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(Received for publication, December 23,
1994; and in revised form, April 20, 1995) From the
We have demonstrated previously that dexamethasone treatment of
HepG2 cells caused an enhancement of insulin's metabolic effects
(Kosaki, A., and Webster, N. J.(1993) J. Biol. Chem. 268,
21990-21996). This correlated with increased expression of the
mRNA encoding the B isoform of the insulin receptor (IR). In the
present study, we have demonstrated that dexamethasone treatment
caused in addition an enhancement in insulin-stimulated immediate-early
gene expression (c-fos and egr-1). Dexamethasone
treatment caused an increase in in vivo IR autophosphorylation
and insulin receptor substrate-1 (IRS-1) phosphorylation both early
events in the insulin signaling pathway. Furthermore, the IRS-1
phosphorylation was distinctly left shifted, although the level of
IRS-1 protein was unchanged. Total cellular tyrosine phosphatase
activity was unaltered when assayed with In conclusion, it appears that the B isoform of the IR
signals more efficiently than the A isoform in HepG2 cells.
The insulin receptor (IR) ( The HepG2 cell is derived
from a human hepatoblastoma and has been useful as a model for liver
one of the major sites of insulin action (16, 17) . We
have shown previously that dexamethasone (dex) causes an increase in
sensitivity and responsiveness for insulin's metabolic effects
(glucose incorporation into glycogen and 2-deoxyglucose transport) in
these cells. This correlated with a switch in expression from the A
(-exon 11) to the B (+exon 11) isoform of the IR similar to
the ratio seen in adult liver(18) . The aim of the present
study was to investigate whether similar enhancements would be seen for
insulin's mitogenic effects and to determine whether the
expression of the B isoform of the IR is responsible for the
enhancement. We show that dex treatment does cause an enhancement in
insulin-stimulated immediate-early gene expression (c-fos and egr-1). The B isoform couples more efficiently to the insulin
receptor substrate 1 (IRS-1) in vivo than the A isoform.
Furthermore, insulin-stimulated autophosphorylation and kinase activity in vitro are greater for the B isoform of the IR. Tryptic
peptide mapping identified a novel phosphopeptide in the B isoform that
may be involved in the enhanced kinase activity and/or signaling.
The c-fos primer pair consisted of oligonucleotides spanning nucleotides
140-169 (sense primer, 5`-GTTCTCGGGTTTCAACGCGGACTACGAGGC-3`) and
276-309 (antisense primer,
5`-GGCACTAGAGACGGACAGATCTGCGCAAAAGTCC-3`) which generate a fragment of
169 base pairs following amplification. The egr-1 primer pair
consisted of oligonucleotides spanning nucleotides 539-568 (sense
primer, 5`-GAGCCGAGCGAACAACCCTACGAGCACCTG-3`) and 763-791
(antisense primer, 5`-GCGCTGAGGATGAAGAGGTTGGAGGGTTGG-3`) which generate
a fragment of 252 base pairs following amplification. The L30 primer
pair consisted of oligonucleotides spanning nucleotides 74-98
(sense primer, 5`-GAAAGTACGTGCTGGGGTACAAACAGACTC-3`) and either
285-309 (antisense primer for c-fos measurement,
5`-ATCGGAATCACCTGGGTCAATGATAGCCAG-3`) or 224-254 (antisense
primer for egr-1 measurement,
5`-CCACACGCTGTGCCCAATTCAATGTTATTGC-3`) which generate a fragment of 238
and 181 base pairs, respectively. Five µl of the cDNA synthesis
reaction was used for polymerase chain reaction (PCR)
For the
IRS-1 phosphorylation assay, equal amounts of WGA-purified receptor
were preincubated with increasing concentrations of insulin for 16 h at
4 °C, followed by the addition of 30 µCi of
[
For the immunoprecipitation study, the
Figure 1:
Insulin-stimulated c-fos and egr-1 gene expression. HepG2 cells were cultured with and
without 1 µM dexamethasone (Dex) in Ham's
F-12 mixture for 4 days and stimulated with increasing concentrations
of insulin for 45 min at 37 °C. The total cellular RNA was
extracted and subjected to RT-PCR to determine c-fos and egr-1 gene expression using the ribosomal protein L30 mRNA as
internal standard. Panel A, insulin stimulation of c-fos gene expression. B, insulin dose-response curves for
c-fos expression. Results are the mean ± S.E. of four
experiments and are normalized to L30 mRNA levels. Maximal insulin
stimulation is 1.6-fold higher in the presence of dexamethasone (p < 0.01, n = 4). The ED
Figure 2:
Insulin-stimulated tyrosine
phosphorylation in whole cells. HepG2 cells were cultured with and
without 1 µM dex (Dex) in Ham's F-12
mixture for 4 days and stimulated with increasing concentrations of
insulin for 1 min at 37 °C. Cells were solubilized and aliquots,
equalized for either insulin binding (panels A-C) or cell
number (panels D and E), were separated by SDS-PAGE,
and transferred to an Immobilon-P membrane. Tyrosine phosphorylated
proteins, IR, and IRS-1 were visualized by immunoblotting followed by
ECL detection. The band densities were quantified by densitometry. Panel A, anti-phosphotyrosine immunoblot. The
One possible explanation for the increase in
sensitivity and maximal phosphorylation of IRS-1 seen in cells treated
with dex is that the level of IRS-1 protein has increased.
Consequently, we measured IR and IRS-1 levels by immunoblotting. Cell
lysates from equal numbers of the cells were immunoblotted with a
polyclonal anti-IR antibody (Fig. 2D). The amount of IR
was 1.5-fold higher in cells treated with dex similar to the increment
in insulin binding that we reported previously(18) .
