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INTRODUCTION |
PKC-
1 is an atypical
member of the protein kinase C family of Ser/Thr kinases that is an
important mediator of the metabolic actions of insulin, such as
translocation of the insulin-responsive glucose transporter GLUT4 and
enhancement of glucose transport (1-4). Activation of PKC-
in
response to insulin stimulation is regulated by PI
3-kinase-dependent pathways and involves PDK-1 phosphorylating Thr410 in the PKC-
activation loop (5,
6). Known substrates of PKC-
include nucleolin (7), heterogeneous
ribonucleoprotein A1 (8), Sp1 (9), Sendai virus phosphoprotein (10),
and IKK
(11). However, direct substrates of PKC-
that
participate in insulin signaling have not been identified to date.
IRS-1 is a substrate of the insulin receptor tyrosine kinase that upon
tyrosine phosphorylation functions as a docking molecule to engage and
activate SH2 domain-containing proteins, including PI 3-kinase and
SHP-2 (12). IRS-1 is also extensively phosphorylated on serine
residues, and modulation of IRS-1 serine phosphorylation is observed in
response to the treatment of cells with a variety of agents, including
insulin (13, 14), tumor necrosis factor
(15-17), PDGF (18),
angiotensin II (19), phorbol esters (20, 21), and okadaic acid (14,
22). Interestingly, IRS-1 has recently been identified as a substrate
for both Akt and GSK-3, Ser/Thr kinases downstream from PI 3-kinase
that participate in the metabolic actions of insulin (23, 24). These
studies suggest that feedback mechanisms in metabolic insulin signaling
pathways may exist at the level of IRS-1 to regulate insulin
sensitivity (23, 24). Although serine phosphorylation of IRS-1 has
generally been associated with impairment in its ability to couple with PI 3-kinase in response to insulin (14, 16-19, 21, 22, 24, 25),
phosphorylation of IRS-1 by Akt may also positively modulate its
function (23). In the present study, we found that IRS-1 may also be a
novel physiological substrate for PKC-
that participates in the
feedback regulation of metabolic insulin signaling pathways.
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MATERIALS AND METHODS |
Reagents
Reagents were obtained from the following sources: monoclonal
anti-HA antibody from Covance Research Products (Denver,
PA); monoclonal anti-phosphotyrosine antibody (4G10), polyclonal
anti-p85, anti-IRS-1, and anti-PDGF
receptor antibodies, recombinant
human IRS-1 protein, and recombinant protein phosphatase 2A from
Upstate Biotechnology, Inc. (Lake Placid, NY); recombinant PKC-
from PanVera Corporation (Madison, WI); goat polyclonal antibodies against
N-terminal and C-terminal regions of PKC-
, sheep polyclonal anti-SHP-2 antibody, and rabbit polyclonal anti-HA antibody from Santa
Cruz Biotechnology, Inc. (Santa Cruz, CA); and protein A- and protein
G-agarose beads and LipofectAMINE Plus from Life Technologies, Inc.
Expression Plasmids
pCIS2--
This is the parental expression vector with a
cytomegalovirus promoter (26, 27).
IRS1-HA--
cDNA for human IRS-1 was subcloned into pCIS2
as described (28), and the QuikChange site-directed mutagenesis
kit (Stratagene, La Jolla, CA) was used to create sequence coding for
an HA-epitope tag fused to the C terminus of IRS-1 (sense primer, 5'-G
CAG CCA GAG GAC CGT CAG TAT CCT TAT GAT GTT CCT GAT TAT GCT
TAG CTC AAC TGG ACA TCA CAG C-3'; antisense primer, 5'-G CTG TGA TGT
CCA GTT GAG CTA AGC ATA ATC AGG AAC ATC ATA AGG ATA CTG ACG
CTC CTC TGG CTG C-3').
IRS1-Y612/Y632--
This construct was derived from
IRS1-HA by using the QuikChange kit to sequentially introduce Phe for
Tyr substitutions in YXXM motifs at positions 465, 662, 941, and 989 with the mutagenic oligonucleotides: 465 sense, 5'-G CTA AGC
AAC TTT ATC TGC ATG GGT GGC-3'; 465 antisense, 5'-GCC ACC
CAT GCA GAT AAA GTT GCT TAG C-3'; 662 sense, 5'-TG GAC CCC
AAT GGC TTC ATG ATG ATG TCC-3'; 662 antisense, 5'-GGA CAT
CAT CAT GAA GCC ATT GGG GTC CA-3'; 941 sense, 5'-GGC ACT
GAG GAG TTC ATG AAG ATG GAC C-3'; 941 antisense, 5'-G GTC
CAT CTT CAT GAA CTC CTC AGT GCC-3'; 989 sense, 5'-AGC CGG
GGT GAC TTC ATG ACC ATG CAG-3'; 989 antisense; 5'-CTG CAT GGT CAT GAA GTC ACC CCG GCT-3'. This mutagenesis left
YXXM sites at Tyr612 and Tyr632 intact.
