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INTRODUCTION |
An early event in insulin action, after receptor binding, is
activation of tyrosine kinase activity in the cytosolic domain of the
receptor (1). Insulin receptor substrate (IRS)1 proteins
are physiological substrates for insulin
receptor tyrosine kinase (2-5), and tyrosyl-phosphorylated IRS
proteins serve as docking proteins by providing binding sites for SH2
domain containing proteins, including PI 3-kinase, Grb2/Sos, SHP2,
c-Fyn, and Nck (2, 3, 6-8). These events lead to activation of
multiple signaling pathways that are required for insulin pleiotropic
action (6, 9). The importance of IRS proteins in insulin action has
been demonstrated by gene disruption experiment. A lack of IRS-1 or
IRS-2 in mice results in hyperinsulinemia and insulin resistance
(10-13), with disruption of IRS-2 also impairing pancreatic
-cell
function, so that diabetes occurs (13). Thus, IRS proteins are
indispensable for normal insulin action.
IRS-1 and IRS-2 contain more than 30 serine/threonine residues in
consensus sequences for many serine/threonine kinases, including casein
kinase II, cAMP-dependent protein kinase, protein kinase C,
cdc2 kinase, MAP kinase, and Akt/protein kinase B (2, 3, 14, 15).
Recently, serine/threonine phosphorylation of IRS-1 has been linked to
attenuation of insulin signaling in a cell culture system. For
instance, okadaic acid is a potent and specific inhibitor for type 1 and 2A protein phosphatase (16-18). It induces hyper-serine/threonine
phosphorylation of IRS-1 and impairs insulin-induced tyrosine
phosphorylation of IRS-1, PI 3-kinase activation, and glucose uptake in
3T3-L1 adipocytes (16, 19, 20). Moreover, chronically treating cells
with insulin or tumor necrosis factor-
induces serine/threonine
phosphorylation of IRS-1 and impairs insulin action (21-25). In
vitro studies have shown that serine phosphorylation of IRS-1
lowers its tyrosine phosphorylation by the insulin receptor. After
dephosphorylation, IRS-1 regains its ability to be
tyrosyl-phosphorylated (25). Furthermore, serine/threonine phosphorylation of IRS-1 was 4-fold increased in cardiomyocytes of
obese rats and was suggested to play a role in the pathogenesis of the
insulin resistance in these animals (26).
The search for serine/threonine kinases has led to the identification
of several candidates based on their ability to phosphorylate IRS-1
in vitro and in vivo. Casein kinase 2 phosphorylates rat IRS-1 mainly on Thr502, and weakly on
Ser99; however, the major site (Thr502) is not
conserved between human and rat (27). PI 3-kinase possesses dual kinase
activity that phosphorylates inositol lipids at the D-3 position of the
inositol ring and phosphorylates IRS-1 and the p85 regulatory subunit
of PI 3-kinase on serine sites (28-33). More recently, glycogen
synthase kinase 3 and ERK2 have been found to phosphorylate IRS-1
in vitro and in vivo, converting IRS-1 into an
inhibitor of insulin receptor tyrosine kinase (34-36). Despite all of
these observations, the physiological relevance of these serine kinases
to insulin resistance has not been established.
The JCR:LA-cp rat, when homozygous for the corpulent gene (cp/cp), is
obese, hyperphagic, and hyperlipidemic (37). It develops a profound
insulin resistance between 4 and 7 months of age with hyperinsulinemia
and impaired glucose tolerance, although fasting plasma glucose is
normal (38). Rats with normal homozygous (+/+) or heterozygous (cp/+)
are lean and metabolically normal. The JCR:LA-cp rat has been used as a
model to study the metabolic and pathophysiological aspect of insulin
resistance (38, 39).
This study was aimed at defining serine/threonine kinase activity in
chronic insulin-treated cells and obese JCR:LA-cp rats based on an
in vitro kinase assay, which used GST-IRS-1 fragments as
substrates and partially purified cell or tissue extracts as the source
of kinase activity. Elevated serine kinase activity was detected both
in the insulin-resistant culture cells, and in livers and muscles from
obese JCR:LA-cp rats. Phosphorylation of IRS-1 was located exclusively
in the 526-859 amino acid region. Phosphopeptide mapping demonstrated
that this serine kinase activity was not related to MAP kinase
activity, suggesting that it is most likely attributable to a unique
and yet to be identified IRS-1 serine kinase.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
CHO cells overexpressing the human insulin
receptor and rat IRS-1 (CHO/IR/IRS-1) (40) were grown in Ham's F-12
medium supplemented with 10% fetal bovine serum (FBS). Twelve h before
each experiment, the culture medium was changed to serum-free
Dulbecco's modified Eagle's medium with high glucose (DMEM/high
glucose) (40). 32D cells overexpressing the human insulin receptor and
rat IRS-1 (32D/IR/IRS-1) or the human insulin receptor and mouse IRS-2
(32D/IR/IRS-2) were grown in RPMI 1640 medium supplemented with 10%
FBS and 5% WEHI-3B conditional medium (3, 41). Both CHO and 32D cells were cultured at 37 °C in a humidified atmosphere composed of 95%
air and 5% CO2. 3T3-L1 cells were maintained as
fibroblasts in DMEM/high glucose containing 10% calf serum in a 10%
CO2 and humidified environment at 37 °C. They were
differentiated into adipocytes by sequentially growing in DMEM/high
glucose containing 10% FBS, 0.4 µg/ml dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine and 5 µg/ml insulin for 2 days, then DMEM/high glucose containing 10% FBS and 5 µg/ml insulin
for 2 days, and finally DMEM/high glucose containing 10% FBS for an
additional 3 days. With this protocol, more than 80% of cells
differentiated into adipocytes (42).
