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INSERM U-145 (L.D., I.M.-S., E.V.O.), 06107 Nice Cedex 2, France; the Research Division, Joslin Diabetes Center and Program in Biological and Biochemical Sciences, Harvard Medical School (M.G.M., M.F.W.), Boston, Massachusetts 02115
Address all correspondence and requests for reprints to: Dr. Laurent Delahaye, INSERM U-145, Faculté de Médecine, avenue de Valombrose, 06107 Nice Cedex 2, France. E-mail: delahaye{at}unice.fr
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Abstract |
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regulatory subunit of
phosphatidylinositol (PI) 3-kinase. In a first series of experiments
using the yeast two-hybrid system, we show that IRS-1 interacts with
p85, and this interaction depends on tyrosine phosphorylation, as
shown with the IRS-1 mutant F18 and 3Y-IRS-1. F18-IRS-1 contains 18
potential tyrosine phosphorylation sites mutated to phenylalanine;
three of them, i.e. Y608, 628, and 658, which are
potential binding sites for p85, have been added back in the
3Y-IRS-1 mutant. The tyrosine phosphorylation of IRS-1, which is
required for the interaction with p85, is thought to occur via
endogenous yeast kinases that phosphorylate IRS-1 at least on these PI
3-kinase-binding sites.
Next, we show that not only p85 but also p55PIK, another
regulatory subunit of PI 3-kinase, interacts with IRS-1 in yeast.
Interestingly, for both regulatory subunits their interaction with
IRS-1 is up-regulated by mutating serines 662 and 731 on IRS-1.
In a previous study we found that insulin-stimulated PI 3-kinase activity was increased not only in the presence of S662A/S731A-IRS-1 but also under resting conditions compared with the activity seen with WT-IRS-1.
Here we demonstrate in 293-EBNA cells overexpressing S662A/S731A-IRS-1 that insulin-stimulated protein kinase B activity is not augmented, whereas without insulin treatment, basal activity is increased compared with that in cells overexpressing wild-type IRS-1. In conclusion, we have shown that 1) potential serine phosphorylation sites on IRS-1, which are adjacent to YXXM binding motifs for PI 3-kinase, negatively regulate binding of IRS-1 to PI 3-kinase regulatory subunits; and 2) these modulations affect protein kinase B activity.
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Introduction |
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-subunits activates the tyrosine kinase activity of the
ß-subunits, which allows subsequent tyrosine phosphorylation of
receptor substrates such as Shc, insulin receptor substrate-1 (IRS-1),
IRS-2, IRS-3, and IRS-4 (1, 2, 3). The IRS proteins then become docking
molecules that interact via their phosphorylated tyrosines with SH2
domain-containing effectors, leading to the biological effects of the
hormone (4, 5, 6).
In addition to its tyrosine phosphorylation, IRS-1 is found to be
heavily phosphorylated on serine residues in intact cells. Mounting
evidence indicates that serine phosphorylation could negatively
regulate insulin action. For example, we and others have reported that
in various insulin-sensitive cells, okadaic acid and angiotensin II are
able to induce IRS-1 serine and threonine phosphorylation, which
correlates with a decrease in IRS-1 tyrosine phosphorylation and a
subsequent decrease in IRS-1/p85 association and IRS-1-associated
phosphatidylinositol (PI) 3-kinase upon insulin stimulation (7, 8, 9). In
3T3-F442A adipocytes, tumor necrosis factor- has also been shown to
induce IRS-1 serine phosphorylation (10). This serine-phosphorylated
IRS-1 is thought to interact differently with the insulin receptor (IR)
compared with wild-type IRS-1 (WT-IRS-1), resulting in
inhibition of IR tyrosine kinase activity. It was recently shown in CHO
cells that in the absence of growth factor or insulin, glycogen
synthase kinase-3 (GSK-3) is activated and phosphorylates IRS-1
on serine residues. This is correlated with a decrease in IRS-1
tyrosine phosphorylation and IR tyrosine kinase activity (11).
Moreover, in 293 cells, phorbol 12-myristate 13-acetate (PMA) treatment
stimulates mitogen-activated protein (MAP) kinase, which phosphorylates
IRS-1 on serine 612, resulting in inhibition of insulin signaling (12, 13).
Taken together, these data indicate that serine phosphorylation of either the IR or IRS-1 modulates insulin action and is implicated in the negative regulation of hormone signaling. Hence, this process is likely to contribute at least in part to the insulin resistance found in disease states such as obesity and type II diabetes.