Immunoblotting with an anti-IRS-1 antibody indicated that dex treatment
did not alter the amount of IRS-1 protein (Fig. 2E). It
should be noted that the increase in IR protein is not the origin of
the increased in vivo autophosphorylation as extracts were
normalized for insulin binding prior to antiphosphotyrosine
immunoblotting (Fig. 2A). However, IRS-1 levels are
unaffected by dex treatment so less IRS-1 is loaded in the
antiphosphotyrosine immunoblot for cells treated with dex. When
adjusted for equal cell number and therefore equal IRS-1 levels, the
maximal insulin-stimulated phosphorylation of IRS-1 is 2.2-fold higher
in cells treated with dex rather than the 1.3-fold shown in Fig. 2A (data not shown).
Figure 3:
In vitro autophosphorylation of
IR isoforms. HepG2 cells were cultured with and without 1 µM dex (Dex) in Ham's F-12 mixture for 4 days. IRs
were purified by WGA affinity chromatography. Equal numbers of IRs,
normalized by insulin binding, were subjected to in vitro autophosphorylation in the presence of
[
Figure 4:
In vitro kinase activity of IR
isoforms. HepG2 cells were cultured with and without 1 µM dex (Dex) in Ham's F-12 mixture for 4 days.
WGA-purified IRs normalized for insulin binding were subjected to an in vitro kinase assay using poly-glu
Figure 5:
Two-dimensional tryptic phosphopeptide
maps of IR isoforms. WGA purified IRs from cells cultured without (panel A) and with (panel B) dex were incubated with
100 nM insulin for 16 h at 4 °C, followed by the addition
of 30 µCi of [
The position of phosphopeptide E suggested that it may be derived
from the carboxyl terminus of the
Figure 6:
Immunoprecipitation of tryptic fragments
of IR. IRs were labeled and digested as in Fig. 5. The
tryptic-phosphopeptides were incubated with an anti-COOH-terminal
peptide antibody for 16 h and then with Protein A-Sepharose for 2 h in
500 µl of 100 mM NMA at 4 °C. Sepharose-bound antibody
was recovered by centrifugation and washed twice with 1 ml of NMA.
Phosphopeptide was eluted by resuspending the complex in 500 µl of
1 M acetic acid containing 10 µg of the cold carrier
peptide at 4 °C for 30 min. Acetic acid was removed by rotary
evaporation and the phosphopeptide was washed extensively with water.
Then
Figure 7:
Two-dimensional tryptic peptide maps of
IRS-1. Equal numbers of IRs from cells cultured without (panel
A) and with (panel B) dex were preincubated with 100
nM insulin for 16 h at 4 °C, followed by the addition of
30 µCi of [
Figure 8:
Effect of dexamethasone on phosphatase
activity in HepG2 Cells. Cells cultured with and without dex (Dex) were homogenized in lysis buffer (see
``Experimental Procedures''). After centrifugation, the
Triton X-100-soluble total fraction was incubated with in vitro
We have published previously that dexamethasone treatment of
HepG2 hepatoma cells causes a switch in insulin receptor isoform
expression from A to B. This alteration is accompanied by an increase
in responsiveness and insulin sensitivity for two of insulin's
metabolic effects, namely glucose incorporation into glycogen and
2-deoxyglucose transport. Changes in isoform expression and insulin
response are also observed during the differentiation of 3T3-L1
adipocytes. In the present study, we demonstrate that similar
enhancements are observed for two of insulin's mitogenic effects,
namely induction of the c-fos and egr-1 genes. This
suggested to us that it was expression of the B isoform of the IR upon
dexamethasone treatment that caused the enhanced signaling as the
metabolic and mitogenic pathways are thought to diverge at a very early
point, perhaps at the insulin receptor itself. Consequently, we
examined two of the earliest events in the signaling pathway, IR
autophosphorylation and IRS-1 phosphorylation in vivo. Maximal
insulin-stimulated phosphorylation of both proteins was elevated in
cells treated with dexamethasone. Furthermore, the sensitivity for
IRS-1 phosphorylation, but not for IR autophosphorylation, was
increased. This increase in sensitivity for IRS-1 phosphorylation is
likely to be the origin for the observed enhancement in signaling as it
has been demonstrated that IRS-1 is involved in both insulin-stimulated
DNA synthesis in Rat-1 fibroblasts and Glut-4 translocation in 3T3-L1
cells(28, 29) . Tyrosine phosphorylation is
regulated in vivo by a balance of kinase and phosphatase
activities. Alteration in either activity could cause the observed
increase in phosphorylation. Therefore, we undertook experiments on
receptors purified by wheat-germ agglutinin affinity chromatography. In vitro, the B isoform of the IR autophosphorylates to a
greater extent than the A isoform and has increased kinase activity
toward the synthetic substrate poly-glu The two receptor isoforms differ by 12 amino acids in the carboxyl
terminus of the extracellular An alternative explanation could be that
the B isoform may be utilizing autophosphorylation sites that are
unavailable in the A isoform due to conformational restraints imposed
by the extracellular In conclusion,
dexamethasone causes an increase in responsiveness and sensitivity for
insulin-stimulated immediate early gene expression in HepG2 cells. The
results presented here indicate that it is the expression of the B
isoform of the IR upon dex treatment that is responsible for this
enhancement. The B isoform has a greater insulin-stimulated kinase
activity, phosphorylates IRS-1 more efficiently in vitro, and
couples to IRS-1 more efficiently in vivo. Autophosphorylated
tyrosine residues on tyrosine kinases are the docking sites for
signaling proteins containing SH2 domains, and the identification of a
new phosphopeptide in the B isoform raises the possibility that this
isoform could interact with signaling molecules that are not available
to the A isoform(32, 33, 34) . It will be
interesting to identify this new autophosphorylation site and determine
whether the B isoform couples to novel SH2-containing proteins. It
should be stated, however, that we cannot rule out the possibility that
dexamethasone could be having other effects on the cell that could
contribute to the enhanced signaling. For example, it has been shown
that dex treatment of IM-9 and Fao cells causes changes in the
carbohydrate composition of the IR, but it is not known whether this
effects signaling(35) . Alternatively, dex may effect membrane
fluidity which could alter the function of the IR. Although these are
possibilities, we believe that the major determinant for enhanced
signaling is the switch in expression of IR isoforms from A to B. A
method of modulating the alternative splicing in vivo that
does not require hormonal treatment will be required to rule out these
alternatives definitively.
