Phosphorylation of PTP1B at Ser50 by Akt Impairs Its Ability to Dephosphorylate the Insulin Receptor
Lingamanaidu V. Ravichandran1,
Hui Chen1,
Yunhua Li and
Michael J. Quon
Cardiology Branch, National Heart, Lung, and Blood Institute,
National Institutes of Health, Bethesda, Maryland 20892
Address all correspondence and requests for reprints to: Michael J. Quon, M.D., Ph.D., Cardiology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Building 10, Room 8C-218, 10 Center Drive MSC 1755, Bethesda, Maryland 20892-1755. E-mail:
quonm{at}nih.gov
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ABSTRACT
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PTP1B is a protein tyrosine phosphatase that negatively
regulates insulin sensitivity by dephosphorylating the insulin
receptor. Akt is a ser/thr kinase effector of insulin signaling that
phosphorylates substrates at the consensus motif RXRXXS/T.
Interestingly, PTP1B contains this motif (RYRDVS50), and
wild-type PTP1B (but not mutants with substitutions for
Ser50) was significantly phosphorylated by Akt
in vitro. To determine whether PTP1B is a substrate
for Akt in intact cells, NIH-3T3IR cells transfected
with either wild-type PTP1B or PTP1B-S50A were labeled with
[32P]-orthophosphate. Insulin stimulation caused a
significant increase in phosphorylation of wild-type PTP1B that could
be blocked by pretreatment of cells with wortmannin or cotransfection
of a dominant inhibitory Akt mutant. Similar results were observed with
endogenous PTP1B in untransfected HepG2 cells. Cotransfection of
constitutively active Akt caused robust phosphorylation of wild-type
PTP1B both in the absence and presence of insulin. By contrast,
PTP1B-S50A did not undergo phosphorylation in response to insulin. We
tested the functional significance of phosphorylation at
Ser50 by evaluating insulin receptor autophosphorylation in
transfected Cos-7 cells. Insulin treatment caused robust receptor
autophosphorylation that could be substantially reduced by coexpression
of wild-type PTP1B. Similar results were obtained with coexpression of
PTP1B-S50A. However, under the same conditions, PTP1B-S50D had an
impaired ability to dephosphorylate the insulin receptor. Moreover,
cotransfection of constitutively active Akt significantly inhibited the
ability of wild-type PTP1B, but not PTP1B-S50A, to dephosphorylate the
insulin receptor. We conclude that PTP1B is a novel substrate for Akt
and that phosphorylation of PTP1B by Akt at Ser50 may
negatively modulate its phosphatase activity creating a positive
feedback mechanism for insulin signaling.
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INTRODUCTION
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PTP1B IS A protein tyrosine phosphatase
(PTPase) that plays an important physiological role to negatively
modulate insulin sensitivity in vivo, at least in part, by
directly dephosphorylating both the insulin receptor and insulin
receptor substrate 1 (IRS-1) (1, 2, 3, 4). PTP1B knockout mice
have increased sensitivity to the metabolic actions of insulin and are
resistant to becoming obese (1, 2). In addition,
overexpression of PTP1B in rat adipose cells impairs metabolic actions
of insulin (5, 6), and alterations in expression of PTP1B
in insulin target tissues have been implicated in the pathophysiology
of insulin resistance in obesity and diabetes (7, 8).
Among the multitude of PTPases identified to date, only a few,
including PTP1B, LAR, PTP-
, and PTP-
, are known to
dephosphorylate insulin receptors in intact cells (3, 9, 10, 11, 12). This implies a high level of specificity for particular
PTPases to dephosphorylate and inactivate the insulin receptor tyrosine
kinase (12). Thus, PTP1B is an attractive therapeutic
target for the treatment of diabetes and obesity. Since PTP1B is the
prototypical PTPase that was the first to be identified
(13) and among the first to be cloned (14, 15), it has been the subject of numerous studies (for reviews
see Refs. 16 and 17). The crystal structure
for PTP1B has been solved (18), and considerable
effort has been devoted to designing specific PTP1B inhibitors
(6, 19, 20, 21, 22, 23). Nevertheless, mechanisms for regulating PTP1B
activity remain poorly understood. The elucidation of potential
regulatory mechanisms may lead to novel strategies for manipulating
PTP1B function that will be useful for treating diabetes and
obesity.