Animals--
JCR:LA-cp rats in this study were males, bred as
described previously (43). The rats were individually housed and fed
with standard rat chow. The lights were maintained on a reversed cycle, with lights on at 8:00 p.m. and off at 8:00 a.m. Two obese (cp/cp) and
two lean male animals (represented as +/? as bred at 2:1 mixture of
+/cp and +/+) were used in this study initially. They were all 6 months
of age, with fasting blood glucose, triglyceride, and insulin levels of
210 ± 1 mg/dl, 56 ± 1 mg/dl and 45 ± 5 microunits/ml for lean and 201 ± 5 mg/dl, 288 ± 24 mg/dl, and 106 ± 6 microunits/ml for obese rats, respectively. All care and treatment of
the animals was in accordance with guidelines of the Canadian Council
on Animal Care and the National Institutes of Health. The protocols
were subjected to prior approval by the Institutional Animal Care and Use Committees of the University of Alberta and the University of Vermont.
GST Fusion Proteins--
The N-terminal (amino acids 2-516,
designated as IRS-1N), middle (amino acids 526-859,
IRS-1M) and C-terminal (amino acids 900-1235,
IRS-1C) portions of rat IRS-1 protein, and N-terminal
(amino acids 526-693, designated as N-IRS-1M) and
C-terminal (amino acids 721-859, C-IRS-1M) of
IRS-1M were expressed as glutathione
S-transferase (GST) fusion proteins with the pGEX-2T vector
(Amersham Pharmacia Biotech) (44). The DNAs encoding these regions of
IRS-1 were synthesized by the polymerase chain reaction, using a rat
IRS-1 cDNA as a template and pairs of oligonucleotide primers that
contain appropriate restriction sites bordering these fragments (8).
The PCR products were isolated, digested with appropriate restriction
enzymes and subcloned into pGEX-2T, which were used to transform
Escherichia coli DH5
. Transformed cells were grown to an
A600 nm of 0.6 in LB medium supplemented with
0.1 mg/ml ampicillin and induced for 4 h with 0.5 mM
isopropyl-
-D-thiogalactopyranoside (BioStart). Fusion
proteins were purified by affinity chromatography on
glutathione-Sepharose column (Amersham Pharmacia Biotech) and eluted by
glutathione as described by Smith and Johnson (44). Glutathione was
removed by dialysis against phosphate-buffered saline (PBS) containing 10 mM dithiothreitol (DTT). All GST fusion proteins had the
expected molecular weight when analyzed by SDS-PAGE.
Preparation of Cell and Tissue Extracts--
Cells were treated
with insulin (100 nM) for the indicated times, and then
frozen in liquid nitrogen and thawed in cold lysis buffer (10 mM Tris-HCl, pH 7.4, 250 mM sucrose, 100 mM NaF, 5 mM EDTA, 5 mM EGTA, 1 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, 10 µg/ml leupeptin
(Sigma), and 10 µg/ml approtinin (Sigma)). In some case, cells were
also pretreated with 50 µM PD98059 (New England Biolabs)
for 1 h before insulin treatment. Livers and muscles from male
JCR:LA-cp obese (cp/cp) and lean (+/?) rats were rapidly minced and
homogenized in lysis buffer (1 g of tissue/10 ml) with a Brinkmam
homogenizer, followed by centrifugation at 10,000 × g
for 10 min (Sorvall RC-5B). The supernatants were centrifuged at
100,000 × g for 30 min in Beckman L8-M
ultracentrifuge. The supernatants were sequentially precipitated with
(NH4)2SO4 at 30, 50, and 90%
saturation, followed by centrifugation at 100,000 × g
for 30 min for each precipitation.
(NH4)2SO4 precipitates were
redissolved in lysis buffer followed by centrifuging at top speed in a
Biofuge (Heraeus) for 15 min. The recovered supernatants were desalted
and used as the source of kinase for the in vitro kinase assay.
