AL). QuickChange DNA mutagenesis kit was from
Stratagene, Inc (La Jolla, CA). Restriction endonucleases and Elongase
mix were from Life Technologies, Inc. or from New England BioLabs
(Beverly, MA). The GSK-3 peptide (GRPRTSSFAEG) was synthesized at
Stanford University (Pan Facility, Stanford University, CA) (24). The
pWZL-hygro retroviral vector was kindly provided by Dr. Garry Nolan
(Palo Alto, CA, Ref. 25), pGEX-4T-3 was from Amersham Pharmacia Biotech.
Cell Culture and Treatments--
3T3-L1 cells were grown in
Dulbecco's modified Eagle's medium (DMEM) containing 10% calf serum
at 37 °C, 5% CO2. The cells were grown to confluency
and serum-starved 2-4 h before each experiment. Insulin was added when
needed as described in the figure legends.
DNA Constructs--
DNA fragments encoding the iSH2 domain of
the p85 subunit of PI 3-kinase with the HA tag were generated by PCR
and inserted into the retroviral vector pWZL-hygro using
EcoRI and SalI sites. The obtained intermediate
pWZL-iSH2-HA plasmid was used for insertion of the various domains of
IRS-1 (PH, PH-PTB, PTB). These amino-terminal parts of the constructs
were generated by PCR using a set of 5' and 3' primers containing
BamHI and EcoRI recognition sites attached to the
downstream sequence of interest. Resultant PCR products were then
ligated into pWZL-iSH2-HA. The iSH2 construct was obtained in a
separate PCR reaction by introducing an ATG codon in front of the iSH2
sequence using pWZL-iSH2-HA as a template. The R28C single amino acid
substitution was obtained by mutagenesis using the QuickChange
mutagenesis kit (Stratagene, Inc.) and the PH-PTB·iSH2 cDNA as a
template. Two additional amino acids changes (K21L and K23L were added
in a single round of mutagenesis using the same kit and the R28C mutant
cDNA as a template to generate the triple mutant (TM). Restriction
endonuclease mapping was used for screening of colonies because an
EcoR47III site was destroyed during the mutagenesis. DNA
sequence analyses were performed to verify the sequences of the
constructs. To generate the GST fusions of PH, PH-PTB, and single and
triple mutant PH-PTB, the appropriate portions of the IRS-1 constructs
of the retroviral plasmids were cut out and inserted into the pGEX-4T-3 vector.
Protein Expression in Escherichia coli and Purification of GST
Fusion Proteins--
E. coli BL-21 cells transfected with
pGEX vectors encoding the IRS-1 constructs were grown to mid-log phase,
induced with 0.1 mM
isopropyl-1-thio-
-D-galactopyranoside and grown for an additional 3-4 h. Cells from one liter of bacterial cultures were harvested by centrifugation at 7700 × g for 10 min at
4 °C (Beckman JA-10 rotor), resuspended in 20 ml of
phosphate-buffered saline buffer containing protease inhibitors and
sonicated on ice in short bursts. Sonicated bacterial extracts were
solubilized for 30 min at 4 °C using 1% Triton X-100 and
centrifuged at 12,000 × g for 10 min. Cleared
supernatants were incubated with glutathione-agarose beads (Sigma) for
2 h at 4 °C. Beads with immobilized complexes were washed
several times with phosphate-buffered saline and then eluted with 10 mM reduced glutathione in 50 mM Tris-HCl, pH
8.0 for 30 min at 4 °C. Purified fusion proteins were dialyzed
against phosphate-buffered saline and analyzed by 15%
SDS-polyacrylamide gel electrophoresis. The purity of the proteins was
assessed by densitometry of the Coomassie-stained gels.
Generation of Stable Cell Lines Expressing the IRS-1·iSH2
Constructs--
3T3-L1 fibroblasts were infected with the retroviruses
expressing IRS-1·iSH2 constructs as previously described (26).
In brief, 70% confluent Phoenix packaging cells were transiently transfected with plasmid DNA (pWZL-constructs) using the calcium phosphate precipitation method as described (26). After the final
medium change, the cells were incubated for 3 days at 30 °C, 5%
CO2 to generate the viral supernatant. Viral infection of
3T3-L1 cells was performed using supernatants collected from the
transfected Phoenix cells. 6-well plates with 50% confluent 3T3-L1
cells were infected with 1 ml of each viral supernatant. The plates
were spun at 2,500 rpm for 90 min and then cultured until confluent.