PKC
-WT--
This is the rat wild type PKC-
with an
N-terminal HA-epitope tag in the pCDNA3 expression vector (29,
30).
PKC
-KD--
This is the kinase-inactive point mutant
of rat PKC-
(L281W) with N-terminal HA-epitope tag in pCDNA3
expression vector (29, 30).
Cell Culture and Transfection
NIH-3T3 fibroblasts stably overexpressing human insulin
receptors (NIH-3T3IR) were maintained in Dulbecco's
modified Eagle's medium containing 10% fetal bovine serum,
L-glutamine (2 mM), penicillin (100 units/ml), and streptomycin (100 µg/ml) in a humidified atmosphere with 5% CO2 at 37 °C. NIH-3T3IR cells were
transiently transfected with PKC-
and/or IRS-1 constructs using
LipofectAMINE Plus according to the manufacturer's instructions. Rat
adipose cells were obtained by collagenase digestion of epididymal fat
pads of male rats as described (27) and were used within 2 h of isolation.
In Vitro PKC-
Kinase Assays
In vitro kinase assays using PKC-
and IRS-1 were
carried out at 30 °C for 30 min in kinase assay buffer containing 50 mM Tris-HCl, pH 7.4, 10 mM MgCl2,
50 µM ATP, 2.5 µCi of [
-32P]ATP/assay,
and 4 µg of phosphatidylserine. The reactions were stopped by adding
Laemmli sample buffer and boiling for 10 min. Samples were subjected to
7.5% SDS-PAGE, and phosphorylated IRS-1 was detected with a
PhosphorImager. In addition, gel contents were transferred to
nitrocellulose and immunoblotted with anti-IRS-1 antibody. Finally, the
activity of PKC-
in each assay was independently verified using
peptide
as a substrate (31). For assays using purified PKC-
and
IRS-1 proteins, 0.5 µg of IRS-1 and 0.1 µg of PKC-
(specific
activity of, 1410 nmol of phosphate transferred to peptide
substrate/min/mg of protein) were used. In some experiments, endogenous
IRS-1 or recombinant PKC-
was immunoprecipitated from lysates of
NIH-3T3IR cells (1 mg of total protein) prepared using
lysis buffer (50 mM Tris-HCl, pH 7.4, 125 mM
NaCl, 1% Triton X-100, 0.5% Nonidet P-40, 1 mM
Na3VO4, 50 mM NaF, 1 mM
sodium pyrophosphate, 10 mM
-glycerophosphate, 0.1 mM okadaic acid, and a complete protease inhibitor mixture
(Roche Molecular Biochemicals)). Lysates precleared with protein
G-agarose beads for 1 h at 4 °C were immunoprecipitated with
antibodies against the HA-epitope (to obtain HA-tagged PKC-
) or
IRS-1 and protein G-agarose beads for 2 h at 4 °C. The
immunocomplexes were then washed four times with kinase assay buffer
and used for in vitro kinase assays as described above.
In Vivo Phosphorylation Experiments
NIH-3T3IR cells transiently transfected with
HA-tagged IRS-1 and PKC-
constructs were serum-starved overnight and
then labeled for 2 h with 75 µCi/ml
[32P]orthophosphate in KRBH buffer, pH 7.4 (107 mM NaCl, 5 mM KCl, 3 mM
CaCl2, 1 mM MgSO4, 20 mM Hepes, 10 mM glucose, 1% bovine serum
albumin, and 7 mM NaHCO3). Cells were then
washed four times with phosphate-buffered saline, and cell lysates were
subjected to immunoprecipitation with anti-HA antibody and protein
A-agarose beads. Immunocomplexes were washed four times with lysis
buffer, boiled in Laemmli sample buffer, and separated by 7.5%
SDS-PAGE. Phosphorylated IRS-1 was detected and quantified with a
PhosphorImager. Gel contents were transferred to nitrocellulose for
immunoblotting with anti-IRS-1 antibody.