Phosphorylation of IRS-1 Fusion Proteins by Cell
Lysates--
The in vitro kinase assay was carried out in a
final volume of 40 µl of kinase buffer (20 mM HEPES, pH
7.4, 1 mM DTT, 10 mM MgCl2, 100 µg/ml bovine serum albumin, 0.5 µg/ml okadaic acid (Sigma), 50 µM cold ATP, 5 µCi of [
-32P]ATP)
containing 10 µg of substrate and 10 µg of
(NH4)2SO4 precipitate at 30 °C
for 20 min. In some cases, protein kinase A inhibitor (Promega),
myristoylated protein kinase C peptide inhibitor (Promega), or
wartmannin (Sigma) were added at final concentrations of 2.5, 100, and
1 µM, respectively. Reactions were stopped by adding 10 µl of 5 × Laemmli buffer containing 0.5 M DTT and
boiled for 5 min. Proteins were separated by 10% SDS-PAGE, stained,
and destained. Phosphorylated proteins were visualized by
autoradiography. In some cases, protein bands were excised, and counted
on a scintillation counter (MINAXI Tri-CarB 4000, Packard Instrument
Co.).
Phosphorylation of IRS-1 by Recombinant ERK2--
IRS-1 GST
fusion proteins were phosphorylated by a linked in vitro
recombinant MAP kinase assay (45). The constitutively active form of
the MEK protein (kindly provided by Dr. James Posada, University of
Vermont) was used in conjunction with either a wild type (ERK2) or a
kinase-inactive form of ERK2 (ERK2*) (kindly provided by Dr. James
Posada) in kinase buffer (20 mM HEPES, pH 7.5, 10 mM MgCl2, 1 mM EGTA, and 10 µM [
-32P]ATP) at 30 °C for 15 min.
The reactions were terminated by addition of Laemmli sample buffer and
proteins were separated by 10% SDS-PAGE. The phosphorylated substrates
were visualized by autoradiography (45).
Tryptic Peptide Maps and Amino Acid Analysis--
Samples for
tryptic peptide maps were phosphorylated in vitro as
described for the in vitro kinase assay with the exception that 25 µCi of [
-32P]ATP and a 2-h incubation time
were used. After SDS-PAGE separation, brief staining and destaining,
and autoradiography, phosphorylated proteins in the gel were excised,
and incubated at 37 °C in 20% methanol for 4 h.
Phosphoproteins were extracted by homogenizing the gel fragments in 1.2 ml of 50 mM ammonium bicarbonate (pH 7.6) containing 0.1%
SDS and 1%
-mercaptoethanol, followed by incubating at 100 °C
for 5 min and 37 °C for 1.5 h. After centrifugation, phosphoproteins in the supernatants were precipitated by 15%
trichloroacetic acid at 4 °C for 1 h, washed with 0.4 ml
ethanol/ether (1:1, v/v) twice and air dried (46).
For tryptic peptide maps, trichloroacetic acid precipitates were
dissolved in 100 µl of 50 mM ammonium bicarbonate (pH
7.6) containing 0.3 mg/ml L-1-tosylamido-2-phenylethyl
chloromethyl ketone trypsin (Worthington Biochemical Corp.), incubated
at 37 °C overnight, dried in a speedvac, and redissolved in Tricine sample buffer (Bio-Rad). Phosphotryptic peptides were resolved by
16.5% Tricine-PAGE and detected by autoradiography (47).
For phosphoamino acid analysis, the trichloroacetic acid precipitates
were dissolved in 100 µl of 6 N HCl, hydrolyzed at
110 °C for 1 h, and dried in the speedvac. Residues were
dissolved in 10 µl of 0.5 mg/ml standards (phosphotyrosine,
phosphoserine, and phosphothreonine mixture). Samples (2 µl) were
spotted on a precoated TLC cellulose plate (EM Science) and separated
in running buffer (acetic acid:pyridine:water, 10:1:189) by
electrophoresis at 1300 V for 40 min on a Hunter thin layer peptide
mapping system (HTLE-7000, C.B.S. Scientific Co.). Standard
phosphoamino acids were visualized by ninhydrin (Sigma) staining
(0.25% in acetone), and radioactive amino acid was visualized by
autoradiography (40).