To investigate the molecular mechanism underlying the modulation of
insulin signaling by IRS-1 serine phosphorylation, we constructed
serine mutants of IRS-1 at the level of YXXMSP
motifs that are potential binding sites for p85. These mutants,
S612A/S632A-IRS-1 and S662A/S731A-IRS-1, correspond to serine mutated
to alanine surrounding tyrosines Y608/Y628 and Y658/Y727, respectively.
Expressing these mutants in 293 cells, we found that these potential
serine phosphorylation sites are negative regulators of basal and
insulin-induced IRS-1-associated PI-3 kinase activities. Indeed,
mutation of these serines increases tyrosine phosphorylation of IRS-1,
its binding to p85, and its associated PI 3-kinase activity (8). PI
3-kinase is a cytosolic enzyme implicated in various cellular
functions, such as insulin-induced glucose transport (14) and membrane
ruffling (15). PI 3-kinase is composed of a regulatory subunit of 85
kDa and a catalytic subunit of 110 kDa, which has lipid kinase and
serine kinase activities (16, 17). p85 SH2 domains recognize
phosphorylated IRS-1 tyrosine residues in YXXM motifs (18, 19).
Recently, several novel regulatory subunits of PI 3-kinase have been
cloned, including 1) p55 and p50, which are alternatively spliced
isoforms of the p85 gene; 2) p62cplx; and 3)
p55PIK (20, 21, 22, 23). Compared with p85, p55PIK
contains a conserved region with p85 in its C-terminal part comprising
two SH2 domains and an inter-SH2 domain, but it lacks several domains
in its N-terminus (i.e. Bcr, SH3, and one proline-rich
domain) that are replaced by a 34-amino acid sequence. In the present
study we used the yeast two-hybrid system to examine the interaction of
p85 and p55PIK with IRS-1. In this system we
investigated the importance of IRS-1 tyrosine phosphorylation using
IRS-1 tyrosine mutants (F18 and 3Y) with or without coexpression of the
IR. Using the IRS-1 serine mutant S662A/S731A, we also determined
whether the interaction of IRS-1 with the regulatory subunits of PI
3-kinase could be modulated by serine sites. Finally, we show in intact
293 cells that overexpression of the S662A/S731A-IRS-1 mutant increases
the basal activity of protein kinase B (PKB) compared with that of
WT-IRS-1. Taken together, our data indicate that IRS-1 serines 662 and
731 could play an important role in modulating downstream effectors of
IRS-1, and hence insulin action.
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Materials and Methods |
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complementary DNA (cDNA) into the pGBT9 vector was a gift from J.
E. Pessin (Iowa City, IA). Oligonucleotides were purchased from
Eurogentec (Seraing, Belgium), restriction enzymes were obtained from
New England Biolabs, Inc. (Beverly, MA), Pwo DNA
polymerase was obtained from Boehringer Mannheim (Meylan,
France), and synthetic defined dropout yeast media lacking the
appropriate amino acids were purchased from BIO-101 (La Jolla, CA).
Cell culture media and geneticin were purchased from Life Technologies, Inc. (Paisley, Scotland). All chemical reagents
used were obtained from Sigma Chemical Co., Inc. (St.
Louis, MO), except protein A-Sepharose, which was obtained from
Pharmacia Biotech, Inc. (Uppsala, Sweden);
2-mercaptoethanol and 3-aminophtalhydrazide were purchased from Fluka
(Buchs, Switzerland). Antibodies to IRS-1 for immunoprecipitation and
Western blotting were raised to the C-terminal (1223–1235) peptide of
rat IRS-1.
Plasmid constructions
WT-IRS-1 cDNA and IRS-1 mutants F18, 3Y, and S662A/S731A
cDNAs (8, 24) were subcloned in-frame into the pACTII vector using
convenient restriction sites, yielding GAD-WT-IRS-1, GAD-F18-IRS-1, and
GAD-3Y-IRS-1, GAD-S662A/S731A-IRS-1 fusion proteins, respectively.