Coimmunoprecipitation Experiments
Using an anti-IRS-1 antibody, IRS-1 was immunoprecipitated from
cell lysates (500 µg of total protein) derived from
NIH-3T3IR cells transiently transfected with the empty
control vector, PKC
-WT or PKC
-KD, as described above. Cell
lysates were precleared with protein G-agarose beads to minimize
nonspecific binding. As an additional control for nonspecific binding,
samples were also immunoprecipitated with preimmune rabbit IgG. Both
cell lysates and immunoprecipitated samples were immunoblotted with
antibodies against IRS-1 and the HA-epitope (to detect HA-tagged
PKC-
). The coimmunoprecipitation of endogenous IRS-1 and PKC-
was
examined in freshly isolated rat adipose cells stimulated without or
with insulin (100 nM, 5 min). Cell lysates were prepared as
described previously using ice-cold TES buffer (containing 1% Triton
X-100, 0.5% Nonidet P-40, and inhibitors as listed above for lysis
buffer) (32). Anti-PKC-
antibodies directed against either the N- or C-terminal regions of PKC-
were used to immunoprecipitate endogenous PKC-
. As an additional control for nonspecific binding, samples were
also immunoprecipitated with preimmune goat IgG. Both cell lysates and
immunoprecipitated samples were immunoblotted with antibodies against
IRS-1 and PKC-
.
Functional Assessment of IRS-1
NIH-3T3IR cells transiently cotransfected with
IRS1-HA and either PKC
-WT or a control vector were serum-starved
overnight and then stimulated with insulin (100 nM) for 0, 2, or 60 min. Cell lysates (300-500 µg of total protein) were
subjected to immunoprecipitation with anti-HA antibody as described
above and separated by 7.5% SDS-PAGE, and gel contents were
transferred to nitrocellulose. Membranes were immunoblotted with
antibodies against IRS-1, p85, and SHP-2 and then stripped and reprobed
with phosphotyrosine antibody. Cell lysates from each group were also
immunoblotted for IRS-1 and PKC-
. To assess IRS-1-associated PI
3-kinase activity, anti-HA immunoprecipitates were washed once with
phosphate-buffered saline containing 1% Nonidet P-40 and 100 µM Na3VO4, twice with 100 mM Tris-HCl, pH 7.5, containing 500 mM
LiCl2 and 100 mM
Na3VO4, and once with 10 mM
Tris-HCl, pH 7.5, containing 100 mM NaCl, 1 mM
EDTA, and 100 mM Na3VO4. For each
reaction, 10 µg of phosphatidylinositol (Sigma) sonicated in 10 µl
of PI 3-kinase reaction buffer (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.3 mM EGTA) and 10 µCi of
[
-32P]ATP in 40 µl of PI 3-kinase reaction buffer
were added along with MgCl2 at a final concentration of 10 mM. The phosphorylation reaction was started by adding 50 µl of the substrate solution with 50 µl of the immune complex.
After incubation for 20 min at 30 °C, the reaction was stopped by
adding 100 µl of 0.1 N HCl and 200 µl of
CHCl3/CH3OH (1:1). The organic phase containing the phosphorylated phospholipid product was extracted and applied to a
silica gel thin layer chromatography plate (Whatman) coated with 1%
potassium oxalate. Thin layer chromatography plates were developed in
CHCl3/CH3OH/H2O/NH4OH
(60:47:11.3:2), dried, and visualized by autoradiography. Assays were
quantified by PhosphorImager and normalized for the amount of IRS-1
recovered in anti-HA immunoprecipitates.
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RESULTS |
IRS-1 Is a Substrate for PKC-
Both in Vitro and in Intact
Cells--
In vitro kinase assays using purified PKC-
and IRS-1 proteins were carried out to determine whether IRS-1 is
capable of functioning as a substrate for PKC-
. Incubation of
PKC-
with IRS-1 in the presence of [32P]ATP
significantly increased the amount of phosphorylated IRS-1 (Fig.
1, lanes 1 and 2).
This phosphorylation was substantially reversed upon treatment
with protein phosphatase 2A (a Ser/Thr-specific phosphatase),
suggesting that IRS-1 is phosphorylated on Ser/Thr residues by PKC-
(Fig. 1, lane 3). Endogenous IRS-1 immunoprecipitated from NIH-3T3IR cells was also specifically
phosphorylated in the presence of PKC-
(Fig. 1, lanes 4 and 5). In addition, only wild-type PKC-
but not
kinase-inactive mutant PKC-
(immunoprecipitated from transfected
NIH-3T3IR cells) was able to phosphorylate purified IRS-1
in vitro (Fig. 1, lanes 6 and 7). We
next performed in vivo labeling experiments in
NIH-3T3IR cells transiently cotransfected with IRS1-HA and
an empty expression vector (pCIS2), PKC
-WT, or PKC
-KD to
determine whether IRS-1 can also function as a substrate for PKC-
in
intact cells. Importantly, the overexpression of PKC-
led to a
significant ~2.5-fold increase in phosphorylation of IRS1-HA (Fig.