Immunoprecipitation and Immunoblot Analysis--
For whole
lysate immunoblot analysis, cells were lysed directly into Laemmli
sample buffer containing 0.1 M DTT, sonicated and boiled
for 5 min. For MAP kinase immunoblotting, partially purified cell
lysates or both wild type recombinant MAP kinase (ERK2) and
kinase-deficient enzyme (ERK2*) were boiled in Lammli sample buffer for
5 min. For immunoprecipitation, cells were lysed in homogenization
buffer (20 mM Tris-HCl (pH 7.5), 137 mM NaCl, 100 mM NaF, 1 mM MgCl2, 1 mM CaCl2, 200 µM sodium
orthovanadate, 0.4 mM phenylmethylsulfonyl fluoride, 50 µg/ml aprotinin, 50 µg/ml leupeptin, 10% glycerol, and 1% Nonidet
P-40 (Calbiochem)) and centrifuged at 13,000 rpm for 15 min (40). The
supernatants were incubated with anti-IRS-1 antibody (
IRS-1) or
anti-
subunit of the insulin receptor antibody (
IR) (C-19, Santa
Cruz), and the immunocomplexes were washed three times with
homogenization buffer, and denatured in Laemmli buffer containing 0.1 M DTT. Proteins in cell lysates or immunoprecipitates were
separated by 6 or 10% SDS-PAGE, and transferred to a nitrocellulose
membrane. The membrane was blocked overnight at 4 °C with 1% milk
and 1% bovine serum albumin in TBS (20 mM Tris-HCl, pH
8.0, 0.15 M NaCl) and incubated with either
anti-phosphotyrosine antibody (PY20, Santa Cruz) at 1:1000,
IRS-1 or
IRS-2 at 1:300,
IR at 1:300, or anti-ERK2 (
ERK2) (C-14, Santa
Cruz) at 1:300 in TBST (TBS with 0.05% Tween-20) for 1 h. The
membrane was washed three times with TBST, and then probed with HRP
conjugated protein A or protein G (Santa Cruz) at 1:3000 for 30 min,
washed three times with TBST and once with TBS. Specific proteins were
visualized by using an enhanced chemiluminescence system
(SuperSignalTM, Pierce).
[32P]Phosphate in Vivo Labeling--
Cells were
plated in 6-well plates, grown to subconfluence and labeled in 2 ml/well RPMI 1640 medium without sodium phosphate (Life Technologies,
Inc.) containing 0.5 mCi/ml [32P]orthophosphate (NEN Life
Science Products) at 37 °C in the absence or presence of 100 nM of insulin for 4 h. The cells were incubated for an
additional 2 min with or without 100 nM insulin before being lysed in homogenization buffer. IRS-1 was immunoprecipitated with
IRS-1, separated by 7.5% SDS-PAGE, and phosphoproteins were visualized by autoradiography (40).
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RESULTS |
Chronic Insulin Treatment Decreases Insulin-induced Tyrosine
Phosphorylation and Increases Serine/Threonine Phosphorylation of IRS
Proteins--
32D cells overexpressing the insulin receptor and IRS-1
or IRS-2 (32D/IR/IRS-1 and 32D/IR/IRS-2) were exposed to control medium or medium containing 100 nM of insulin for 4 or 12 h
and then acutely treated with insulin for 2 min. Acute insulin exposure increased tyrosine phosphorylation of both IRS-1 and IRS-2 in the cells
exposed to control medium (Fig. 1A,
upper panel, lanes a versus b and g versus
h), but failed to induce tyrosine phosphorylation of IRS
proteins after exposure to 4 or 12 h of insulin (Fig. 1A, upper panel, lanes c-f and i-l). IRS-1 and IRS-2
protein levels were assessed by
IRS-1 or
IRS-2 immunoblotting.
There was no significant change of either with the insulin treatment
(Fig. 1A, lower panel). The same results were obtained when
CHO/IR/IRS-1 cells were exposed to 4 h of insulin although IRS-1
protein level also appeared to slightly decrease (Fig.
1B).

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Fig. 1.
The insulin-stimulated tyrosine
phosphorylation of IRS-1/IRS-2 in chronic insulin-treated cells.
32D/IR/IRS-1 cells and 32D/IR/IRS-2 cells (A) and
CHO/IR/IRS-1 cells (B) were chronically treated without or
with insulin (100 nM) for indicated times, and acutely
stimulated or left unstimulated for 2 min before being lysed in Laemmli
sample buffer. Proteins were separated by 6% SDS-PAGE, transferred to
nitrocellulose paper, and immunoblotted (IB) with the
indicated antibodies. C, the insulin receptor was
immunoprecipitated (IP) from insulin-treated or untreated
CHO/IR/IRS-1 cells, separated by 7.5% SDS-PAGE, and immunoblotted with
either anti-phosphotyrosine antibody ( PY) or IR.
D, 32D/IR/IRS-1 and CHO/IR/IRS-1 cells were labeled with
[32P]orthophosphate for 4 h in the presence or
absence of 100 nM insulin and stimulated with 100 nM insulin for 2 min before being lysed. IRS-1 was
immunoprecipitated with IRS-1 and separated by 7.5% SDS-PAGE.
Phosphorylated IRS-1 was visualized by autoradiography.
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To see whether the reduced tyrosine phosphorylation of IRS proteins is
mediated at the level of the insulin receptor, autophosphorylation and
the protein level of IRs were measured in CHO/IR/IRS-1 cells by
immunoprecipitation with
IR and immunoblotting with
IR or anti-phosphotyrosine antibody. Both were unaffected by 4 h of insulin treatment (Fig. 1C). Thus, impaired insulin-induced
tyrosine phosphorylation of IRS-1 is not due to the defect in the
insulin receptor.
Overall phosphorylation of IRS-1 was determined by 32P
in vivo labeling of CHO/IR/IRS-1 and 32D/IR/IRS-1 cells,
followed by
IRS-1 immunoprecipitation (Fig. 1D) (40). In
contrast to the dramatically decreased tyrosyl phosphorylation of IRS-1
(Fig. 1, A and B), overall phosphate content of
IRS-1 was increased in both cell lines after chronic insulin treatment,
suggesting that serine/threonine phosphorylation was increased (Fig.