Full-length p55PIK cDNA was amplified by PCR using the
following primers: sense, 5'-ccggaattcgaccgcgatgacgcagactg-3'; and
antisense, 5' tcccccgggttatctgcagagcgtaggc-3'. Then the fragment was
subcloned in-frame into the polylinker of pBTM116 using the
EcoRI (5'-side) and SmaI (3'-side) restriction
sites. The fusion protein obtained (LexA-p55PIK)
corresponded to the LexA DNA binding domain (1–147) fused to
p55PIK. LexA-p85 was obtained by PCR amplification of
p85 cDNA using the following set of primers: sense,
5'-gccgaggggtacgaattccgggcgctg-3'; antisense,
5'-atcgcctcggatccgcgtacactgggtagg-3', and then subcloning the PCR
product in-frame into EcoRI and BamHI sites of
pBTM116. Correct in-frame fusion between LexA and p55PIK or
p85 cDNA was verified by sequencing with a primer corresponding to a
LexA sequence (5'-cttcgtcagcagagcttc-3'), using the T7 sequencing kit
(Pharmacia Biotech).
Constructs encoding hemagglutinin (HA)-tagged PKB in the mammalian expression vector pECE were gifts from Brian Hemmings and have been described previously (25).
Yeast transformation and reporter gene activity
The yeast strain L40 was cotransformed with pBTM116 and pACTII
plasmids expressing hybrid proteins of interest, using the lithium
acetate method (26). L40 were grown for 48 h on plates containing
Trp-, Leu- complete supplemented (CS) medium
to select clones containing both plasmids (pBTM116 and pACTII carry the
Trp+ and Leu+ selection markers, respectively).
Suppression of IRß gene expression carried on the pBTM116 vector was
accomplished by addition of L-methionine (Sigma Chemical Co.) at 20 mM to the medium. The histidine
reporter gene was tested by replicating the clones expressing the
different sets of plasmids on plates containing CS medium without
tryptophan, leucine, and histidine and by growing them at 30 C for
48 h. Double transformants were also assayed for ß-galactosidase
activity, using a color filter assay as previously described (27) or a
liquid culture assay. Briefly, three clones of each transformation were
grown for 24 h in Trp-, Leu- CS medium,
then diluted 10-fold in 2 ml of the same CS medium. After 24 h of
additional growth, 1 ml of cells was used for determination of OD at
600 nm; 100–500 µl of cells were used for colorimetric assay at 574
nm. Cells were pelleted, resuspended in 500 µl Z buffer (60
mM Na2HPO4, 40 mM
NaH2PO4, 40 mM KCl, and 1
mM MgSO4)-25 µl chloroform, and vortexed for
15 sec. After a 10-min incubation at 30 C, 100 µl of the chromogenic
substrate chlorophenol red-ß-D-galactopyranoside at 50
mM were added. The reaction was performed at 30 C, and
ß-galactosidase activity was measured according to Millers’ method
(28). One unit of ß-galactosidase activity was defined as follows:
(A574 x 1000)/[ A600 x volume (ml) x time
(min)].
Protein expression in yeast
Cotransformed yeast on plates expressing hybrid proteins were
grown for 10 h at 30 C in 1 ml Trp-,
Leu- CS medium, then for an additional 12 h in
the same medium adjusted to 50 ml. One milliliter of yeast diluted
10-fold was used for OD quantification at 600 nm. The cells were washed
in PBS, frozen in nitrogen for 10 min, and placed at -20 C for another
10 min. Then the cells were lysed on ice for 20 min with buffer A (50
mM HEPES, 150 mM NaCl, 10 mM EDTA,
10 mM Na4P2O7, 2
mM sodium orthovanadate, and 100 mM NaF, pH
7.5) supplemented with 1% (vol/vol) Triton X-100 and protease
inhibitors: 100 U/ml aprotinin, 1 mM PhMeSO2F
(phenylmethylsulfonylfluoride), 20 mM leupeptin, 2
mM pepstatin, and 4 mM benzamidine. Then glass
beads (425–600 µm; Sigma Chemical Co.) were added, and
the tubes were vortexed for 30 sec and maintained on ice. This step was
repeated three times. After the tubes were shaken for 20 min at 4 C and
centrifuged for 30 min at 13,000 rpm, immunoprecipitation with rabbit
polyclonal antibodies to IRS-1 was performed for 2.5 h at 4 C.
Samples were washed alternatively five times with buffer A supplemented
with 1% (vol/vol) Triton X-100 and buffer A supplemented with 0.2%
(vol/vol) Triton X-100, 0.1% (wt/vol) SDS, and 0.5 M NaCl.
Samples were resuspended in Laemmli buffer (29), loaded on 7.5%
polyacrylamide gel, and subjected to SDS-PAGE under reducing
conditions. Proteins were transferred to an Immobilon membrane
(Millipore Corp., Milford, MA). The membrane was blocked
with 10 mM Tris-HCl and 140 mM NaCl, pH 7.4,
supplemented with 5% (wt/vol) BSA and probed with either rabbit
polyclonal antibodies to IRS-1 or a mouse monoclonal antibody to
phosphotyrosine, followed by incubation with
[125I]protein A.