Cells were selected in complete medium containing 500 µg/ml
hygromycin for a total of 8 days. The total pool of selected cells was
used in all subsequent experiments.
Western Blot Analyses--
3T3-L1 fibroblasts overexpressing
IRS-1·iSH2 constructs were grown in 10-cm plates to confluence and
lysed in Akt lysis buffer (see Akt kinase assay below). After removal
of the insoluble matter by centrifugation, the cell lysates were
immunoprecipitated using anti-HA epitope antibodies (12CA5) bound to
protein A-Sepharose beads. Precipitated complexes were washed three
times with cold buffer containing 50 mM Hepes, pH 7.6, 150 mM NaCl, and 0.1% Triton X-100. Twenty microliters of gel
loading buffer were added to each immunoprecipitate, and the samples
were boiled for 5 min and loaded on 10% SDS-polyacrylamide gels. The
proteins were transferred to nitrocellulose membranes and blotted with
12CA5 antibodies, and bound mouse antibodies were visualized using an
anti-mouse antibody conjugated to horseradish peroxidase and ECL.
3T3-L1 cells stably transfected with the empty pWZL vector were used as
a negative control.
Subcellular Fractionation--
3T3-L1 cells expressing the
PH-PTB·iSH2 constructs were grown to confluence and serum-deprived
for 2 h. The cells were washed rapidly with ice-cold TES (20 mM Tris, pH 7.4, 5 mM EDTA, 250 mM
sucrose), and then harvested in 1 ml of homogenization buffer containing TES with 10 µg/ml aprotinin, 10 µg/ml leupeptin, 250 µM phenylmethylsulfonyl fluoride, 2 mM sodium
vanadate, 10 mM sodium fluoride, and 2 mM
sodium pyrophosphate. The cell suspension was homogenized by passaging
10 times through a 25-gauge needle, incubated on ice for 10 min, and
then spun at 4 °C for 10 min at 3000 rpm on a table top centrifuge.
Supernatants were removed and subjected to ultracentrifugation at
100,000 × g (60,000 rpm) for 1 h at 4 °C using
a TLA-100.3 rotor (Beckman Instruments Inc.). Supernatants obtained in
the high-speed spin, designated cytosol (C), were
immunoprecipitated with anti-HA antibodies. Pellets were resuspended in
1 ml of the homogenization buffer and spun at 100,000 × g for 1 h. Supernatants obtained in the second
high-speed spin were discarded, and pellets were solubilized on ice in
homogenization buffer containing 0.5 M NaCl for 30 min.
Solubilized pellets were then recentrifuged at 14,000 rpm for 15 min at
4 °C. Supernatants obtained in this spin, designated as soluble
particulate (SP), were also immunoprecipitated. The pellets
from this spin (designated as insoluble particulate, IP)
were dissolved by boiling in SDS and directly loaded onto 10%
SDS-polyacrylamide gels. Immunoprecipitates of cytosol (C)
and soluble particulate (SP) were loaded on the same gel,
and all three subcellular fractions were analyzed by immunoblotting
with 12CA5 antibodies and ECL. Protein bands were quantified using an
NIH image program. Ratios of the total particulate fraction over
cytosol were calculated for each construct.