Akt is a ser/thr kinase downstream from PI3K in insulin signaling
pathways (24, 25, 26) that has been implicated as an effector
of metabolic actions of insulin (27, 28). The discovery of
a number of substrates for Akt has been facilitated by the
identification of a robust Akt consensus phosphorylation motif RXRXXS/T
(for reviews see Refs. 29 and 30).
Interestingly, PTP1B contains this motif
(RYRDVS50), and amino acids immediately preceding
the putative Akt phosphorylation site at Ser50
(Tyr46, Arg47,
Asp48 and Val49) play
important roles in stabilizing substrates in the catalytic cleft of
PTP1B (18, 22, 31, 32, 33, 34). Thus, it is plausible that
phosphorylation at Ser50 may alter the ability of
PTP1B to engage and dephosphorylate its substrates. In the present
study, we evaluated PTP1B as a novel substrate for Akt and investigated
the potential role of phosphorylation at Ser50 in
regulating PTP1B function related to insulin signaling.
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RESULTS
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To identify potential regulatory phosphorylation sites on PTP1B,
we scanned the PTP1B amino acid sequence for the Akt consensus
phosphorylation motif RXRXXS/T and discovered a matching sequence in
PTP1B (RYRDVS) with a predicted phosphorylation site at
Ser50. Even though this region of PTP1B is highly
conserved among PTPases and homologous sequences are contained in many
intracellular and receptor-like PTPases, the exact Akt phosphorylation
motif is present in this region in only 2 of 30 other PTPases examined
(Fig. 1A
). Moreover, the Akt
phosphorylation motif in PTP1B is completely conserved among many
different species, raising the possibility that PTP1B may be a bona
fide substrate for Akt (Fig. 1B
). To help evaluate PTP1B as a substrate
for Akt, we constructed point mutants with substitutions of Ala or Asp
for Ser50, a catalytically inactive substrate
trapping mutant with substitution of Ser for
Cys215, and constructs that contained mutations
at both positions 50 and 215 (Fig. 1C
).

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Figure 1. Akt Consensus Phosphorylation Motif in PTP1B
A, Amino acid sequence alignment of other PTPases with the region of
human PTP1B containing the Akt consensus phosphorylation motif (RXRXXS)
(adapted from Ref. 32 ). The putative Akt phosphorylation
site at Ser50 in PTP1B is present in homologous sequences
of only 2 of the other 30 PTPases examined. B, The Akt consensus
phosphorylation motif in PTP1B is conserved among different species. C,
Illustration of PTP1B point mutants that were constructed to substitute
Ala or Asp for Ser50 at the putative Akt phosphorylation
site or Ser for Cys215 in the catalytic domain of PTP1B.
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Akt Phosphorylates PTP1B in Vitro at
Ser50
To determine whether PTP1B is capable of functioning as a direct
substrate for Akt, we first performed in vitro Akt kinase
assays using activated recombinant Akt and purified PTP1B (truncated
protein containing amino acids 1322) in the presence of
[32P]-ATP. In these assays, autophosphorylation
of Akt was evident in both the presence and absence of PTP1B (Fig. 2A
, lanes 1 and 3). However, significant
phosphorylation of PTP1B was only observed in the presence, but not in
the absence, of Akt (Fig. 2A
, lanes 23). We next evaluated
Ser50 as a potential Akt phosphorylation site by
using wild-type and mutant PTP1B proteins immunoprecipitated from
lysates of transfected NIH-3T3IR cells as the
substrate in our in vitro kinase assays. Note that little,
if any, endogenous PTP1B was detected in PTP1B immunoprecipitates of
untransfected cells while comparable amounts of recombinant wild-type
and mutant PTP1B were recovered (Fig. 2B
). Consistent with the previous
results, full-length wild-type PTP1B underwent significant
phosphorylation in the presence, but not in the absence, of Akt (Fig. 2A
, lanes 56). Similarly, PTP1B-C215S (catalytically inactive mutant
with Ser50 intact) was also substantially
phosphorylated in the presence of Akt (Fig. 2A
, lane 9). Importantly,
phosphorylation of PTP1B-S50A or PTP1B-S50D (mutants missing the
putative Akt phosphorylation site) was negligible when compared with
either PTP1B-WT or PTP1B-C215S (Fig. 2A
, lanes 69) and was similar to
PTP1B-WT incubated in the absence of Akt (Fig. 2A
, lane 5). These
results suggest that Akt is capable of directly phosphorylating PTP1B
at Ser50 and raise the possibility that PTP1B may
be a novel substrate for Akt.