Cell culture and transfection
HA-tagged WT-PKB, WT-IRS-1, and S662A/S731A-IRS-1 were
transiently expressed in 293 EBNA cells, which are human embryo kidney
cells constitutively expressing the EBNA-1 protein from the
Epstein-Barr virus (Invitrogen, San Diego, CA). The cells
were cultured in DMEM containing 5% (vol/vol) FCS and 500 µg/ml
geneticin. Transfection was performed by the calcium phosphate
precipitation method of Chen and Okayama (30) (3 µg
DNA/9.5-cm2 dish). Eighteen hours after transfection, the
calcium phosphate-DNA precipitates were removed, and cells were
incubated in DMEM supplemented with 0.2% (wt/vol) BSA for 20 h
before the experiment.
Immunoblot, immunoprecipitation, and in vitro kinase assay of
PKB
293-EBNA cells were stimulated or not with 10-6
M insulin for 5 min and washed in PBS. Then they were lysed
in a buffer containing 50 mM HEPES, 150 mM
NaCl, 100 mM NaF, 10 mM EDTA, 10 mM
Na4P2O7, 2 mM vanadate,
0.5 mM phenylmethylsulfonylfluoride, 100 IU/ml aprotinin,
20 µM leupeptin, and 1% (vol/vol) Triton X-100. The
lysates were centrifuged at 15,000 x g at 4 C for 15
min, and 90% of each sample was immunoprecipitated using anti-HA
antibody (12CA5) coupled to protein G-Sepharose. After 2.5 h, the
pellets were washed, and phosphorylation buffer containing 50
mM Tris, 10 mM MgCl2, 1
mM dithiothreitol, 5 µM ATP, 3.3 µCi
[
-32P]ATP, and 30 µM Crosstide
(Neosystem, Strasbourg, France) as substrate was added. After 30 min at
room temperature, the reaction was stopped by spotting 40 µl onto
Whatman p81 papers, and immersing in 1% (vol/vol)
orthophosphoric acid. After three washes, the papers were air-dried,
and radioactivity was determined by Cerenkov counting. We subtracted
the background obtained from phosphorylation alone. Ten percent of each
lysate was analyzed by SDS-PAGE under reducing conditions, and proteins
were transferred to an Immobilon membrane (Immobilon polyvinylidene
difluoride, Millipore Corp.). The membrane was cut and
immunoblotted with antibodies to either IRS-1 or PKB (provided by B.
Hemmings and raised against a peptide containing amino acids 469–480
of PKB), followed by [125I]protein A and
autoradiography.
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Results |
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with IRS-1 in yeast two hybrid system: role
of IRS-1 tyrosine phosphorylation and IRS-1, respectively. These two hybrid
proteins, LexA-p85 and GAD-IRS-1, were coexpressed in yeast, and a
colorimetric experiment was performed to measure ß- galactosidase
activity, which reflects interaction between the two proteins. As the
interaction between IRS-1 and p85 depends on IRS-1 tyrosine
phosphorylation, we coexpressed IRß, IRS-1, and p85 in yeast. To
do so, we constructed a modified pLex vector in which receptor
ß-subunit (IRß) is under control of a promoter repressible by
methionine to increase phosphorylation of IRS-1 (31). We verified that
the IR intracellular region is expressed in the absence of methionine,
and that addition of the repressing molecule prevents expression. As
negative controls, we coexpressed LexA-p85 and GAD-IRS-1 with the
unrelated proteins GAD-ras and LexA-lamin, respectively.
LexA-p85 and GAD-IRS-1 did not induce detectable ß-galactosidase
activity corresponding to nonspecific interaction (data not shown). As
shown in Fig. 1
, in the absence of
methionine, i.e. when IRß was expressed, a 4-fold increase
in the interaction between IRS-1 and p85 was seen. However,
ß-galactosidase activity due to interaction between IRS-1 and p85
was also detectable when IRß was not expressed, suggesting that 1)
yeast may contain tyrosine kinases able to tyrosine phosphorylate
IRS-1; or 2) IRS-1 may interact with p85 in a
phosphotyrosine-independent manner. To further address this issue, we
used two IRS-1 mutants, F18 and 3Y. In F18 IRS-1, the 18 tyrosines
corresponding to potential phosphorylation sites by IR and insulin-like
growth factor I receptor were replaced by phenylalanines. In
3Y-IRS-1, three tyrosines, i.e. Y608, Y628, and
Y658, were added back to F18-IRS-1, resulting in an IRS-1 protein
containing only potential binding sites for PI 3- kinase. We first
examined the interaction between the WT or IRS-1 mutants and the IR.