Volume 270,
Number 35,
Issue of September 01, pp. 20816-20823, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
P-labeled IR and
IRS-1. Studies in vitro on wheat-germ agglutinin-purified
receptors showed that the B isoform of the IR had increased kinase
activity both toward itself and the exogenous substrates
poly-glu
:tyr
and recombinant IRS-1 protein. In
addition, two-dimensional tryptic phosphopeptide maps indicated that
the B isoform has an additional phosphopeptide that is not seen for the
A isoform.
)protein is a
heterotetrameric protein composed of two 
-subunits that confer the
ability to bind insulin and two 
-subunits that contain the
membrane spanning and the tyrosine kinase
domains(1, 2, 3) . The -subunit is
entirely extracellular and is linked by disulfide bounds to the
extracellular portion of the -subunit. Following binding of
insulin to the -subunits, the first observable event is
autophosphorylation on the cytoplasmic portion of the -subunit
leading to an increase in the receptor's intrinsic tyrosine
kinase activity toward other
substrates(1, 4, 5) . The human IR is encoded
by a single gene that is located on chromosome 19 and composed of 22
exons(6) . The mature IR, however, exists as two isoforms,
designated A and B, which result from alternative splicing of the
primary transcript(6, 7, 8) . The A isoform
lacks exon 11, is expressed ubiquitously, and is the only isoform in
lymphocytes, brain, and spleen; the B isoform contains exon 11 and is
expressed predominantly in liver, muscle, adipocytes, and kidney (9, 10, 11, 12) . Exon 11 is
composed of 36 nucleotides that encode a 12-amino-acid segment
(residues 717-728) of the carboxyl terminus of the -subunit
of IR. It has been reported that the affinity of the A isoform for
insulin is higher than that of the B isoform causing a left shift in
the insulin dose-response curves for insulin stimulation of
autophosphorylation, glycogen synthesis, and thymidine
uptake(12, 13, 14) . Two groups found that
the A isoform exhibits a higher insulin internalization and recycling
rate than the B isoform(14, 15) , whereas a third
found no difference in either the rate of internalization or the rate
of degradation(13) . However, all of these studies have been
performed in Chinese hamster ovary or Rat-1 fibroblast cell lines,
which are not good models for insulin-sensitive tissues. The fact that
the B isoform is expressed primarily in insulin-sensitive tissues
indicates that the B isoform of the IR must play an important role in
signaling in insulin-sensitive tissues.
Materials
Monocomponent pork insulin was kindly
provided by Eli Lilly (Indianapolis, IN). Cell culture reagents were
purchased from Life Technologies Inc., and calf serum and fetal calf
serum were from Gemini Bioproducts (Calabasas, CA).
[-P]dCTP (3,000 Ci/mmol) and
[
-
P]ATP (4,500 Ci/mmol) were purchased from
ICN (Costa Mesa, CA). Anti-IR antibodies (83-14 and against
COOH-terminal peptide) were kindly provided by Dr. K. Siddle
(Cambridge, United Kingdom). Polyclonal anti-IRS-1 antibodies for
immunoblotting and immunoprecipitation were kindly provided by Dr. C.
R. Kahn (Boston, MA) and Dr. H. Maegawa (Shiga, Japan), respectively.
All other chemicals were purchased from Sigma or Fisher Scientific. Taq DNA polymerase (Amplitaq) was purchased from
Perkin-Elmer-Cetus.
Cell Culture
HepG2 cells were maintained routinely
in minimum essential medium plus Earle's salts with 10% fetal
calf serum at 37 °C under 5% CO
. The cells were plated
at a density of 1
10
cells/well in 12-well
plates. After 3 days, the medium was replaced with differentiation
medium (Ham's F-12 plus 0.5% calf serum, 1 µM
triiodothyronine, and 20 mM glucose) with and without 1
µM dexamethasone for a further 4 days. Medium was replaced
every 2 days.Reverse Transcription and Amplification of
cDNA
Total cellular RNA was prepared using RNAzol B (Tel-Test,
Inc., Friendswood, TX) according to the manufacturer's protocol.
First-strand cDNA was prepared by reverse transcription using 0.5
µg of total RNA in a volume of 20 µl (250 pmol of random
hexamer primers, 1 unit of Inhibit-ACE RNase inhibitor (5`-3`Inc.,
Boulder, CO), 200 units of Moloney murine leukemia virus reverse
transcriptase (Life Technologies Inc.), 50 mM Tris-HCl, pH
8.3, 75 mM KCl, 3 mM MgCl, 1 mM dithiothreitol, and 1 mM dNTPs) at 42 °C for 1 h.
DNA/RNA hybrids were denatured at 95 °C for 2 min. amplification in a 50-µl final reaction volume (0.5
µM each oligonucleotide primer, 10 mM Tris-HCl,
pH 8.3, 50 mM KCl, 1.5 mM MgCl, 0.1
mM dNTPs, 2 units of Taq DNA polymerase, and 1
µCi of [-P]dCTP). Twenty-five cycles of
amplification were performed using a Perkin-Elmer DNA thermal cycler
System 9600. Each cycle consisted of a 30-s denaturation at 94 °C,
a 30-s annealing at 55 °C, and a 60-s extension at 72 °C. The
number of cycles was optimized to ensure that the amplification lay
within the exponential phase. The products of the PCR amplification
were resolved by electrophoresis on 8% polyacrylamide gels. The gels
were dried and exposed to film at room temperature. The band densities
were quantified using a PhosphorImager (Molecular Dynamics, Sunnyvale,
CA). The counts of c-fos and egr-1 were normalized to
L30 as a internal standard.