2). By contrast, the overexpression of
the kinase-inactive mutant PKC
-KD did not result in increased phosphorylation of IRS1-HA (Fig. 2, lane 3). Taken together,
these results raise the possibility that IRS-1 may be a novel
physiological substrate for PKC-
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Fig. 1.
PKC- phosphorylates
IRS-1 in vitro. Kinase assays were carried out
in vitro in the presence of [ -32P]ATP using
either purified PKC- (lanes 1-5) or HA-tagged PKC-
constructs immunoprecipitated from lysates of transfected cells
(lanes 6 and 7). Purified IRS-1 protein
(lanes 1-3, lanes 6 and 7) or IRS-1
immunoprecipitated from cell lysates (lanes 4 and
5) were used as the substrate. Top panel, IRS-1
is phosphorylated by PKC- (lanes 2, 5, and
6) but not by kinase-inactive mutant PKC- (lane
7). Treatment with protein phosphatase 2A (30 min, 30 °C)
reversed the phosphorylation of IRS-1 by PKC- (lane 3).
Middle panel, anti-IRS-1 immunoblot (IB)
demonstrates comparable amounts of IRS-1 within each kinase assay.
Lower panel, anti-HA immunoblot (IB) demonstrates
comparable recovery of HA-tagged wild-type and mutant PKC-
(lanes 6 and 7). Representative results are shown
for experiments that were independently repeated at least five times.
PP2A, protein phosphatase 2A; Ippt,
immunoprecipitate.
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Fig. 2.
IRS-1 is a substrate for
PKC- in intact cells.
NIH-3T3IR cells transiently transfected with HA-tagged
IRS-1 and pCIS2 (empty vector), PKC -WT, or PKC -KD were labeled
with [32P]orthophosphate. Recombinant IRS-1
immunoprecipitated from cell lysates with anti-HA antibody was
subjected to 7.5% SDS-PAGE and autoradiography. A,
top panel, autoradiogram from a representative in
vivo labeling experiment. A, lower panel,
anti-IRS-1 immunoblot (IB) demonstrating comparable
recovery of HA-tagged IRS-1 in each group. B, PhosphorImager
quantification of three independent autoradiograms (mean ± S.E.).
Ippt, immunoprecipitate.
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Association between IRS-1 and PKC-
in Intact Cells--
To
provide further support for the hypothesis that IRS-1 is a direct
substrate for PKC-
in vivo, we next investigated the ability of PKC-
to interact with IRS-1 in NIH-3T3IR
cells overexpressing either wild-type or kinase-inactive mutant PKC-
. Interestingly, both wild-type and mutant PKC-
coimmunoprecipitated with endogenous IRS-1 under our experimental
conditions (Fig. 3). Neither wild-type
nor mutant PKC-
was detected in control immunoprecipitation
experiments performed with preimmune rabbit IgG (Fig. 3, lanes
3 and 4). We studied these interactions in a more
physiological context by examining the ability of endogenous IRS-1 to
coimmunoprecipitate with endogenous PKC-
in freshly isolated rat
adipose cells. Lysates of adipose cells stimulated without or with
insulin were immunoprecipitated using antibodies against PKC-
and
then immunoblotted for both IRS-1 and PKC-
. Consistent with our
results in NIH-3T3IR cells, IRS-1 coimmunoprecipitated with
PKC-
in the basal state (absence of insulin) (Fig.
4A, lanes 1 and
3). Upon insulin stimulation, we observed a significant
~2-fold increase in the amount of IRS-1 associated with PKC-
(Fig.
4, A (lanes 2 and 4) and
B). Comparable results were obtained using antibodies
directed against either the N- or C-terminal regions of PKC-
. No
IRS-1 was detected in control immunoprecipitation experiments performed
with preimmune goat IgG (Fig. 4A, lanes 5 and
6). These results suggest that regulated interactions
between PKC-
and IRS-1 may potentially contribute to insulin action
in bona fide insulin target cells.

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Fig. 3.