GAD-F18-IRS-1, GAD-3Y-IRS-1, or GAD-WT-IRS-1 was coexpressed with the
IR (LexA-IRß). This interaction occurs through the PTB domain of
IRS-1 and the phosphorylated NPXY960 of the IR (32). The
three IRS-1 molecules interact similarly with IR, suggesting that no
major conformational changes due to mutation of tyrosine to
phenylalanine residues have occurred, and that the three IRS-1 proteins
are similarly expressed and properly folded in yeast. As shown in Fig. 1, no or weak interaction was seen between F18-IRS-1 and p85.
Interestingly, interaction between 3Y-IRS-1 and p85 was almost
completely restored, as 85% of the ß-galactosidase activity seen
with wild-type IRS-1 was obtained. Moreover, this interaction was
increased 4-fold when IRß was present compared with the interaction
in the absence of receptor.
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were not due to variations in IRS-1
expression but, rather, reflected the tyrosine phosphorylation level,
we measured expression of WT, F18, and 3Y-IRS-1 (Fig. 2). No IRS-1 was detected when yeast was
transformed by pACT II alone. Further, F18 and 3Y-IRS-1 mutants were
expressed at similar levels as WT-IRS-1. We then determined the
phosphorylation state of IRS-1 in yeast in the presence or absence of
IRß. We failed to detect tyrosine phosphorylation when IRß
expression was repressed (not shown). However, as shown in Fig. 2
(lower panel), IRß expression clearly induced tyrosine
phosphorylation of WT-IRS-1. Although F18-IRS-1 was expressed (Fig. 2, upper panel), no tyrosine phosphorylation was detected, as
expected. 3Y-IRS-1 with Y608, 628, and 658 was tyrosine phosphorylated,
as shown in Fig. 2 (lower panel), to a lesser extent than
WT-IRS-1, indicating that other tyrosine residues are phosphorylated on
WT-IRS-1.
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in our yeast two-hybrid system; the
fact that IRß is not required may indicate either that yeast kinases
could phosphorylate IRS-1 on PI 3-kinase binding motifs or IRS-1 could
interact in a phosphotyrosine-independent manner; 2) this interaction
is increased by expression of IRß due to increased IRS-1
phosphorylation; and 3) IRS-1 tyrosine phosphorylation by endogenous
yeast kinases would occur at specific sites because F18-IRS-1 shows no
or weak interaction with p85, and 3Y-IRS-1 restored the association
with the PI 3-kinase regulatory subunit compared with WT-IRS-1, showing
that at least one of the tyrosines, 608, 628, or 658, is phosphorylated
and interacts with p85.
Interaction of p55PIK with IRS-1
Next we tested whether p55PIK, a PI 3-kinase
regulatory subunit different from p85, could interact with IRS-1 in
the yeast two-hybrid system. Insulin promotes the tyrosine
phosphorylation of p55PIK on residue 341 in intact cells
(20). In CHO/IR cells, insulin stimulates the binding of
p55PIK to IRS-1 and also the PI 3-kinase activity
associated to p55PIK (20, 22).
p55PIK cDNA was subcloned into pBTM116 vector to obtain the
LexA-p55PIK fusion protein. Then, LexA-p55PIK
or LexA-p85 were coexpressed with GAD-WT-IRS-1, GAD-F18-IRS-1, or
GAD-3Y-IRS-1, and ß-galactosidase activity was measured for each
cotransformant (Fig. 3). As the
interaction occurs in absence of IRß expression, the experiments were
performed without IR using the pLex vector, which has no IRß cDNA. We
found that p85 interacts with WT-IRS-1. This interaction strongly
decreased with F18-IRS-1. With 3Y-IRS-1 mutant, the interaction with
p85 was partially restored compared with WT-IRS-1. Similar to
p85, p55PIK interacted with GAD-WT-IRS-1, and this
interaction was significantly decreased with F18-IRS-1. Finally,
GAD-3Y-IRS-1 interacted with p55PIK, indicating that
phosphorylation of the three YXXM binding sites is sufficient to allow
for the interaction.