In Vitro Autophosphorylation of IR
To measure in vitro autophosphorylation, IRs from HepG2 cells were
partially purified by wheat germ agglutinin (WGA) affinity
chromatography(19) . Equal amounts of IR were incubated with
increasing concentrations insulin for 16 h at 4 °C, followed by the
addition of 30 µCi of [-
P]ATP in
reaction buffer (25 mM HEPES, pH 7.4, 50 µM ATP,
5 mM MnCl
, and 50 mM NaF) for 15 min at 4
°C. The reaction was terminated by the addition of equal volume of
50 mM ATP, 4 mM sodium orthovanadate, 200 mM NaF, 20 mM sodium pyrophosphate, 10 mM EDTA, and
25 mM HEPES, pH 7.6. The receptors were immunoprecipitated
with anti-IR antibody ) for 16 h at 4 °C followed by precipitation
with Pansorbin (Calbiochem). Phosphorylated -subunits were
visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(PAGE) and autoradiography and were quantified with the PhosphorImager. In Vitro Kinase Activity for Exogenous
Substrates
The ability of IRs to phosphorylate the exogenous
substrates poly-glu:tyr and recombinant IRS-1
protein was determined(20) . Briefly, for the
poly-glu:tyr assay, equal amounts of
WGA-purified receptor were preincubated with increasing concentrations
of insulin for 16 h at 4 °C. The substrate
poly-glu:tyr was added for 15 min to a final
concentration of 2 mg/ml. Phosphorylation was initiated by the addition
of 5 µCi of [-
P]ATP in reaction buffer
(25 mM HEPES, pH 7.4, 50 µM ATP, 5 mM MnCl
, and 12 mM MgCl, final
concentration) for 30 min at 4 °C. The labeling was terminated by
the addition of 10 µl of cold ATP (70 mM, final
concentration). Then, the mixture was applied to Whatman 3MM paper,
washed with 10% trichloroacetic acid, 10 mM sodium
pyrophosphate and counted in a liquid scintillation counter.P]ATP in reaction buffer (25 mM HEPES,
pH 7.4, 50 µM ATP, 5 mM MnCl
, and 50
mM NaF) for 15 min at 23 °C. Recombinant IRS-1 protein
(0.3 µg) was added and the incubation continued for a further 90
min at 23 °C. The reaction was terminated by the addition of an
equal volume of 50 mM ATP, 4 mM sodium orthovanadate,
200 mM NaF, 20 mM sodium pyrophosphate, 10 mM EDTA, and 25 mM HEPES, pH 7.6. The IRS-1 proteins were
immunoprecipitated with an anti-IRS-1 antibody for 16 h at 4 °C
followed by precipitation with Pansorbin. Phosphorylated IRS-1 proteins
were visualized by SDS-PAGE and autoradiography and were quantified
using the PhosphorImager.Anti-phosphotyrosine Immunoblotting
Cells were
grown to confluence in 12-well plates. Cells were stimulated with
increasing concentrations of insulin in Krebs-Ringer phosphate-HEPES
buffer (131 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl, 1.2 mM MgSO, 2.5 mM NaHPO, 10 mM HEPES, pH 7.5, 0.1%
bovine serum albumin (BSA)) for 1 min at 37 °C. The cells were
washed with ice-cold phosphate-buffered saline and solubilized in 2
Laemmli's sample buffer (21) containing 2 mM sodium orthovanadate and 200 mM sodium fluoride. The
proteins were denatured by boiling for 5 min, then were separated by
electrophoresis on 7.5% SDS-PAGE, and transferred to Immobilon
membranes (Millipore, Bedford, MA). The filter was blocked with 3% BSA
in T-TBS (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1%
Tween-20) for 30 min and incubated with mouse monoclonal antibody PY-20
(ICN) in blocking buffer for 2 h. The filter was washed with T-TBS for
30 min and then incubated with a sheep anti-mouse horseradish
peroxidase-conjugated antibody (Amersham Corp.) for 30 min. After
washing with T-TBS for 60 min, a chemiluminescent detection kit (ECL,
Amersham Corp.) was used to visualize the tyrosine-phosphorylated
proteins. The band densities were quantified using a Stratascan-7000
densitometer (Stratagene, La Jolla, CA).
Anti-IR and -IRS-1 Immunoblotting
Proteins in
WGA-purified receptors or total cellular extracts were separated by
electrophoresis on 7.5% SDS-PAGE and transferred to Immobilon
membranes. The filters were blocked with 5% BSA in TBS and incubated
with polyclonal antibodies against the COOH terminus of either the IR
(-IRCt) or IRS-1 (-IRS-1). The filters were washed in T-TBS
for 30 min, incubated with sheep anti-rabbit horseradish-peroxidase
conjugated antibody (Amersham) in blocking buffer for 60 min, washed
for 60 min in T-TBS, then the proteins were visualized using the ECL
chemiluminescent kit (Amersham).Insulin Binding
Insulin binding was measured as
described previously(18) . Cells were incubated with 33.3
pMI-insulin in Krebs-Ringer phosphate-HEPES
buffer for 3 h at 12 °C. The cells were washed in ice-cold
phosphate-buffered saline, solubilized, and counted. Nonspecific
insulin binding was determined in the presence of 1 µM insulin. Binding to WGA-purified receptors was performed as
published previously(22) . WGA-purified protein was incubated
with 33.3 pM
I-insulin for 16 h at 4 °C in a
solution containing 50 mM HEPES, pH 7.5, 150 mM NaCl,
0.1% Triton X-100, and 0.1% BSA. Free and bound insulin were separated
by polyethylene glycol precipitation.