1D).
Elevated Serine Kinase Activity in Chronic Insulin-treated Cells
and in Liver and Muscle of JCR:LA-cp Rats--
To determine
serine/threonine kinase activity in chronic insulin-treated cells,
lysates prepared from CHO/IR/IRS-1 cells were fractionated by
(NH4)2SO4 precipitation into 30, 50, and 90% fractions, which were used as sources of kinase to
phosphorylate GST-IRS-1 fragments (the N-terminal region of 2-516
amino acids designated IRS-1N, the middle region of
526-859 amino acids designated IRS-1M, and the C-terminal
region of 900-1235 amino acids designated IRS-1C (Fig.
2A)) in an in vitro
kinase assay. GST-IRS-1N, GST-IRS-1M, and
GST-IRS-1C were mainly phosphorylated by the 50%
(NH4)2SO4 fractions and very weakly
by the 30% and 90% fractions (Fig. 2B). There was no
significant difference in the phosphorylation of GST-IRS-1N
and GST-IRS-1C by any of the three fractions when the
insulin-treated and untreated cells were compared (Fig. 2B,
left and right panels). In contrast, insulin treatment
(4 and 12 h) significantly enhanced kinase activity in the 50%
fraction for the GST-IRS-1M fragment (Fig. 2B, middle
panel, lanes e and f versus d).
When 3T3-L1 adipocytes, a more physiological relevant cell line, were examined for the effect of chronic insulin treatment, the same result
was obtained (Fig. 3A).

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Fig. 2.
Phosphorylation of IRS-1 fragments by cell
lysates. A, construction of GST fusion proteins
containing various regions of IRS-1. IRS-1N contains the
amino acid 2-516 region, IRS-1M the amino acid 526-859
region, and IRS-1C the amino acid 900-1235 region of rat
IRS-1 protein. The pleckstrin homology domain (PH),
phosphotyrosine binding domain (PTB), and tyrosine
phosphorylation region (PY) are indicated. B,
CHO/IR/IRS-1 cells were chronically treated with insulin for 4 and
12 h or left untreated. Cell lysates were prepared and
precipitated sequentially by
(NH4)2SO4 at the indicated
saturations. GST-IRS-1N, GST-IRS-1M, and
GST-IRS-1C were phosphorylated by
(NH4)2SO4 fractions in an in
vitro kinase assay, separated by 10% SDS-PAGE, and visualized by
autoradiography. The result is a representative of three separate
experiments. GST fusion proteins are indicated by arrows.
C, GST-IRS-1M was phosphorylated by the 50%
(NH4)2SO4 lysates fraction in the
absence or presence of indicated inhibitors in duplicates. The result
is a representative of two experiments.
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Fig. 3.
Phosphorylation of IRS-1 fragments by lysates
from 3T3-L1 adipocytes, liver and muscle of JCR: LA-cp rats and
phosphoamino acid analysis. Lysates were prepared from 3T3-L1
adipocytes (A) and liver and muscle isolated from lean and
obese rats (B) as described under "Experimental
Procedures," precipitated at the 50% saturation of
(NH4)2SO4, and re-suspended in
lysate buffer. GST-IRS-1 fragments were phosphorylated with the
(NH4)2SO4 precipitates by the
in vitro kinase assay. Phosphorylated proteins were
separated by 10% SDS-PAGE and visualized by autoradiography. The
result is a representative of at least three experiments. C,
phosphorylated GST-IRS-1M was extracted from gel, and
hydrolyzed by 6 N HCl at 110 °C for 1 h.
Phosphoamino acids, together with standards, were separated on a TLC
plate and visualized by Ninhydrin staining, and radioactive
phosphoamino acids were detected by autoradiography. Phosphoamino acid
standards are indicated. The result is a representative of at least two
experiments.
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Phosphorylation of GST-IRS-1M in vitro by the
50% (NH4)2SO4 fraction was tested
in the presence of a protein kinase C inhibitor, protein kinase A
inhibitor, and wortmannin, a potent inhibitor for PI 3-kinase. None of
the inhibitors altered the chronic insulin-induced serine kinase
activity (Fig. 2C).