Akt and PI 3-Kinase Assays--
3T3-L1 cells expressing
IRS-1·iSH2 constructs were grown to confluence, serum-deprived for
2 h and stimulated with insulin as indicated. Cells were then
washed once with cold HBS buffer (50 mM Hepes, pH 7.6, 150 mM NaCl) and lysed in either PI 3-kinase lysis buffer (137 mM NaCl, 20 mM Tris-HCl, pH 8.0, 1 mM MgCl2, 1 mM CaCl2,
10% glycerol, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 400 µM vanadate, 1 mM
dithiothreitol) or Akt lysis buffer (50 mM Hepes, pH 7.6, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM vanadate, 2 mM sodium
pyrophosphate, 30 mM sodium fluoride, 1 mM
EDTA, 1 mM dithiothreitol, 100 nM okadaic
acid). For the PI 3-kinase assays, cell lysates were immunoprecipitated with the anti-HA epitope antibodies (12CA5) captured on protein A-Sepharose beads. The precipitates were assayed for PI 3-kinase activity as described (27) using phosphatidylinositol as a substrate. The amounts of PI 3-kinase activity in the cells transfected with pWZL
vector were considered as background (always less than 5% of the
signal) and subtracted from experimental values. Data shown are the
means of three independent experiments. In the Akt kinase assays,
endogenous Akt was immunoprecipitated from cell lysates using an
antibody to the PH domain of Akt bound to protein A-Sepharose. Akt
assays were performed as described in Ref. 28 using the GSK-3 peptide
(GRPRTSSFAEG) as a substrate. The phosphorylated peptide was separated
on a 40% urea gel from unincorporated radioactive ATP. Phosphopeptide
bands were excised, and radioactivity associated with each band was
counted using a scintillation counter (Beckman LS). Data shown are
means of three independent experiments. Akt activity in nonstimulated
cells transfected with the pWZL vector were considered as background
and subtracted from experimental values.
Phospholipid Binding Assays--
Twenty microliters (containing
0.2 µCi) of PI 4,5-bisphosphate [2-3H]inositol (2-10
Ci/mmol) were mixed with 20 µl of 2 mg/ml phosphatidylcholine (dissolved in chloroform/methanol mix (1:1) containing 0.1% HCl) and
evaporated under a stream of N2. Dried phospholipids were reconstituted in 200 µl of HNE buffer (30 mM Hepes, pH
7.0, 100 mM NaCl, 1 mM EDTA) containing 0.02%
Nonidet P-40 and sonicated with three bursts of 3 min each. Forty
microliters of the reconstituted phospholipids were incubated with 0.5 µg of each protein in 100 µl of HNE buffer containing 0.02%
Nonidet P-40. The mixtures were incubated for 1 h at room
temperature. Then 35 µl of the glutathione-agarose slurry were added
to each reaction and incubated for 30 min at 4 °C to capture the
protein-phospholipid complexes. Beads were washed once with 1 ml of the
cold HNE buffer containing 0.5% Nonidet P-40, and radioactivity
retained on the beads was measured by liquid scintillation counting
(Beckman LS).
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RESULTS |
Construction and Expression of Chimeric IRS-1 Molecules--
To
generate IRS-1 molecules that would constitutively bind and activate PI
3-kinase without being tyrosine phosphorylated, we constructed
cDNAs that encode different portions of the IRS-1 molecule fused in
frame with the inter-SH2 domain of the p85 regulatory subunit of the PI
3-kinase because this peptide (22) has previously been documented to be
sufficient to bind to the 110-kDa catalytic unit of the PI 3-kinase
(Fig. 1). In addition, each chimera
contained an HA tag at the carboxyl terminus. Stable cell lines
expressing each of these constructs were generated, and the amount of
the expressed construct was examined by immunoprecipitation and Western blotting (Fig. 2). The construct only
encoding the inter-SH2 domain was found to be poorly expressed and not
studied further (Fig. 2). In contrast, constructs encoding either the
PH domain alone or both the PH and PTB domains were found to be well
expressed although the construct encoding the PH domain alone was
expressed at a level of about one-half that of the PH-PTB construct
(Fig. 2). In addition, point mutants in the PH domain of the PH-PTB construct were made. These included both a single mutant in which Arg28 was changed to Cys (called PH-(R28C)-PTB) or a triple
mutant containing the Arg28 mutation as well as mutations
in Lys21 and Lys23 (called PH-(TM)-PTB). Both
of these mutants were found to be expressed at levels comparable with
the nonmutant PH-PTB construct (Fig. 2). These mutations were designed
based on the findings that mutations in comparable residues of other PH
domains impairs their ability to function (7). Finally, a construct
that only contained the PTB domain fused to the inter-SH2 domain was
found to be expressed although at a lower level then the constructs containing either both the PH and PTB domains or only the PH domain (Fig. 2).

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Fig. 1.
Schematic of the IRS-1 chimeric
molecules. The HA epitope (dark oval) and the
inter-SH2 domain (iSH2) (open rectangle) of the p85 subunit
of the PI 3-kinase (amino acids 466-567) were fused to the carboxyl
terminus of all the different molecules. Additional domains added
include the pleckstrin homology domain of IRS-1 (PH, amino
acids 1-140; striped rectangle) and the phosphotyrosine
binding domain of IRS-1 (PTB, amino acids 141-307; solid
rectangle). Mutated residues in the PH domain are indicated by an
asterisk and include Arg28 (changed to a Cys),
Lys21 (changed to Leu) and Lys23 (changed to
Leu). The triple mutant (TM) contains all three
mutations.