Two-dimensional Tryptic Phosphopeptides
Mapping
Polyacrylamide-gel pieces containing P-labeled IR and IRS-1 following in vitro phosphorylation were excised and electroeluted in 20 mM Tris-HCl, pH 8.0, 2 mM EDTA, 0.1% SDS, and 0.1%
2-mercaptoethanol for 4 h. Eluted protein was precipitated with 4
volumes of acetone at -80 °C for 60 min followed by
centrifugation at room temperature for 10 min at 10,000
g. The pellet was dried and digested with 10 µg of
TPCK-treated trypsin (Worthington Diagnostic Systems, Freehold, NJ) in
100 µl of 100 mMN-ethylmorpholine acetate (NMA),
pH 8.2, for 24 h at 37 °C. A further 10 µg of TPCK-treated
trypsin was added and digestion continued for 12 h. The peptides were
lyophilized, resuspended with water, and relyophilized at least three
times. The
P-labeled tryptic peptides were then
resuspended in 5 µl of electrophoresis buffer and spotted onto thin
layer cellulose plates. High voltage electrophoresis was performed in
1:3.5:40.5 formic acid/acetic acid/water, pH 1.9, using a Hunter thin
layer electrophoresis system (C. B. S. Scientific, Del Mar, CA). Plates
were subjected to ascending thin layer chromatography in the second
dimension in 75:15:50:60 n-butanol/acetic acid/pyridine/water,
dried, and then subjected to autoradiography at -80
°C(23) .
P-labeled IR was resuspended in 100 µl of 100 mM NMA, pH 8.3, following trypsin digestion. The sample was boiled
for 5 min and 0.5 mM phenylmethylsulfonyl fluoride added to
inactivate any remaining trypsin. The digested receptor was incubated
with an anti-COOH-terminal peptide (residues 1322-1341) antibody
for 16 h and then with Protein A-Sepharose for 2 h in 500 µl of NMA
at 4 °C. Sepharose-bound antibody was recovered by centrifugation
and washed twice with 1 ml of NMA. Phosphopeptides were eluted by
resuspending the complex in 500 µl of 1 M acetic acid
containing 10 µg of the cold carrier peptide, and mixing at 4
°C for 30 min. Acetic acid was removed by rotary evaporation, and
the phosphopeptide was washed extensively with water before analysis by
two-dimensional mapping as described above(24) .
Protein Tyrosine Phosphatase Activity
Protein
tyrosine phosphatase activity was measured using P-labeled
WGA-purified IR and recombinant IRS-1(25) . WGA-purified IR and
recombinant IRS-1 were labeled with [
-
P]ATP
as described above. The
P-labeled IR and IRS-1 were
immunoprecipitated with anti-IR) and anti-IRS-1 antibodies for 16 h at
4 °C followed by precipitation with Pansorbin and washed with
EBG-buffer (25 mM HEPES, pH 7.4, 120 mM NaCl, 5
mM KCl, 1 mM MgSO
, 1 mM
MgCl, 1 mM CaCl, 0.05% Triton X-100,
and 10% glycerol). For the preparation of whole cell homogenates, cells
were rinsed with phosphate-buffered saline, sonicated for 20 s in 10
volumes of buffer (25 mM imidazole, pH 7.2, 2 mM EGTA, 2 mM EDTA, 0.1% -mercaptoethanol, 2 mM MgCl, 2.1 mM benzamidine, 0.025%
phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, 20 µg/ml
aprotinin, 250 mM sucrose, 10 mM dithiothreitol, and
2% Triton X-100), and set on ice for 30 min. Insoluble proteins were
removed by centrifugation at 14,000 g for 10 min.
Supernatants (7.1 µg protein/80 µl) were incubated with
P-labeled IR and IRS-1 at 30 °C for the indicated
period. The reaction was terminated by addition of Laemmli's
sample buffer and boiled for 5 min. Dephosphorylated IR and IRS-1 were
separated by SDS-PAGE and visualized by autoradiography.
Effect of Dexamethasone on Insulin-stimulated c-fos and
egr-1 Gene Expression
We have published previously that
dexamethasone treatment of HepG2 cells causes an increase in insulin
sensitivity for glucose transport and glucose incorporation into
glycogen(18) . To determine whether insulin's mitogenic
effects were similarly enhanced, we attempted to measure
insulin-stimulated thymidine incorporation. However, we were unable to
measure any effect of insulin due to high basal incorporation (data not
shown). This appears to be a characteristic of HepG2 cells as they
continue to proliferate in serum-free medium. Consequently, we measured
insulin-stimulated c-fos and egr-1 expression as
components of the mitogenic pathway. Reverse transcription and
amplification by PCR (RT-PCR) of total cellular RNA was used to measure
c-fos and egr-1 mRNA levels following stimulation by
insulin. The ribosomal L30 protein mRNA was co-amplified as an internal
control (Fig. 1, A and C). In the cells
cultured with dex, maximal insulin stimulation was 1.6-fold higher for
both c-fos (p < 0.01, n = 4) and egr-1 (p < 0.02, n = 4) gene
expression. Moreover, in both cases the ED for insulin
stimulation was distinctly left shifted in the cells cultured with
dexamethasone (Fig. 1, B and D; ED
9.8 to 5.6 nM for c-fos, p < 0.05, n = 4; 5.0 to 2.1 nM for egr-1, p < 0.02, n = 4).
values were
9.8 and 5.6 nM for cells cultured without and with dex,
respectively, and are indicated by arrows (p <
0.05, n = 4). Panel C, Insulin stimulation of egr-1 gene expression. D, insulin dose-response
curves for egr-1 expression. Results are the mean ±
S.E. of four experiments and are normalized to L-30 mRNA levels.
Maximal insulin stimulation is 1.6-fold higher in the presence of
dexamethasone (p < 0.02, n = 4). The
ED
values were 5.0 and 2.1 nM for cells cultured
without and with dex, respectively, and are indicated by arrows (p < 0.02, n =
4).