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Figure 2. Akt Phosphorylates PTP1B in Vitro at
Ser50
In vitro kinase assays were carried out in the presence
of [ -32P]-ATP using purified active Akt (lanes 1, 3,
4, and 69) and either truncated purified PTP1B (a.a. 1322) (lanes
23) or full-length PTP1B immunoprecipitated from lysates of
NIH-3T3IR cells transiently transfected with empty vector,
PTP1B-WT, PTP1B-S50A, PTP1B-S50D, or PTP1B-C215S mutants (lanes 49).
A, Purified PTP1B is phosphorylated only in the presence of Akt
(compare lanes 2 and 3). Phosphorylation of immunoprecipitated PTP1B-WT
and PTP1B-C215S by Akt (lanes 6 and 9) is significantly greater than
that observed for PTP1B-S50A or PTP1B-S50D (lanes 7 and 8).
Autophosphorylation of Akt is evident and consistent with the presence
of active Akt (lanes 1, 3, 4, and 69). B, Anti-PTP1B immunoblot
demonstrating comparable recovery of PTP1B in anti-PTP1B
immunoprecipitates used for the kinase assays (lanes 59).
Representative results are shown for experiments that were repeated
independently at least four times.
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Phosphorylation of PTP1B at Ser50 in Intact
Cells
To determine whether PTP1B can be phosphorylated at
Ser50 in intact cells, we performed in
vivo labeling experiments in NIH-3T3IR cells
transiently cotransfected with Akt and either PTP1B-WT or PTP1B-S50A.
After transfection and labeling with
[32P]-orthophosphate, cells were treated
without or with insulin, and the amount of label incorporated into
PTP1B was assessed in PTP1B immunoprecipitates using a PhosphorImager.
Insulin treatment caused a significant 70% increase in phosphorylation
of PTP1B-WT in intact cells (Fig. 3
, lanes 12). By contrast, no significant increase in PTP1B
phosphorylation was observed with insulin treatment of cells expressing
PTP1B-S50A (Fig. 3, lanes 34). Note that the PTP1B recovered by
immunoprecipitation in these experiments was predominantly recombinant
PTP1B since there is very little endogenous PTP1B present in
NIH-3T3IR cells (cf. Fig. 2B
). Since
Akt is downstream from PI3K in insulin signaling pathways, we evaluated
the effects of pretreatment of cells with the PI3K inhibitor
wortmannin. Interestingly, we found that the insulin-stimulated
increase in phosphorylation of PTP1B-WT was completely inhibited by
wortmannin (data not shown). To examine the insulin-stimulated
phosphorylation of PTP1B in a more physiological context, we repeated
the in vivo labeling studies in untransfected HepG2 cells.
Importantly, insulin also stimulated a significant increase in
phosphorylation of endogenous PTP1B in this liver cell line (Fig. 4
, lanes 12) that was inhibited by
wortmannin pretreatment (Fig. 4
, lanes 34). Although a previous
report suggested that PTP1B undergoes tyrosine phosphorylation in
response to insulin stimulation in transfected rat 1 fibroblasts
(35), we were unable to detect any increased tyrosine
phosphorylation of PTP1B under our experimental conditions by
immunoblotting with an antiphosphotyrosine antibody (data not shown).
We next evaluated the effects of constitutively active and dominant
inhibitory mutants of Akt on insulin-stimulated phosphorylation of
PTP1B. In control cells transfected with only PTP1B-WT (no Akt
cotransfection), insulin stimulation resulted in an increase in
phosphorylated PTP1B (Fig. 5, lanes
12). This phosphorylation was inhibited by coexpressing the dominant
inhibitory mutant Akt-AAA (Fig. 5
, lanes 34). Coexpression of the
constitutively active Akt-myr resulted in robust phosphorylation of
PTP1B in both the absence and presence of insulin (Fig. 5
, lanes 56).