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Fig. 2.
Expression of the chimeric IRS-1 molecules in
3T3-L1 cells. Stable cell lines expressing the different
constructs were obtained by retroviral infections. Cell lysates were
immunoprecipitated with anti-HA epitope antibodies, and the
precipitates were analyzed by SDS-polyacrylamide gel electrophoresis
and Western blotting with the anti-HA antibodies. Precipitates from
lysates of cells infected with the empty virus (pWZL) are included as a
control. The positions of the PTB, PH, and PH-PTB chimeric molecules as
well as molecular weight markers are indicated. Also indicated are the
Ig heavy chain (Hc) and light chain (Lc) of the
anti-HA antibodies used in the precipitations.
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Measurement of the Amount of PI 3-Kinase Activity Associated with
the Different Chimeric IRS-1 Molecules--
To determine whether the
chimeric IRS-1 molecules did contain an associated PI 3-kinase
activity, lysates of the stable cell lines described above were
immunoprecipitated with an antibody to the HA epitope, and the amount
of PI 3-kinase activity in these precipitates was determined (27). The
construct containing both the PH and PTB domains of IRS-1 had the
maximal amount of PI 3-kinase activity associated with it (Fig.
3). The amount of PI 3-kinase activity
associated with the point mutants in the PH domain of the PH-PTB
construct (both PH-(R28C)-PTB and PH-(TM)-PTB) were found to be not
significantly different then the nonmutant PH-PTB domain containing
chimera (Fig. 3). The chimeric construct containing only the PH domain
of the IRS-1 had ~50% of the PI 3-kinase activity of the PH-PTB
molecule, whereas the PTB and iSH2 constructs had lower amounts of PI
3-kinase activity. The PI 3-kinase activities associated with these
constructs (Fig. 3) were proportional to the amounts of each protein
expressed (Fig. 2).

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Fig. 3.
Measurement of the amount of PI 3-kinase
activity associated with the different chimeric IRS-1 molecules.
Cells expressing the chimeric IRS-1 molecules were lysed, and the
anti-HA antibodies were used to precipitate these molecules. The amount
of associated PI 3-kinase was measured in an in vitro
reaction as described. Results shown are mean ± S.E. from three
experiments normalized to the amount of activity present in the
precipitates from the cells expressing the PH-PTB domain construct. The
asterisk indicates that these values differ from those of
the PH-PTB construct with a p < 0.01.
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Measurement of the Amount of Active Akt Induced by the Chimeric
IRS-1 Molecules--
To test the role of the different domains of
IRS-1 in the ability of the chimeric molecule to stimulate a downstream
response, we examined the amount of active endogenous Akt in the cell
lines expressing the different constructs. Confluent cells were
serum-deprived for 2 h, and then cells expressing the pWZL vector
were stimulated with insulin to determine the maximal levels of active
endogenous Akt in these cells. Cells were lysed, and the Akt was
immunoprecipitated and assayed for enzymatic activity (28). Cells
expressing the PH-PTB domain had the highest levels of endogenous Akt
activity, a value ~60% of that observed after stimulation with
insulin (Fig. 4). Mutation of either a
single residue in the PH domain (the PH-(R28C)-PTB construct) or 3 amino acids (the PH-(TM)-PTB construct) in the PH-PTB construct
significantly impaired the ability of these constructs to activate the
endogenous Akt (Fig. 4). In cells expressing a construct that only
contained the PH domain, the endogenous Akt was also activated to a
level of ~40% of that observed after insulin stimulation (Fig. 4).
The construct containing only the PTB domain of IRS-1 exhibited a low
ability to activate the endogenous Akt, consistent with its low
expression and poor activation of the PI 3-kinase.

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Fig. 4.
Measurement of the amount of active Akt
induced by the chimeric IRS-1 molecules. Cells expressing the
chimeric IRS-1 molecules were lysed, and the endogenous Akt was
precipitated and assayed for enzymatic activity. Results shown are
mean ± S.E. from three independent experiments normalized to the
amount of activity present in the precipitates from control cells
treated for 5 min with 1 µM insulin. The
asterisk indicates that these values differ from those of
the PH-PTB construct with a p < 0.01.