In Vivo Autophosphorylation of IR and Kinase
Activity
Results from the study of insulin receptor mutants
generated in vitro have suggested that the insulin-stimulated
metabolic and mitogenic pathways diverge at a very early point in the
signaling pathway, perhaps at the receptor itself. As dex enhances
components of both the metabolic and mitogenic pathways, we decided to
look at very early steps in the insulin signaling pathway, namely
receptor autophosphorylation and endogenous substrate phosphorylation
in intact cells. Cells, cultured in the presence or absence of dex,
were stimulated with increasing concentrations of insulin, and tyrosine
phosphorylation was assessed on whole cell extracts by immunoblotting
with an anti-phosphotyrosine antibody. Samples were normalized for
insulin binding to allow a direct comparison of receptor
autophosphorylation. Insulin stimulated the phosphorylation of two
major proteins; one protein at 95 kDa corresponds to the IR
-subunit and the other at 185 kDa to IRS-1 (Fig. 2A). In spite of loading equal numbers of
receptors, maximal insulin stimulation of IR autophosphorylation was
1.5-fold higher in the cells treated with dex (Fig. 2B, p < 0.01, n = 8). A smaller but significant
1.3-fold increase in IRS-1 phosphorylation was observed also (Fig. 2C, p < 0.01, n = 8).
Eight independent pair-matched experiments were performed on different
passages of cells and at different times to ensure significance. The
insulin sensitivity for IR autophosphorylation was unchanged (Fig. 2B). However, the ED for
insulin-stimulated phosphorylation of IRS-1 was distinctly left-shifted
in the cells treated with dex (5.6 to 2.7 nM) (Fig. 2C, p < 0.04, n = 8).
This is most easily seen by comparing lanes 2 and 3 with 8 and 9 in Fig. 2A. Greater
phosphorylation is evident at both 1 and 3 nM insulin in the
presence of dex.
-subunits
of the IR and IRS-1 are indicated by arrows. Panel B,
insulin dose-response curves for autophosphorylation of IR
-subunits. Results are the mean ± S.E. of eight
experiments. Maximal insulin stimulation is 1.5-fold higher in the
presence of dex (p < 0.01, n = 8). The
ED values of the curves were 5.5 and 8.8 nM for
cells cultured without and with dex, respectively, and are indicated by
the arrows (p > 0.05, n = 8). Panel C, insulin dose-response curves for IRS-1
phosphorylation. Results are the mean ± S.E. of eight
experiments. Maximal insulin stimulation is 1.3-fold higher in the
presence of dex (p < 0.01, n = 8). The
ED
values of the curves were 5.4 and 2.6 nM for
cells cultured without and with dex, respectively, and are indicated by arrows (p < 0.04, n = 8). Panel D, effect of dex on IR protein levels. IRs were
visualized using a polyclonal anti-IR antibody. Dex treatment causes a
1.5-fold increase in IR level. Panel E, effect of dex on IRS-1
protein levels. IRS-1 was visualized using a polyclonal antibody to
IRS-1. Dex treatment has no effect on IRS-1
levels.
In Vitro Autophosphorylation of IR
The increase in
maximal autophosphorylation of the IR seen in whole cells could be a
result of an increase in the inherent kinase activity of the receptor
itself. Therefore, we determined the ability of insulin to stimulate
autophosphorylation of purified IRs. Receptors were partially purified
from cells, untreated or treated with dex, by WGA affinity
chromatography and then equal numbers of IRs, adjusted by insulin
binding, were subjected to in vitro autophosphorylation. As
shown in Fig. 3A, insulin stimulated
autophosphorylation of the IR -subunit in a dose-dependent manner.
In the cells treated with dex, however, maximal insulin stimulation was
1.7-fold higher (p < 0.01, n = 4) with no
change in sensitivity (ED: 9.8 nM for -Dex;
10.2 nM for +Dex, Fig. 3B). This is
similar to the results obtained by antiphosphotyrosine immunoblotting
of whole cell extracts (Fig. 2B). We verified that we
had loaded equal amounts of IR by immunoblotting (Fig. 3C). Previously, we have shown that treatment of
HepG2 cells with dex causes a switch in isoform expression from 20:80
to 80:20 A:B by RT-PCR. Assuming that the protein expression mirrors
the mRNA ratio, these numbers can be used to determine the activities
of each isoform. This analysis predicts that the B isoform incorporates
2.4-fold more
P. This is in agreement with the results of
Kellerer et al.(26) who documented a 2.5-fold
increase in
P incorporation for the B isoform IR purified
from Rat 1 fibroblasts.
-
P]ATP. The receptors were
immunoprecipitated with an anti-IR antibody for 16 h at 4 °C
followed by precipitation with Pansorbin. Phosphorylated
-subunits
were visualized by SDS-PAGE and autoradiography and quantified on a
PhosphorImager. Panel A, insulin stimulation of IR
autophosphorylation. PanelB, insulin dose-response
curves for in vitro autophosphorylation. Results are the mean
± S.E. of four experiments. Maximal insulin-stimulated
autophosphorylation is 1.7-fold higher in the presence of dex (p < 0.01, n = 4). The ED values are
indicated by arrows (ED
: 9.8 nM for
-Dex; 10.2 nM for +Dex). Panel C,
partially purified IRs normalized for insulin binding were separated by
SDS-PAGE, transferred to an Immobilon-P membrane and subjected to
immunoblotting using an anti-IR antibody to verify equal receptor
number.
In Vitro Kinase Activity of IR
To test whether the
B isoform of the IR has enhanced kinase activity in vitro,
phosphorylation assays with equal numbers of WGA-purified IRs, adjusted
by insulin binding, were performed using either the exogenous substrate
poly-glu:tyr or recombinant IRS-1 protein.