We next examined kinase activity in lysates prepared from the liver and
muscle of JCR:LA-cp lean and obese rats (6 months of age). Lysates were
precipitated with 50% saturation of
(NH4)2SO4, and kinase activity was
measured by the phosphorylation of GST-IRS-1 fragments in our in
vitro kinase assay. Consistent with the results from CHO/IR/IRS-1
cells, increased kinase activity of GST-IRS-1M was detected
both in the liver and muscle of obese rats, when compared with that of
lean animals (Fig. 3B, lanes f versus e and h
versus g). In contrast, kinase activity was not
different in the lean and obese rats for the GST-IRS-1N and
GST-IRS-1C fragments (Fig. 3B, lanes a-d and
i-l). This enhanced serine kinase activity is not due to a
different amount of proteins applied to our in vitro kinase
assay, because 1) the same amounts of total proteins from obese and
lean tissues were applied in the assay, 2) Coomassie Blue staining of
SDS-PAGE actually showed the same levels of proteins in both obese and
lean samples (data not shown), and 3) phosphorylation of
GST-IRS-1N or GST-IRS-1C with the lysates
preparation from obese and lean tissues showed no significant
difference (Fig. 3B, lanes a-d and i-l). One
note of caution: the results with liver were reproducible on each
determination (n = 6) as opposed to muscle, which at
times failed to show observable increased kinase activity. We felt the
latter reflects difficulty in preparing and working with tissue
extracts from skeletal muscle.
The nature of phosphoamino acids was determined by phosphoamino acid
analysis for the GST-IRS-1M phosphorylated by the 50%
(NH4)2SO4 fraction prepared from
chronic insulin-treated cells and JCR:LA-cp rats. Phosphoserine was the phosphoamino acid in all samples as opposed to virtually no
phosphothreonine or phosphotyrosine (Fig. 3C). Phosphoserine
content was increased in GST-IRS-1M phosphorylated by
chronic insulin-treated cells or tissues of obese rat (Fig. 3C,
lanes b, d, f and h versus lanes a, c, e and g). Thus, the enhanced serine kinase activity that
phosphorylate the 526-859 amino acid region of IRS-1 was correlated
with insulin-resistant cells and animal models.
The in Vitro Phosphorylation of IRS-1 in the Insulin-resistant
State Stems from a Serine Kinase Activity Other Than a MAP
Kinase--
MAP kinase has been shown to phosphorylate IRS-1 on
Ser612, which is included in the GST-IRS-1M
fragment together with another potential MAP kinase site
(Ser632) (35, 36, 48). To determine whether or not the
elevated kinase activity is MAP kinase, two new GST fusion proteins
were created, one containing the N-terminal portion of
GST-IRS-1M amino acids 526-693 containing both MAP kinase
sites (designated as GST-N-IRS-1M), and another, the
C-terminal portion of GST-IRS-1M amino acids 721-859
lacking any MAP kinase sites (designated as GST-C-IRS-1M)
(Fig. 4A).
GST-IRS-1M together with GST-N-IRS-1M and
GST-C-IRS-1M were then phosphorylated in vitro
by recombinant MAP kinases (wild type ERK2 or kinase-deficient ERK2*)
and the 50% (NH4)2SO4 fraction
prepared from chronic insulin-treated cells and tissues from JCR:LA-cp
rats (Fig. 4B). As expected, wild type ERK2 phosphorylated GST-IRS-1M and GST-N-IRS-1M (Fig. 4B,
lanes 5 and 15), whereas phosphorylation of
GST-C-IRS-1M was barely detected (Fig. 4B, lane
25). None of the substrates were phosphorylated by
kinase-deficient ERK2* (Fig. 4B, lanes 6, 16, and
26). In contrast, the GST-N-IRS-1M and
GST-C-IRS-1M both increased its phosphorylation with 50%
(NH4)2SO4 fractions from chronic
insulin-treated CHO/IR/IRS-1 cells (Fig. 4B, lanes 1, 2, 11, 12, 21, and 22). Pretreatment with PD98059, a potent MEK
inhibitor (49, 50) to block the activation of MAP kinase prior to
chronic insulin treatment decreased serine kinase activity toward
GST-IRS-1M and GST-N-IRS-1M, but had little
effect on the phosphorylation of GST-C-IRS-1M (Fig.
4B, lanes 3 and 4 versus 1 and
2, 13 and 14 versus 11 and 12, and
23 and 24 versus 21 and 22). The
enhanced serine kinase activity in muscle of obese JCR:LA-cp rats also
phosphorylated both GST-N-IRS-1M and
GST-C-IRS-1M (Fig. 4B, lanes 19-20, 29, and
30). In contrast, the enhanced serine kinase in liver of
obese rat only phosphorylated GST-C-IRS-1M, and barely
phosphorylated GST-N-IRS-1M (Fig. 4B, lanes 17, 18, 27, and 28). Note that the protein level of ERK2 used
in the in vitro kinase assay was very similar between CHO/IR/IRS-1 cells and recombinant enzymes, but lower in rat liver and
muscle as measured by Western blotting analysis (Fig. 4C). Together, these results clearly show that a serine kinase other than a
MAP kinase is activated in the chronic insulin-treated cells and
insulin-resistant animals to phosphorylate
GST-C-IRS-1M.

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Fig. 4.