PKC-
coimmunoprecipitates with IRS-1 in transfected
NIH-3T3IR cells. Lysates from cells transiently
transfected with HA-tagged PKC- constructs (lanes 5 and
6) were immunoprecipitated using an anti-IRS-1 antibody
(lanes 1 and 2) or preimmune rabbit IgG as a
control (lanes 3 and 4). In the representative
blots shown, immunoprecipitations with the IRS-1 antibody and preimmune
IgG were done concurrently on the same lysates and run in adjacent
lanes on the same gel. Top panel, anti-IRS-1 immunoblot
(IB) demonstrating comparable recovery of IRS-1 in
both anti-IRS-1 immunoprecipitates and cell lysates. Lower
panel, anti-HA immunoblot demonstrating coimmunoprecipitation of
PKC- with IRS-1 (lanes 1 and 2) and expression
of PKC- constructs in transfected cells (lanes 5 and
6). Representative results are shown for experiments that
were independently repeated at least three times. Ippt,
immunoprecipitate.
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Fig. 4.
Association of IRS-1 with
PKC- in rat adipose cells is increased upon
insulin stimulation. Freshly isolated rat adipose cells were
treated with insulin (100 nM, 5 min) as indicated, and cell
lysates (lanes 7 and 8) were immunoprecipitated
with antibodies against either N-terminal (lanes 1 and
2) or C-terminal (lanes 3 and 4)
regions of PKC- or preimmune goat IgG as a control (lanes
5 and 6). In the representative blots shown,
immunoprecipitations with the C-terminal antibody (CT-Ab)
and preimmune IgG were done concurrently on the same lysates and run in
adjacent lanes on the same gel whereas the samples immunoprecipitated
with the N-terminal antibody (NT-Ab) were run on a separate
gel. A, top panel, anti-IRS-1 immunoblot
(IB) demonstrating increased association between
IRS-1 and PKC- upon insulin stimulation. A, lower
panel, anti-PKC- immunoblot. B, quantification of
IRS-1 coimmunoprecipitation (mean ± S.E.) based on scanning
densitometry of four independent immunoblots normalized for
PKC- recovery. Ippt, immunoprecipitate.
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Overexpression of PKC-
Impairs Insulin-stimulated Tyrosine
Phosphorylation of IRS-1 and Associated PI 3-Kinase Activity--
To
assess the functional consequences of IRS-1 Ser/Thr phosphorylation by
PKC-
, the time course of IRS-1 tyrosine phosphorylation after
insulin stimulation was examined in NIH-3T3IR cells
transiently cotransfected with IRS1-HA and either a control vector or
PKC
-WT (Fig. 5). In control cells,
insulin stimulation resulted in significant tyrosine phosphorylation of
IRS1-HA by 2 min that decreased ~60% by 60 min (Fig. 5, A
(lanes 1-3) and B). Interestingly, when compared
with results from control cells, insulin-stimulated tyrosine
phosphorylation of IRS1-HA recovered from cells overexpressing PKC-
was significantly reduced (Fig. 5A, lanes 5 and
6). Quantification of results from three independent experiments normalized for IRS1-HA recovery showed that the
overexpression of PKC-
was associated with an ~40% decrease in
tyrosine phosphorylation of IRS-1 at both the 2- and 60-min time points
when compared with control cells (Fig. 5B). Thus, Ser/Thr
phosphorylation of IRS-1 by PKC-
may negatively modulate IRS-1
function by impairing insulin-stimulated tyrosine phosphorylation of
IRS-1.

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Fig. 5.
Overexpression of PKC-
in NIH-3T3IR cells impairs insulin-stimulated
tyrosine phosphorylation of IRS-1. A, cells transiently
transfected with HA-tagged IRS-1 and either a control vector or PKC-
were treated with insulin (100 nM) for 0, 2, or 60 min.
Cell lysates were then immunoprecipitated with an anti-HA antibody
followed by immunoblotting with an anti-phosphotyrosine antibody
(top panel) or an anti-IRS-1 antibody (lower
panel). B, quantification of IRS-1 phosphotyrosine
content (mean ± S.E.) based on scanning densitometry of three
independent immunoblots normalized for IRS-1 recovery. Ippt,
immunoprecipitate.
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Because tyrosine phosphorylation of IRS-1 is required to engage and
activate downstream SH2 domain-containing effectors, we studied the
effects of the overexpression of PKC-
on insulin-stimulated interactions between IRS-1 and PI 3-kinase or SHP-2.