Taken together with our in vitro data, these results in
intact cells suggest that phosphorylation of PTP1B at
Ser50 by Akt can occur in vivo
and that this phosphorylation may be regulated by insulin.

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Figure 3. Insulin Stimulates Phosphorylation of PTP1B at
Ser50 in Intact Cells
NIH-3T3IR cells transiently cotransfected with Akt and
either PTP1B-WT or PTP1B-S50A were labeled with
[32P]-orthophosphate and then stimulated without or with
insulin (100 nM, 10 min). PTP1B immunoprecipitated from
cell lysates was subjected to 10% SDS-PAGE and Phosphor-Imager
analysis. A, PhosphorImager scan of a representative in
vivo labeling experiment showing that insulin stimulates
phosphorylation of PTP1B-WT (lanes 1 and 2) but not PTP1B-S50A (lanes 3
and 4). B, Anti-PTP1B immunoblot demonstrating comparable recovery of
PTP1B in all anti-PTP1B immunoprecipitates. C, Quantification of
results by PhosphorImager were normalized for recovery of PTP1B in the
immunoprecipitates. Results shown are the mean ± SEM
of three independent experiments.
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Figure 4. Insulin-Stimulated Phosphorylation of Endogenous
PTP1B in Untransfected HepG2 Cells Is Blocked by Wortmannin
Pretreatment
HepG2 cells were labeled with [32P]-orthophosphate and
pretreated without or with wortmannin. Cells were then stimulated
without or with insulin (100 nM, 10 min) and PTP1B
immunoprecipitates of cell lysates were subjected to 10% SDS-PAGE and
PhosphorImager analysis. A, Phosphor-Imager scan of representative
in vivo labeling experiment showing that insulin
stimulates phosphorylation of PTP1B only in the absence of wortmannin
pretreatment (lanes 1 and 2) but not after wortmannin pretreatment
(lanes 3 and 4). B, Anti-PTP1B immunoblot demonstrating comparable
recovery of PTP1B in all anti-PTP1B immunoprecipitates. C,
Quantification of results by PhosphorImager was normalized for recovery
of PTP1B in the immunoprecipitates. Results shown are the mean ±
SEM of three independent experiments.
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Figure 5. Effect of Akt Mutants on Phosphorylation of PTP1B
NIH-3T3IR cells transiently transfected with PTP1B-WT and
an empty vector (control), Akt-AAA, or Akt-myr were labeled with
[32P]-orthophosphate and stimulated without or with
insulin (100 nM, 10 min). PTP1B immunoprecipitated from
cell lysates was subjected to 10% SDS-PAGE and PhosphorImager
analysis. A, Image from PhosphorImager analysis of a representative
in vivo labeling experiment that was repeated
independently five times. B, Anti-PTP1B immunoblot demonstrating
comparable recovery of PTP1B in the immunoprecipitates for each
group.
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Role of Ser50 in Regulating Function of PTP1B
To examine functional consequences of phosphorylation of PTP1B by
Akt in response to insulin stimulation, we used an in vitro
phosphatase assay to directly assess PTP1B activity.
NIH-3T3IR cells cotransfected with PTP1B-WT and
either a control vector or Akt-myr were stimulated without or with
insulin, and anti-PTP1B immunoprecipitates were tested for their
ability to dephosphorylate a tyrosine-phosphorylated peptide substrate
derived from the insulin receptor. Interestingly, the activity of PTP1B
to dephosphorylate the peptide substrate was significantly decreased by
approximately 25% after either insulin stimulation or cotransfection
of Akt-myr (Fig. 6
). Thus,
phosphorylation of PTP1B by Akt in response to insulin stimulation may
impair the catalytic activity of PTP1B.

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Figure 6. Catalytic Activity of PTP1B Is Impaired After
Insulin Stimulation or Cotransfection with Constitutively Active Akt
NIH-3T3IR cells transiently cotransfected with
PTP1B-WT and either a control vector or constitutively active Akt
(Akt-myr) were serum starved overnight and then treated without or with
insulin (100 nM, 5 min). Phosphatase activity in PTP1B
immunoprecipitates was assessed using a tyrosine-phosphorylated
insulin receptor peptide as described in Materials and
Methods. A, Phosphatase activity was normalized for PTP1B
recovery and plotted as a percent of the sample from untreated control
cells (mean ± SEM of three independent experiments).