Insulin stimulated phosphorylation of both
poly-glu:tyr and recombinant IRS-1 protein in a
dose-dependent manner (Fig. 4). In the cells treated with dex,
however, maximal insulin-stimulated phosphorylation was 1.6- and
1.5-fold higher for poly-glu:tyr (p < 0.01, n = 4; Fig. 4A) and
recombinant IRS-1 protein (Fig. 4B), respectively, with
no difference in sensitivity. Using the isoform ratios mentioned
earlier, the contributions of the individual isoforms can be
calculated. Thus the B isoform has 2.2- and 2.0-fold higher activity
toward poly-glu:tyr and IRS-1, respectively.
These results parallel the increase in maximal phosphorylation of IRS-1 in vivo (Fig. 2C). However, no differences in
sensitivity were observed for either substrate. Again these results
agree with those of Kellerer et al.(26) who observed
a 2.0-fold increase in GluTyr phosphorylation for the B isoform.
:tyr or recombinant IRS-1 protein as a substrate. Panel A,
insulin dose-response curves for phosphorylation of
poly-glu:tyr plotted as phosphorylation above
basal versus insulin concentration. Samples were normalized
for insulin binding. Results are the mean ± S.E. of four
experiments. Maximal insulin-stimulated phosphorylation is 1.6-fold
higher in the presence of dex (p < 0.01, n = 4). The ED values are 21.7 and 19.7 nM for -Dex and +Dex, respectively, and are indicated by
the arrows. Panel B, insulin-stimulated
phosphorylation of recombinant IRS-I. Equal numbers of IRs were used to
phosphorylate recombinant IRS-1 in vitro. Phosphorylated IRS-1
proteins were immunoprecipitated then visualized by SDS-PAGE and
autoradiography. A representative autoradiogram is shown. Maximal
insulin-stimulated phosphorylation is 1.5-fold higher in the presence
of dex. Panel C, insulin-stimulated of IR autophosphorylation.
Following immunoprecipitation of the IRS-1 proteins, the supernatants
were subjected to SDS-PAGE and autoradiography to visualize the IR
autophosphorylation.
Two-dimensional Tryptic Phosphopeptide Maps of IR
Isoforms
To determine whether the increased autophosphorylation
of the IR following dex treatment is due to utilization of additional
phosphorylation sites in the B isoform, two-dimensional tryptic
phosphopeptide mapping was performed (Fig. 5). Equal numbers of
WGA-purified IRs, adjusted by insulin binding, were allowed to
autophosphorylate in the presence of
[-
P]ATP, purified by gel electrophoresis,
digested with trypsin, and separated by electrophoresis and then
ascending chromatography on a thin layer cellulose plate.
Phosphopeptides A-D have been assigned by Tavare and Denton (23, 27) based on charge and digestion by V8 protease
and are derived from the triple tyrosine region of the IR (tyrosines
1158, 1162, and 1163). Peptides from the tyrosine 960 region are more
hydrophobic and run at a position higher than peptide B in the
chromatographic axis. Peptide F has been assigned as the peptide
containing the two tyrosines from the COOH terminus of the
-subunit. Peptide G remains unassigned. The intensity of
phosphopeptides A, B, C, D, and F were all increased in cells treated
with dex. Moreover, an additional phosphorylated peptide E (Fig. 5B) was observed for the B isoform of the IR.
P]ATP in reaction buffer for
30 min at 4 °C. The reaction was terminated by the addition of
excess cold ATP with phosphatase inhibitors. The IRs were
immunoprecipitated with an anti-IR antibody for 16 h at 4 °C
followed by precipitation with Pansorbin and subjected to SDS-PAGE.
Phosphorylated
-subunits were eluted from gel and digested with
TPCK-treated trypsin. P-Labeled tryptic phosphopeptides
were separated on thin layer cellulose plates by electrophoresis at pH
1.9 followed by ascending chromatography. A representative
autoradiogram is shown.
-subunit. We attempted to
confirm this assignment by immunoprecipitation with an antibody that
had been raised against the COOH-terminal peptide (residues
1322-1341) that contains the two known auto-phosphorylation sites
(tyrosines 1328 and 1334) (Fig. 6). Peptide F was precipitated
along with peptide G and a fraction of peptide D (Fig. 6B). However, none of fragment E was precipitated
so it is unlikely the E is derived from the COOH terminus. Peptide D
contains the doubly phosphorylated tryptic peptide (amino acids
1156-1168) having an approximate charge of +1.5 at pH 1.9.
The marker dye dinitrophenyl lysine has a charge of +1.7 at pH
1.9. The doubly phosphorylated COOH-terminal peptide (amino acids
1327-1341) is predicted to have an approximate charge of
+1.5 whereas the singly phosphorylated form has a charge of
+2.5. Thus these two peptides should migrate either side of the
dinitrophenyl lysine marker. Only a fraction of D is immunoprecipitated
by the antibody, so D most likely represents a mixture of the doubly
phosphorylated COOH terminus peptide (amino acids 1327-1341) and
the doubly phosphorylated peptide from the triple tyrosine region
(amino acids 1156-1168). The singly phosphorylated peptide
derived from the COOH-terminal peptide is thus G by analogy with
peptides A and B and C and D(27) . Phosphopeptide F is likely
to be a doubly phosphorylated tryptic peptide that contains a single
additional lysine or arginine residue due to incomplete cleavage (amino
acids 1326-1341 or 1327-1342). This would cause the peptide
to have an additional +1 charge. The identity of peptide E is
unknown.
P-labeled tryptic phosphopeptides were separated on
thin layer cellulose plates by electrophoresis at pH 1.9 followed by
ascending chromatography. A representative autoradiogram is shown. Panel A, total phosphopeptides. Panel B,
immunoprecipitated phosphopeptides.