NIH-3T3IR cells transiently cotransfected with IRS1-HA and
either a control vector or PKC
-WT were stimulated with insulin, and
anti-HA immunoprecipitates were immunoblotted for the p85 regulatory
subunit of PI 3-kinase. As expected, insulin stimulation resulted in a
significant increase in association between IRS1-HA and p85 in control
cells (Fig. 6A, top
panel, lanes 1-3). Interestingly, the overexpression
of PKC
-WT did not appear to alter the amounts of p85 associated with
IRS1-HA in response to insulin when compared with controls (Fig.
6A, top panel, lanes 4-6).
Nevertheless, when we examined PI 3-kinase activity in the anti-HA
immunoprecipitates, the overexpression of PKC
-WT was associated with
a significant ~65% decrease in IRS-1-associated PI 3-kinase activity
at the 0-, 2-, and 60-min time points when compared with the control
group (Fig. 6, A (middle panel, lanes
1-6) and B). Thus, impairment in insulin-stimulated IRS-1 tyrosine phosphorylation caused by the overexpression of PKC-
was accompanied by a significant decrease in IRS-1-associated PI
3-kinase activity. The overexpression of PKC-
did not alter the
amount of SHP-2 coimmunoprecipitated with IRS-1 in response to insulin
stimulation (data not shown).

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Fig. 6.
Effects of overexpression of
PKC- on the ability of IRS-1 to bind and
activate PI 3-kinase. NIH-3T3IR cells transiently
transfected with HA-tagged IRS-1 or IRS1-Y612/Y632 and either a control
vector or PKC- were treated with insulin (100 nM) for 0, 2, or 60 min. A, cell lysates were immunoprecipitated with
anti-HA antibody followed by immunoblotting with anti-p85 antibody
(top panel) or anti-IRS-1 antibody (lower
panel). IRS-1-associated PI 3-kinase activity was assessed in a
parallel set of HA immunoprecipitates by measuring the
32P-labeled phosphatidylinositol phosphate (PIP) product
(middle panel). B, PhosphorImager quantification
of [32P]phosphatidylinositol phosphate product. Results
are mean ± S.E. of three independent experiments. IB,
immunoblot; Ippt, immunoprecipitate.
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Human IRS-1 contains six tyrosine residues (Tyr465,
Tyr612, Tyr632, Tyr662,
Tyr941, and Tyr987) in YXXM motifs
that may bind the SH2 domains of p85 (12). We recently showed that the
presence of Tyr612 and Tyr632 (in the absence
of Tyr465, Tyr662, Tyr941, and
Tyr987) is sufficient to mimic the ability of wild-type
IRS-1 to activate PI 3-kinase and mediate translocation of GLUT4 in rat
adipose cells whereas IRS-1 missing all six of these YXXM
motifs is unable to engage and activate PI 3-kinase (33). Therefore, we
also examined the effects of the overexpression of PKC-
on the
ability of IRS1-Y612/Y632 to associate with p85 and activate PI
3-kinase in response to insulin. Similar to results we obtained with
wild-type IRS-1, the overexpression of PKC-
was not associated with
alteration in p85 association with IRS1-Y612/Y632 but did cause
significant impairment in IRS-1-associated PI 3-kinase activity at all
time points (Fig. 6, A (lanes 7-12) and
B). These results suggest that phosphorylation of IRS-1 by
PKC-
specifically interferes with the function of Tyr612
or Tyr632. To rule out the possibility that PKC-
was
directly inhibiting PI 3-kinase activity, we compared PI 3-kinase
activity in anti-PDGF receptor immunoprecipitates from cells
cotransfected with PDGF
receptor and either a control
vector or PKC
-WT. We did not observe any significant
ability of PKC-
to impair PDGF-stimulated PI 3-kinase activity associated with the PDGF receptor (Fig.
7).

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Fig. 7.
Overexpression of PKC-
does not inhibit PI 3-kinase activity per
se. NIH-3T3IR cells transiently
cotransfected with PDGF receptor and either a control vector or
PKC- were treated without or with PDGF BB (100 ng/ml, 2 min). Cell lysates were immunoprecipitated with anti-PDGF receptor
antibody followed by immunoblotting with anti-p85 antibody (top
panel). PI 3-kinase activity associated with the PDGF receptor
(PDGFR) was assessed in a parallel set of
anti-PDGF -receptor immunoprecipitates by measuring the
32P-labeled phosphatidylinositol phosphate (PIP)
product (lower panel). Representative results are shown from
experiments that were repeated independently three times.