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Testing the Effect of the Mutations in the PH Domains on its Lipid
Binding Activities--
To test the lipid binding activities of the
various mutant PH domains, we produced bacterial GST fusion proteins
with the wild-type PH-PTB molecule (lacking the inter-SH2 domain of the p85 subunit of PI 3-kinase) as well as the single point mutant (R28C)
and the triple mutant (R28C/K21L/K23L). The purified GST fusion
proteins were incubated with labeled PI 4,5-P2, washed, and
counted. The PH-PTB molecule bound substantially more lipid then the
control GST protein (Fig. 5). In
addition, the two mutant GST·PH-PTB proteins bound ~75% less lipid
than the wild-type PH-PTB protein.

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Fig. 5.
Measurement of the lipid binding activities
of the IRS-1 molecules. GST fusion proteins of the wild-type
PH-PTB domain as well as the single (R28C) mutant and triple mutant
(TM, R28C/K21L/K23L) were purified, and equivalent amounts
of protein were incubated with phospholipid vesicles containing labeled
PI 4,5-P2. The fusion proteins were captured on
glutathione-agarose, washed, and eluted. The amount of bound PI
4,5-P2 was determined by liquid scintillation counting. A
control of the GST protein alone was included. Results shown are
mean ± S.E. from three independent experiments.
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Testing the Effect of the Mutations in the PH Domains on the
Subcellular Distribution of the Chimeric IRS-1 Molecules--
To test
the effect of the point mutations in the PH domains on the subcellular
localization of the chimeric IRS-1 molecules, we fractionated cells
expressing either wild-type PH-PTB construct, PH-(R28C)-PTB or
PH-(TM)-PTB. The fractions were tested for the presence of the
constructs by Western blotting. We found that the wild-type PH-PTB
IRS-1 chimera was present in the particulate fraction to a much greater
degree then either the single mutant (PH-(R28C)-PTB) or the triple
mutant (PH-(TM)-PTB); the particulate to cytosolic ratio of the
wild-type PH-PTB chimera was 2.5-fold greater then the comparable ratio
for either PH-(R28C)-PTB or PH-(TM)-PTB (Fig.
6).

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Fig. 6.
Subcellular localization of the different
chimeric IRS-1 molecules. Cells expressing the PH-PTB·iSH2
constructs were homogenized and subjected to differential fractionation
to obtain cytosolic (C), soluble particulate (SP)
and insoluble particulate (IP) fractions as described under
"Experimental Procedures." The insoluble particulate and
immunoprecipitates of cytosolic and solubilized particulate fractions
were analyzed by SDS-polyacrylamide gel electrophoresis and
immunoblotting using anti-HA antibodies (A). The amounts of
the different chimeric molecules in the subcellular fractions were
quantified using NIH image program and are plotted in B as
the ratio of the particulate (SP+IP) to cytosolic fraction
(C). Data shown are representative of two independent
experiments.
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DISCUSSION |
The principal mechanism whereby the insulin receptor activates PI
3-kinase appears to be via its ability to stimulate the tyrosine
phosphorylation of various endogenous proteins including the different
IRS molecules, IRS-1 to 4 (1, 2). Activation of PI 3-kinase plays a
critical role in subsequent biological responses, such as stimulation
of glucose uptake and regulation of gene transcription (5, 6). A number
of downstream targets of the PI 3-kinase and the lipids it generates
have been identified. These include several Ser/Thr kinases such as the
PDK-1, the family of Akt/PKB kinases, and several atypical protein
kinase C including PKC
and
. The exact role of these different
kinases in eliciting subsequent biological responses has been debated
(5, 6).
Extensive studies have investigated the mechanism whereby the IRS
proteins are localized to the insulin receptor. All four IRS molecules
contain both a PH domain and a PTB domain (1, 2). The PH domain binds
various phospholipids, including PI 4,5-P2 as well PI
3,4,5-P3 (9, 10). In addition some studies have suggested
that the PH domain may also interact with various proteins (11). In
contrast, the PTB domains bind phosphotyrosines, in particular, a
phosphotyrosine in the juxtamembrane region of the insulin receptor
(12, 13). Based on the crystal structure of the PH and PTB domains of
the IRS-1 molecule, a model has been proposed of the two domains
binding cooperatively to localize IRS-1 at the membrane in association
with the insulin receptor (14).