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, p55PIK is
able to interact with WT-IRS-1 in a yeast two-hybrid system. For both
PI 3-kinase regulatory subunits, interaction with IRS-1 depends on
phosphotyrosines residues. In the absence of IRß expression in yeast,
we favor the idea that IRS-1 could be phosphorylated on tyrosines
comprised in YXXM motifs, leading to binding of p85 and
p55PIK SH2 domains to those phosphotyrosines.
Role of IRS-1 serines in modulating interaction between IRS-1 and
PI 3-kinase p55PIK and p85 subunits in the
yeast two-hybrid system
In a previous study, we showed that IRS-1 serines 612, 632, 662,
and 731 modulate insulin action in intact 293-EBNA cells. Indeed, their
mutations resulted in increased IRS-1 tyrosine phosphorylation and
association with p85, which was correlated with enhanced IRS-1
associated PI 3-kinase activity (8). The strongest effect was seen with
the double mutant S662A/S731A-IRS-1, in which serines 662 and 731 were
replaced by alanines. Therefore, we were interested in analyzing the
involvement of these serines in the interaction with p85 in the
yeast two-hybrid system. To do this, GAD-WT-IRS-1 or
GAD-S662A/S731A-IRS-1 was coexpressed with LexA-p85, and the
resulting ß-galactosidase activity was quantified. As shown in Fig. 4A, S662A/S731A-IRS-1 expression resulted
in a 4.5-fold increase in ß-galactosidase activity compared with
WT-IRS-1 when coexpressed with p85. This suggests that for binding
p85, S662A/S731A-IRS-1 was more efficient than the WT protein.
Interestingly, we found that when p55PIK was coexpressed
with S662A/S731A-IRS-1, a 4-fold increase in ß-galactosidase activity
was seen compared with that obtained with wild-type IRS-1. As expected,
IRS-1 serine mutations had no effect on IRS-1 interaction with IR
ß-subunit (Fig. 4B). We also verified that these findings were not
due to a difference between WT or S662A/S731A-IRS-1 expression levels
(Fig. 4C).
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and p55PIK, association with IRS-1 is negatively
regulated by serines 662 and 731 of the latter molecule.
Roles of IRS-1 serines 662 and 731 in insulin-stimulated PKB
activity
We have previously shown in 293-EBNA cells overexpressing
S662A/S731A-IRS-1 that insulin-stimulated PI 3- kinase activity is
increased compared with WT-IRS-1-overexpressing cells. We actually
found that both the insulin-stimulated and the basal PI 3-kinase
activities are increased about 2-fold with the double serine mutant. To
investigate further the effects of these IRS-1 serines on downstream
effectors of PI 3-kinase, we wanted to know whether protein kinase B
activity is modified in the presence of the double serine mutant
compared with WT-IRS-1.
In brief, we overexpressed in 293-EBNA cells WT-PKB with
S662A/S731A-IRS-1 or WT-IRS-1; the cells were stimulated or not with
insulin before lysis, and the lysates were subjected to
immunoprecipitation with antibody to PKB. Finally, a PKB kinase assay
on the pellets was performed (Fig. 5).
With S662A/S731A or WT-IRS-1 we found increased stimulation of
endogenous PKB. However, after transfection of only PKB, insulin
stimulated PKB activity about 4 times more. When PKB and WT-IRS-1 were
coexpressed, we observed insulin stimulation of PKB activity that
increases to about 40% compared with PKB alone, whereas cells
incubated with buffer did not show any increased PKB activity. When we
coexpressed WT-PKB and S6623A/S731A-IRS-1, increased insulin-stimulated
PKB activity compared with PKB alone was seen, although the activity is
not different from that in WT-IRS-1- and PKB-coexpressing cells.
Finally, basal PKB activity was increased about 2 times with the double
serine mutant compared with WT-IRS-1. Taking these results together, we
conclude that under basal conditions (i.e. without insulin
treatment), IRS-1 serines 662 and 731 surrounding tyrosines 658 and
727, which are potential binding sites for PI 3-kinase, are negatively
regulating PKB activity. In contrast, those serines do not affect
insulin-stimulated PKB activity.
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Discussion |
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or p55PIK binding to
IRS-1.
We found that both regulatory subunits of PI 3-kinase were able to
interact with IRS-1 in the presence as well as the absence of IRß. At
first glance this observation could indicate that IRS-1 can interact
with p85 in a fashion that does not require tyrosine phosphorylation
of IRS-1. However, as IRS-1/PI 3-kinase interaction is known to be
dependent on tyrosine phosphorylation, this result would rather suggest
that in the absence of IRß, an endogenous yeast kinase could
phosphorylate IRS-1 on tyrosine residues located in YXXM motifs.