IB, immunoblot; Ippt,
immunoprecipitate.
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DISCUSSION |
PKC-
is an effector of PI 3-kinase signaling pathways that
plays an important role in metabolic actions of insulin (1-4). In
addition, PKC-
mediates a number of other biological actions of
insulin, including activation of p70 S6 kinase (34), ERK2 (35), and
NHE1 (36) and stimulation of protein synthesis (37). However,
downstream substrates of PKC-
in insulin signaling pathways have not
been identified to date. Similarly, direct substrates of PKC-
upstream of PI 3-kinase that may be involved with feedback regulation
of PKC-
have not previously been reported. Among the known
substrates for PKC-
that are unrelated to insulin signaling (7-11),
specific phosphorylation sites have only been identified for
IKK
(11). Unlike the identification of substrates for Akt, where many physiological substrates and phosphorylation sites have been
discovered because of a specific consensus sequence for Akt
phosphorylation (38), identification of substrates for PKC-
has been
difficult. This is because conventional consensus phosphorylation
motifs for protein kinase C ((R/K)XX(S/T)X(K/R)) (39) have not proven robust or specific for all protein kinase C
isoenzymes (40). For example, in IKK
, the amino acid sequence surrounding PKC-
phosphorylation sites at Ser177 and
Ser181 (11, 41) does not conform to either the general
protein kinase C consensus motif (39) or an optimal PKC-
consensus
motif based upon screening of a peptide library (40).
IRS-1 May Be a Novel Physiological Substrate for
PKC-
--
IRS-1 is a substrate of the insulin receptor tyrosine
kinase that plays an important role in coupling signaling by the
insulin receptor with activation of multiple downstream pathways,
including PI 3-kinase and SHP-2 (4, 12). Increased serine
phosphorylation of IRS-1 has been implicated in impairment of IRS-1
tyrosine phosphorylation and may be one mechanism underlying acquired
insulin resistance (16, 17, 42). Feedback control of IRS-1 may exist
because Ser/Thr kinases downstream from PI 3-kinase, such as GSK-3 and Akt, are capable of phosphorylating IRS-1 and modulating its function (23-25). When we examined IRS-1 as a potential substrate for PKC-
, we found that wild-type PKC-
but not kinase-inactive mutant PKC-
phosphorylated IRS-1 under in vitro conditions, suggesting
that IRS-1 is capable of functioning as a direct substrate for PKC-
. Moreover, our in vivo labeling experiments demonstrating
that IRS-1 is a substrate for PKC-
in intact cells suggest that
IRS-1 is a target of feedback regulation not only by GSK-3 and Akt but also by PKC-
. The fact that the interaction between endogenous IRS-1
and PKC-
in rat adipose cells was increased upon insulin stimulation
is also consistent with an important regulatory role for
phosphorylation of IRS-1 by PKC-
in insulin signaling pathways.
Functional Consequences of IRS-1 Phosphorylation by
PKC-
--
Because PKC-
is downstream from PI 3-kinase, feedback
regulation of IRS-1 through phosphorylation by PKC-
might be
expected to alter the ability of IRS-1 to engage and activate PI
3-kinase. The overexpression of PKC-
was sufficient to significantly
increase serine phosphorylation of IRS-1 in intact cells. Consistent
with a negative feedback role, we observed that the overexpression of
PKC-
significantly impaired insulin-stimulated tyrosine
phosphorylation of IRS-1. These results are in keeping with studies
showing that increased serine phosphorylation of IRS-1 secondary to
other stimuli is accompanied by impairment in the ability of IRS-1 to
undergo tyrosine phosphorylation after insulin stimulation (15-22,
43). In addition, there was some specificity to the impairment of IRS-1 function because the interaction between IRS-1 and SHP-2 appeared to be
unaffected by the overexpression of PKC-
.