In the present studies, we have tested the role of these two domains in
the ability of the IRS-1 molecule to elicit a subsequent biological
response, the activation of the Ser/Thr kinase Akt. To accomplish this,
we produced novel chimeric molecules, which contain these two domains
fused to the inter-SH2 domain of the p85 subunit of the PI 3-kinase.
This peptide has previously been shown to be sufficient to bind and
activate the PI 3-kinase (22). By adding these residues to the
different domains of the IRS-1 molecule, we were able to produce
chimeric molecules, which were constitutively bound to active PI
3-kinase. This was necessary because prior studies have shown that the
PH domain of the IRS-1 molecule was critical for its subsequent
tyrosine phosphorylation and association with the PI 3-kinase. By
adding the inter-SH2 domain to the different domains of the IRS-1
molecule, we were able to produce chimeric molecules that were active
in the absence of insulin stimulation and receptor-mediated tyrosine phosphorylation.
In the present work we have shown that the PH domain is critical for
the subsequent ability of these constitutively active chimeric
molecules to activate Akt. The most convincing data for this conclusion
comes from the studies of the point mutants of the PH-PTB domain
chimeric molecule. The wild type and mutants were expressed to
comparable levels and had almost identical associated PI 3-kinase
activities. However, the wild type activated the endogenous Akt whereas
both the single and triple PH domain mutants had a dramatically
decreased ability to activate the enzymatic activities of the
endogenous Akt (i.e. the introduction of either a single mutation or 3 mutations in the PH domain caused an approximate 75%
decrease in the ability of the PH-PTB construct to activate Akt). Both
mutations also had a similar decreased ability to bind to PI
4,5-P2 in comparison to the wild-type protein when they were expressed as PH-PTB·GST fusion proteins. The simplest
interpretation of these data are that the PH domain function
(i.e. binding to PI 4,5-P2 or a negatively
charged protein) is critical to elicit the downstream functions. This
conclusion is also supported by the finding that a chimeric molecule
with only the PH domain of IRS-1 was also capable of stimulating the
activation of the endogenous Akt. The somewhat weaker activity of this
chimera in comparison to the PH-PTB molecule is consistent with its
lower expression and somewhat decreased amount of associated PI
3-kinase activity. Because the construct, which only contained the PTB
domain was expressed at lower levels and therefore had lower associated
PI 3-kinase activity, it is not possible to conclude from this
construct alone whether the PTB domain alone would have been capable at higher levels to induce the activation of Akt. However, the finding that the PH-PTB domain chimeric molecules whose PH domain function were
impaired by either single or triple point mutations were incapable of
eliciting the activation of the Akt argue that the PTB domain alone
would not be sufficient to elicit this response because the PTB domain
function in these chimeric molecules is still intact.
A potential role of the PH domain of IRS-1 is to help localize the
protein in the correct intracellular compartment. To test this
hypothesis, we examined the subcellular localization of the wild-type
and two mutant chimeric molecules by biochemical fractionation of the
cells. The wild-type PH-PTB chimeric molecule was found to be in the
particulate fraction to a much greater degree then the chimeric
molecules containing the mutations in the PH domain. These results are
consistent with the recent report that the IRS-1 molecule is at least
partially present in a particulate fraction of the cell, possibly
because of its association with the cytoskeletal complex (29) as well
as with a recent report that the same mutant we have studied (R28C)
blocks the insulin induced translocation of a IRS·GFP construct to
the membrane (10). Thus, the PH domain may serve to localize the
chimeric molecules in the subcellular localization required for the
subsequent activation of Akt.
In conclusion, the present work presents the first evidence that a
functional PH domain of the IRS-1 molecule is required for its ability
to signal to downstream molecules such as activation of Akt. This
requirement is presumably because of the ability of the PH domain of
IRS-1 to direct the PI 3-kinase to the correct subcellular compartment.
Compromising the function of the PH domain, for example in
insulin-resistant states, could therefore both decrease the ability of
IRS-1 to be tyrosine phosphorylated by the insulin receptor as well as
to link to subsequent downstream targets (2).