Importantly, we show that the F18-IRS-1 mutant, lacking 18 potential
tyrosine phosphorylation sites, was less efficient than wild-type IRS-1
in binding to p85 and p55PIK. Further, addition to
F18-IRS-1 of three tyrosines corresponding to the YXXM motif, which are
binding sites for PI 3-kinase (3Y-IRS-1), resulted in
increased association with regulatory subunits of PI 3-kinase compared
with that of F18-IRS-1. Taken as a whole, these results suggest that in
yeast IRS-1 could be phosphorylated at least in part on tyrosine
residues by endogenous yeast kinases. This phosphorylation would
probably occur on a few residues, as we failed to reveal IRS-1
phosphorylation in yeast by immunoprecipitating IRS-1 followed by
Western blotting with antibodies to phosphotyrosine when IRß
expression is repressed by L-methionine (not shown). As no
classical tyrosine kinases have yet been cloned in yeast, possible
candidate kinases for IRS-1 tyrosine phosphorylation are likely to be
dual specificity kinases, which would possess activity toward threonine
and tyrosine. Indeed, such kinases have actually been identified in
yeast (e.g. Wee1) (33). Those kinases must have the same
specificity as IR toward IRS-1, since they phosphorylate PI
3-kinase-binding sites that are crucial for interaction with PI
3-kinase. However, we cannot rule out the possibility that IRS-1
interacts with p85 in a phosphotyrosine-independent manner, allowing
the p85 SH2 domain to bind IRS-1 nonphosphorylated YXXM motifs.
As 3Y-IRS-1 does not fully restore the interaction with p85 compared
with WT-IRS-1, some other tyrosine-containing sites on IRS-1 are likely
to be phosphorylated and involved in the interaction with p85. When
IRß is expressed, IRS-1 tyrosine phosphorylation is augmented,
leading to increased interaction with the PI 3-kinase regulatory
subunit. Together, our results obtained with 3Y-IRS-1 suggest that 1)
at least one of the tyrosines, 608, 628, or 658, is phosphorylated; and
2) tyrosine residues other than tyrosines 608, 628, and 658 are
involved in the interaction of IRS-1 with p85 and p55PIK
in yeast, because with 3Y-IRS-1 the interaction is not completely
restored compared with that with WT-IRS-1.
Concerning modulation of IRS-1 interaction with PI 3- kinase,
we demonstrate that mutation of IRS-1 serines 662 and 731 to
alanine (S662A/S731A-IRS-1) resulted in increased interaction of IRS-1
mutant with p85 compared with WT-IRS-1. These serines are adjacent
to binding sites for p85 (tyrosine 658 and 727 in YMXM motifs) and
are potential phosphorylation sites for MAP kinases as they are part of
a YMXMSP sequence. Our results extend at the
molecular level our previous work in mammalian cells, where we showed
increased insulin-induced coimmunoprecipitation of p85 with
S662A/S731A-IRS-1 compared with wild-type IRS-1 (8). Moreover, we found
in the yeast two-hybrid system that p55PIK interacted with
S662A/S731A-IRS-1 more efficiently than with WT-IRS-1, suggesting that
the mutated serine residues are also able to modulate binding of
p55PIK to IRS-1. As mutation of serines 662 and 731 does
not affect IRß binding to IRS-1, we favor the idea that this
regulation is specific for association of regulatory PI 3-kinase
subunits with IRS-1. We previously suggested that phosphorylation of
serines 662 and 731 by MAP kinases or related kinases could be
responsible for the inhibitory effect of these residues on IRS-1
binding to p85. Indeed, phosphorylation of these sites would at
least partially prevent insulin-induced tyrosine phosphorylation of
IRS-1 and/or binding of p85 to phosphotyrosine-containing motifs.
Nevertheless, we cannot exclude the possibility that substitution of
serine to alanine by itself allows binding of IRS-1 to PI 3-kinase
regulatory subunits.