Two-dimensional Tryptic Phosphopeptide Maps of
IRS-1
To determine whether IRS-1 is phosphorylated on different
sites by the two receptor isoforms, we generated two-dimensional
phosphopeptide maps of IRS-1 that had been phosphorylated in vitro by either isoform of the IR as above (Fig. 7).
Phosphorylation by the B isoform of the IR causes greater P incorporation into IRS-1 (Fig. 4), and this is
reflected by the increased intensity of the phosphopeptide spots (Fig. 7, A and B). However, no differences in
the pattern of spots are observed suggesting that the receptors
interact with IRS-1 in the same manner in vitro.
P]ATP in reaction buffer for 20
min at 23 °C. Recombinant IRS-1 protein (0.3 µg) was added and
the incubation continued for a further 90 min at 23 °C. The
reaction was terminated by the addition of excess cold ATP with
phosphatase inhibitors. The IRS-1 proteins were immunoprecipitated with
anti-IRS-1 antibody for 16 h at 4 °C followed by precipitation with
Pansorbin. Phosphorylated IRS-1 proteins were separated by SDS-PAGE,
eluted from gel, and digested with TPCK-treated trypsin.
P-Labeled tryptic phosphopeptides were separated on thin
layer cellulose plates by electrophoresis at pH 1.9 followed by
ascending chromatography. A representative autoradiogram is
shown.
Protein Tyrosine Phosphatase Activity
The increase
in insulin-stimulated whole cell phosphorylation in cells treated with
dex could result from decreases in the activities of the tyrosine
phosphatases that act on the IR or IRS-1. At the present time it is not
known which phosphatases are involved. Given this limitation, we
attempted to determine whether dex treatment induced any alterations in
total tyrosine phosphatase activity using IRs and IRS-1 as substrates.
Tyrosine phosphatase activity was measured on Triton X-100-soluble
cellular extracts. B isoform IRs and recombinant IRS-1 were
phosphorylated in vitro in the presence of
[-
P]ATP and immunoprecipitated. Cellular
extracts were incubated with the
P-labeled proteins for
the indicated periods, boiled in sample buffer to stop the reaction,
and the extent of dephosphorylation measured by SDS-PAGE and
autoradiography (Fig. 8). Dex treatment had no effect on the
extent of IR and IRS-1 dephosphorylation. This result suggests but does
not prove that tyrosine phosphatase activity is not grossly altered by
dex treatment. Identification of the phosphatases that can act on the
IR and IRS-1 will be required before this question can be answered
definitively.
P-labeled IR and IRS-1 for indicated period at 30
°C. The reaction was terminated by boiling, and samples were
separated by SDS-PAGE to determine dephosphorylation of IR and IRS-1. A
representative autoradiogram is shown.
:tyr and
recombinant IRS-1 protein. Conversely, we were not able to detect any
alterations in total cellular tyrosine phosphatase activity when
assayed with in vitroP-labeled IR and IRS-1.
Thus it appears that the enhanced phosphorylation seen in vivo is due to differences in the intrinsic kinase activities of the IR
isoforms. There is still a discrepancy between the in vitro and in vivo results, however, as there is no difference
in insulin sensitivity for IRS-1 phosphorylation in vitro.
Although IRS-1 is a direct substrate for the IR kinase in
vitro, there may be additional factors in vivo that
enhance the interaction between the two molecules causing the
left-shift in the insulin dose-response curve. Alternatively, the
receptor may require the intact plasma membrane for correct function.
-subunit. The intracellular kinase
domains of the two isoforms are identical. How is it possible that the
two isoforms have different kinase activities? Kellerer and co-workers (26) found that if the receptors are activated by trypsin
cleavage rather than insulin stimulation then no differences in
activity are observed. So how is insulin able to activate one kinase
more than the other? The B isoform autophosphorylates to a greater
extent than the A isoform both in vivo and in vitro.
There are two possible explanations for this finding. It has been shown
that following insulin stimulation only 43% of insulin receptors
isolated from human adipocytes are phosphorylated on
tyrosine(30) . This has lead to the proposal that there are
distinct pools of kinase competent and incompetent receptors. In
non-insulin-dependent diabetes mellitis, fewer than 15% of IRs are
phosphorylated on tyrosine suggesting that the proportion of
incompetent receptors is greater leading to a kinase
defect(30) . However, these findings can be interpreted
differently as the phosphorylation reaction is inherently reversible.
The observed autophosphorylation is a balance between the forward
phosphorylation and the reverse dephosphorylation reactions. So an
increase in the forward reaction due to an increase in V
would shift the equilibrium toward a more
fully phosphorylated state.-subunits. This latter alternative can be
tested by tryptic phosphopeptide mapping. Kellerer et al.(26) used HPLC analysis of the tryptic peptides but were
not able to observe any differences. However, direct comparison of HPLC
and thin layer peptide mapping of insulin receptor phosphopeptides has
shown that HPLC lacks the sensitivity and resolution of two-dimensional
thin layer mapping(27) . Therefore, we utilized two-dimensional
tryptic phosphopeptide mapping (Fig. 5). The intensity of all
phosphopeptides was higher in the B isoform suggesting that the
equilibrium has been shifted to a more highly phosphorylated state
consistent with the increase in V. More
interestingly, an additional phosphopeptide E was detected in the B
isoform that is not seen in the A isoform. The exact identity of
peptide E remains to be determined, but two possibilities are the
peptides spanning amino acids 1086-1092 and 1102-1127,
which include tyrosines 1087 and 1122, respectively. Both these
peptides are predicted to have a charge of +1.5 at pH 1.9 if
phosphorylated on tyrosine. Interestingly, both peptides are derived
from domains that are conserved among tyrosine kinases; however neither
are known autophosphorylation sites(31) .
)
We thank Dr. Jerrold Olefsky for helpful advice and
encouragement and Drs. K. Siddle, C. R. Kahn, and H. Maegawa for
antibodies.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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