Phosphorylation of IRS-1 fragments by
enhanced serine kinase activity and recombinant MAP kinase, and
phosphotryptic peptide mapping analysis. A,
construction of GST fusion proteins containing N- and C-terminal
portion of IRS-1M. Potential serine phosphorylation sites
for MAP kinase are indicated. B, GST fusion proteins were
phosphorylated in an in vitro kinase assay by a wild type
ERK2 or a kinase-deficient ERK2* conjunction with active MEK, and the
50% (NH4)2SO4 fraction prepared
from chronic insulin-treated CHO/IR/IRS-1 cells pretreated with or
without PD98059, and liver and muscle of JCR:LA-cp rats. Phosphorylated
GST fusion proteins were separated by 10% SDS-PAGE and visualized by
autoradiography. Positions of corresponding GST fusion proteins are
indicated by arrows. C, MAP kinase immunoblotting
analysis. Proteins in 50%
(NH4)2SO4 fractions or recombinant
ERK2 used for the in vitro kinase assay were separated by
10% SDS-PAGE and transferred to nitrocellulose membrane. The membrane
was immunoblotted with anti-ERK2 and visualized by an enhanced
chemiluminescence system. D, phosphotryptic peptide mapping
analysis. Phosphorylated GST fusion proteins were extracted from gels
and subjected for complete tryptic digestion. Phosphotryptic peptides
were separated by 16.5% Tricine-PAGE and visualized by
autoradiography. Major phosphopeptides were marked on both side by
arrows. The result is a representative of at least two
separate experiments.
|
|
Furthermore, if the elevated serine kinase activities in the
insulin-resistant state is not MAP kinase, presumably it phosphorylates IRS-1 at different serine sites. To confirm this, we performed phosphopeptide map analysis on phosphorylated GST-IRS-1 fusion proteins. Three major phosphopeptides were identified in phosphorylated GST-IRS-1M, designated P1, P2, and P3, which displayed
increased phosphorylation after chronic insulin treatment in
CHO/IR/IRS-1 cells with P2 being predominant relative to P1 and P3
(Fig. 4D, lanes c and d). Recombinant ERK2
phosphorylated P1 and P2, but not P3 (Fig. 4D, lane i).
Consistent with this data, pretreatment of cells with PD98059 (MEK
inhibitor) decreased phosphorylation of P1 and P2 without affecting P3
phosphorylation (Fig. 4D, lanes g and h versus e
and f), suggesting that P1/P2 are MAP kinase phosphorylation sites and P3 is likely to be phosphorylated by another serine kinase.
Importantly, P3 was observed in all the samples tested, and was
predominant in liver of the obese rat and 3T3-L1 adipocytes, but
relatively low in muscle of the obese rat relative to P1/P2 (Fig.
4D, lanes a-b and j-m). Interestingly, P1/P2
were not phosphorylated by the enhanced serine kinase from liver of
obese rat (Fig. 4D, lanes j and k).
Phosphopeptide maps were also analyzed for the GST-N-IRS-1M
and GST-C-IRS-1M phosphorylated by the enhanced serine
kinase activity from JCR:LA-cp rats and recombinant ERK2. As expected,
P1/P2 in GST-N-IRS-1M were strongly phosphorylated by
recombinant ERK2 and weakly by the serine kinase from muscle of obese
rat, whereas the phosphorylation of P1/P2 was undetectable in liver of
obese rat (Fig. 4D, lanes n-r). In contrast,
GST-C-IRS-1M contained P3, which is not phosphorylated by
recombinant ERK2 at all, but strongly phosphorylated by the
liver of obese rat, and weakly by the muscle of obese rat(Fig.
4D, lanes s-x). Taken together, these results
suggest that phosphorylation of P1/P2 reflects the MAP kinase
activity that is not active at lease in the liver of JCR:LA-cp
obese rats, whereas P3 is the common phosphopeptide in our
insulin-resistant cell and animal models and is phosphorylated by a yet-to-be-identified serine kinase.
 |
DISCUSSION |
Serine/threonine phosphorylation of IRS-1 has been implicated in
attenuation of insulin action (16, 19-26). To identify
serine/threonine kinase activity in insulin-resistant models, we have
developed an in vitro kinase assay that is based on using
GST-IRS-1 fragments as substrates and partially purified cell extracts
as the source of kinase. This avoided the high background encountered
with in vivo phosphorylation studies (40) and enabled us to
measure the changes of serine kinase activity in tissues originating
from insulin-resistant animals. By combining the in vitro
kinase assay with phosphotryptic peptide mapping analysis, we have
identified a novel serine kinase activity designated as P3 serine
kinase because it phosphorylates the P3 peptide of IRS-1. This serine kinase activity was elevated in chronic insulin-treated CHO/IR/IRS-1 cells and 3T3-L1 adipocytes, and liver and muscle tissues from JCR:LA-cp obese rats, strongly suggesting that it is a feature of
insulin resistance in general. Finally, we provided strong evidence
that the P3 serine kinase is neither a MAP kinase nor a PI
3-kinase.