Recently, it has been shown that stimulation of protein kinase C by PMA activates MAP kinase, ERK-2, which is able to phosphorylate a synthetic peptide containing the IRS-1 sequence comprising serine 612; this phosphorylation results in inhibition of IR to further phosphorylate this peptide (12, 13). This is particularly interesting because the serine residue, which is indeed included in a MAP kinase consensus sequence, YXXMSP, is just upstream tyrosine 608, a recognized PI 3-kinase motif-binding site. Our findings with the IRS-1 serine mutants in yeast suggest that serine-induced negative regulation could occur in this species. Enzymes homologous to mammalian MAP kinases have been cloned in yeast, and they are likely to be able to phosphorylate IRS-1 on serine/threonine (34). Further, we revealed phosphoserine on IRS-1 immunopurified from yeast using phosphoserine immunoblotting, indicating that IRS-1 is, in fact, phosphorylated on serine residues in yeast (not shown). Hence, our results show that association of regulatory subunits of PI 3-kinase with IRS-1 in yeast can be negatively regulated by serine mutation to alanine. The fact that we did not detect IRS-1 tyrosine phosphorylation in yeast while not expressing IRß, although we detected IRS-1 serine phosphorylation, is probably due to the presence of 35 potential serine/threonine phosphorylation sites on IRS-1 compared with 10 putative tyrosine phosphorylation sites, among which 6 are comprised in YMXM. It has been also shown that IRS-1 is heavily phosphorylated on serine under basal conditions compared with tyrosine phosphorylation (8). Moreover, we can hypothesize that only specific tyrosine residues in the YMXM motif on IRS-1 are phosphorylated by endogenous kinase(s) in yeast, whereas multiple serine sites are phosphorylated.
We further investigated whether serines 662 and 731 that affect the
IRS-1 and PI 3-kinase regulatory subunits interactions would provide
the same regulation on a downstream effector of PI 3-kinase such as
PKB. We show that although serines 662 and 731 of IRS-1 negatively
regulate basal and insulin-stimulated PI 3-kinase activities, only
basal PKB activity is affected by the double serine mutant. We
conclude, therefore, that serines 662 and 731 of IRS-1 negatively
regulate PI 3-kinase and PKB activities in resting cells and are
involved in PI 3-kinase, but have no impact on PKB activity in
insulin-stimulated cells. Phospholipids generated by PI 3-kinase are
thought to bind to the PH (pleckstrin homology) domain of PKB,
leading to membrane targeting and/or activation of PKB. Moreover, these
phospholipids are also involved in activating another protein
downstream of PI 3-kinase that is upstream of and directly activates
PKB, i.e. phospholipid- dependent kinase-1 (35). We can
hypothesize that under our insulin-stimulated conditions, the increased
PI 3-kinase activity seen with the IRS-1 serine mutants that is not
correlated with increased PKB activity is due to the fact that above a
certain threshold level, an increase in phospholipids does not result
in additional activation of phospholipid-dependent kinase-1 and/or PKB.
293 cells pretreated with wortmannin display no or little PKB activity
in the presence or absence of IRS-1 (not shown), demonstrating that in
these cells, PKB activity relies mostly on PI 3-kinase activity. PKB
appears to be involved in key insulin-induced metabolic effects,
including glycogen synthesis, glucose transport, and protein synthesis
(36). As evidence has been gathered suggesting that serine
phosphorylation is implicated in insulin resistance related to tumor
necrosis factor- (10), IRS-1 serine phosphorylation could correspond
to a means for regulating key enzymes in insulin signaling, such as PI
3-kinase and/or PKB.
Roth et al. (13) reported that phorbol ester-stimulated protein kinase C activates Erk kinases, which subsequently phosphorylates an IRS-1 peptide containing serine 612. This peptide then becomes less able to be phosphorylated on tyrosine by IRs.
In insulin treated-cells, inhibition of MAP kinases blocked the PMA-induced negative regulation of IRS-1-associated PI 3-kinase activity. Keeping in mind these observations together with ours, we are tempted to speculate that a large number of agents or growth factors, while activating MAP kinases, could at the same time down-regulate insulin signaling for other specific signals delivered into the cell.
In conclusion, our present work provides illustration at the molecular level of the role of serine/threonine phosphorylation of signaling molecules as a mechanism leading to the modulation of insulin action. Whether such a process is operational at the level of the different IRS molecules and impinges on all aspects of the vast repertoire of insulin actions remains to be investigated.
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Acknowledgments |
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Received March 12, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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- and obesity-induced
insulin resistance. Science 271:665–668[Abstract]
gene. J Biol Chem 271:5317–5320 gene generates three isoforms of
regulatory subunit for phosphatidylinositol 3-kinase (PI 3-kinase),
p50, p55, and p85, with different PI 3-kinase activity
elevating responses to insulin. J Biol Chem 272:7873–7882This article has been cited by other articles:
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