The tyrosine-phosphorylated YXXM motifs on IRS-1 that are
predicted to engage and activate PI 3-kinase represent only a
fraction of the total number of phosphotyrosine motifs in IRS-1 that
couple to downstream signaling molecules (12). Therefore, we
specifically examined the consequences of impaired IRS-1 tyrosine
phosphorylation on coupling with PI 3-kinase by assessing the
coimmunoprecipitation of the p85 regulatory subunit of PI 3-kinase with
IRS-1 in the presence or absence of PKC-
. Interestingly, the amount
of p85 associated with IRS-1 after insulin stimulation was similar in both the presence and absence of PKC-
. However, the PI 3-kinase activity associated with IRS-1 was significantly reduced in the presence of PKC-
after both 2 and 60 min of insulin treatment. It is
unlikely that the overexpression of PKC-
is directly inhibiting PI
3-kinase activity because we did not observe any significant ability
of PKC-
to impair PDGF-stimulated PI 3-kinase activity in
anti-PDGF receptor immunoprecipitates. In a previous study, we showed
that a mutant human IRS-1 with substitutions of Phe for Tyr at
positions 465, 612, 632, 662, 941, and 987 did not bind and activate PI
3-kinase and was incapable of mediating translocation of GLUT4 in rat
adipose cells (33). However, an add-back mutant with Tyr612
and Tyr632 mimicked the effects of wild-type IRS-1 to
activate PI 3-kinase in response to insulin and to mediate
translocation of GLUT4 (33). To further define the functional
consequences of IRS-1 phosphorylation by PKC-
, we examined the
effects of the overexpression of PKC-
on the ability of
IRS1-Y612/Y632 to bind and activate PI 3-kinase. Similar to our results
with wild-type IRS-1, the overexpression of PKC-
did not impair
insulin-stimulated association of p85 with IRS1-Y612/Y632 but was
accompanied by a significant decrease in associated PI 3-kinase
activity. These results suggest that phosphorylation of IRS-1 by
PKC-
specifically impairs insulin-stimulated tyrosine
phosphorylation at residues 612 and 632 and that this somehow
interferes specifically with the ability to activate PI 3-kinase. It is
likely that phosphorylation of additional tyrosine residues is also
affected because total IRS-1 tyrosine phosphorylation in response to
insulin was substantially decreased by the overexpression of PKC-
.
Nevertheless, the functional impairment of IRS-1 caused by the
overexpression of PKC-
did not appear to interfere with insulin-stimulated interactions between IRS-1 and SHP-2.
It is somewhat surprising that the decrease in insulin-stimulated
tyrosine phosphorylation of IRS-1 caused by the overexpression of
PKC-
was accompanied by a decrease in IRS-1-associated PI 3-kinase
activity without a change in p85 binding. In some studies, serine
phosphorylation of IRS-1 causes both decreased association of p85 with
IRS-1 and decreased PI 3-kinase activity (14, 22, 25). However, many
studies examining the effects of IRS-1 serine phosphorylation report
either p85 association with IRS-1 or IRS-1-associated PI 3-kinase
activity alone but do not correlate p85 binding with PI 3-kinase
activity (15, 18, 21, 43). When YXXM motifs in IRS-1 are
tyrosine-phosphorylated in response to insulin, the SH2 domains of p85
bind to these sites on IRS-1, resulting in activation of the
preassociated p110 catalytic subunit of PI 3-kinase (12, 44).
Therefore, one might expect p85 binding to correlate with PI 3-kinase
activity. However, the tandem SH2 domains of p85 must be occupied
simultaneously for full activation of PI 3-kinase (44-47). It is
possible that Ser/Thr phosphorylation of IRS-1 by PKC-
may affect
the geometry of interactions between tandem SH2 domains in p85 and
YXXM motifs in IRS-1 such that differences in
coimmunoprecipitation of p85 with IRS-1 are undetectable but full
activation of PI 3-kinase activity is impaired.
Feedback regulation of IRS-1 function by downstream Ser/Thr kinases may
be an important general property of insulin signal transduction. Direct
phosphorylation of IRS-1 by GSK-3 negatively modulates IRS-1 function
(24) whereas phosphorylation of IRS-1 by Akt may have both positive and
negative effects on signaling by IRS-1 (23, 25). Paz et al.
(23) suggest that Akt phosphorylation of IRS-1 on four serine residues
in the PTB domain of IRS-1 enhances IRS-1 tyrosine
phosphorylation in response to insulin. On the other hand, Li et
al. (25) have implicated Akt pathways in negative modulation of
IRS-1 function involving serines 632, 662, and 731. It will be of great
interest to identify specific PKC-
phosphorylation sites on IRS-1 in
future studies.
Conclusions--
We have identified IRS-1 as a potential
physiological substrate of PKC-
. Phosphorylation of IRS-1 by PKC-
represents an additional example of negative feedback regulation of
IRS-1 by a Ser/Thr kinase downstream from PI 3-kinase. The existence of multiple feedback regulatory mechanisms at the level of IRS-1 emphasizes the critical importance of IRS-1 in insulin signaling and
may have implications for understanding the pathophysiology of insulin
resistance in diseases such as diabetes and obesity.