Chronic insulin treatment (mimicking hyperinsulinemia) induces insulin
resistance in 3T3-L1 adipocytes, normal rats and healthy human subjects
(22, 51-53). In order to investigate serine phosphorylation of IRS-1
in insulin resistance, we first measured tyrosine phosphorylation of
IRS-1 and serine kinase activity in culture cells that were chronically
treated with insulin. Phosphotyrosine content in IRS proteins was
drastically reduced in 32D/IR/IRS-1, 32D/IR/IRS-2, and CHO/IR/IRS-1
cells. Furthermore, IRS-1 and IRS-2 failed to respond to an acute
insulin stimulation in terms of tyrosyl phosphorylation. This
insensitivity was not due to a defect in the insulin receptor, because
receptor autophosphorylation and the receptor protein level were not
altered by the chronic insulin treatment. IRS protein levels were
unaffected in 32D cells, suggesting that changes in the IRS protein
levels does not account for the insensitivity. In contrast, overall
phosphate content in IRS-1 was increased by the chronic insulin
treatment, suggesting the increased serine and/or threonine
phosphorylation of IRS-1. Consistent with this finding, serine kinase
activity was elevated in chronically insulin-treated CHO/IR/IRS-1cells
and 3T3-L1 adipocytes. Of greatest potential significance, high serine
kinase activity was also detected in liver and muscle tissues from
insulin-resistant JCR:LA-cp rats. Viewed together, our results show a
consistent finding of IRS-1 serine phosphorylation in both in
vitro and in vivo insulin-resistant states.
We also investigated possible candidates for the serine kinase.
Activation of protein kinase C induces serine phosphorylation of IRS-1
and attenuates insulin action (48). This stems from the activation of
MAP kinase, which phosphorylates IRS-1 at Ser612 in
vitro and in vivo (35, 36). Our results showed that the enhanced serine kinase activity in the insulin-resistant cells and
tissues specifically phosphorylated GST-IRS-1M, but not
GST-IRS-1N and GST-IRS-1C.
GST-IRS-1M contains two consensus sequences for MAP kinase
(PxSP), Ser612 and Ser632. By phosphopeptide
mapping, three major phosphopeptides (P1, P2, and P3) were identified
in GST-IRS-1M that could be phosphorylated by the serine
kinase activity in cells and tissues of insulin-resistant models. P1
and P2, but not P3, were phosphorylated by recombinant MAP kinase
(ERK2), and therefore, they most likely represent MAP kinase activity. However, P1 and P2 phosphorylation did not perfectly correlate with the
serine phosphorylation in all of the tested models, particularly in the
liver of insulin-resistant animal, which argues against MAP kinase
being the serine kinase that phosphorylates IRS-1 in insulin-resistant states.
The serine kinase that phosphorylates P3 is unknown and was tentatively
designated P3 serine kinase. P3 serine kinase was significantly
enhanced in all of the tested insulin-resistant samples and in fact is
the most consistent observation across all of our systems. The current
study provides definitive evidence against MAP kinase being operative
in the phosphorylation of P3 because 1) the P3 phosphopeptide was
identified in amino acids 721-859 region of IRS-1
(GST-C-IRS-1M), which contains no phosphorylation sites for
MAP kinase, 2) recombinant MAP kinase failed to phosphorylate
GST-C-IRS-1M and P3 peptide, and 3) pretreatment of cells
with MEK inhibitor did not decrease P3 serine kinase activity in
chronic insulin-treated CHO/IR/IRS-1 cells. We do not believe that it
is PI 3-kinase, either, because wortmannin had no effect on the serine
kinase activity. Moreover, activation of MAP kinase (22, 54) and PI
3-kinase (22, 55-57) has been reported to be impaired in
insulin-resistant cultured cells, insulin-resistant mice, and diabetes.
Thus, the identity of P3 serine kinase remains unknown.
In summary, we found a novel elevated serine kinase activity that
phosphorylates IRS-1 in lysates from insulin-resistant animals and cell
models. The serine phosphorylation site was located in the 721-859
region of IRS-1. We speculate that serine phosphorylation of this
region of IRS-1 impairs its functional role in insulin action perhaps
through altering tyrosyl phosphorylation of IRS-1. Serine
phosphorylation of IRS-1 seems to be important for the binding of IRS-1
to 14-3-3 protein (58-60). Interestingly, the amino acids 516-865
region of IRS-1, which has been found to interact with 14-3-3, is
similar to the corresponding region of IRS-1M (amino acids
526-859). 14-3-3 proteins have been found to associate with a number
of signaling proteins, including Raf, Bcr-Abl, polyoma virus middle T
antigen, PI 3-kinase, the proto-oncogene product Cbl, and cdc25
phosphatase, implicating an important role in mitogenesis or cellular
transformation (60). Thus, the serine phosphorylation of IRS-1 in
insulin-resistant states may lead to altering the binding of 14-3-3 proteins. We were currently investigating this possibility.
Purification of P3 serine kinase and determination of the
phosphorylation sites within the 721-859 region responsible for the
attenuation of insulin signaling should greatly facilitate the
investigation of the role of serine phosphorylation of IRS proteins in
insulin action.