Both insulin treatment and coexpression of Akt-myr resulted in a
significant decrease in PTP1B catalytic activity (P
< 0.03). B, Anti-PTP1B immunoblot demonstrating comparable recovery of
PTP1B in the immunoprecipitates for each group.
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We next investigated the ability of wild-type and mutant PTP1B to
dephosphorylate the insulin receptor in intact cells. Cos-7 cells have
low levels of endogenous insulin receptor and PTP1B. By transiently
cotransfecting these cells with human insulin receptor and various
PTP1B constructs, we were able to evaluate effects of recombinant PTP1B
on receptor phosphorylation in the transfected cells without
interference from untransfected cells. Cell lysates of cotransfected
cells were immunoblotted with antiphosphotyrosine antibody to
evaluate autophosphorylation of the insulin receptor (Fig. 7
). In control cells overexpressing only
human insulin receptor, insulin treatment caused robust receptor
autophosphorylation (Fig. 7
, lanes 12). As expected, coexpression of
wild-type PTP1B significantly decreased insulin-stimulated receptor
phosphorylation while overexpression of the catalytically inactive
PTP1B-C215S mutant had no detectable effect (Fig. 7
, lanes 34 and
910). Overexpression of PTP1B-S50A, a mutant with substitution of Ala
in the putative Akt phosphorylation site at
Ser50, resulted in decreased insulin-stimulated
receptor phosphorylation similar to that observed with overexpression
of PTP1B-WT (Fig. 7
, lanes 78). By contrast, overexpression of
PTP1B-S50D, a mutant designed to mimic phosphorylation at
Ser50, was associated with a level of receptor
phosphorylation intermediate between that observed in control cells and
cells expressing PTP1B-WT or PTP1B-S50A (Fig. 7
, lanes 56).

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Figure 7. Mutations at Ser50 Affect the Ability
of PTP1B to Dephosphorylate the Insulin Receptor
Cos-7 cells transiently cotransfected with human insulin receptor (hIR)
and either empty vector (control) or various PTP1B constructs were
treated without (-) or with (+) insulin (100 nM, 3 min).
Whole-cell lysates (30 µg total protein) were subjected to 10%
SDS-PAGE and immunoblotted with antibodies against phosphotyrosine,
insulin receptor, or PTP1B. Representative blots are shown for
experiments that were repeated independently four times.
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In addition to the insulin receptor, IRS-1 is also a substrate for
PTP1B (4). Therefore, we evaluated the ability of our
PTP1B mutants to affect insulin-stimulated tyrosine phosphorylation of
IRS-1. In experiments similar to those shown in Fig. 7
, hemagglutinin
(HA)-tagged IRS-1 was immunoprecipitated from lysates of Cos-7 cells
transiently cotransfected with PTP1B constructs, insulin receptor, and
IRS1-HA; these samples were then immunoblotted with antiphosphotyrosine
antibody (Fig. 8
). As with the insulin
receptor, insulin treatment of control cells resulted in significant
tyrosine phosphorylation of IRS1-HA (Fig. 8, lanes 12). This
phosphorylation was significantly decreased in the presence of PTP1B-WT
or PTP1B-S50A (Fig. 8
, lanes 34 and 78). IRS1-HA in
insulin-stimulated cells overexpressing PTP1B-S50D was largely, but not
completely, dephosphorylated (
15% of the level observed in the
insulin-stimulated control) (Fig. 8
, lanes 56). Thus, PTP1B-S50D may
have an impaired ability to dephosphorylate both the insulin receptor
and IRS-1.

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Figure 8. Mutations at Ser50 Affect the
Ability of PTP1B to Dephosphorylate IRS-1
Cos-7 cells transiently cotransfected with human insulin receptor
(hIR), HA-tagged IRS-1 (IRS1-HA), and either empty vector (control) or
various PTP1B constructs were treated without (-) or with (+) insulin
(100 nM, 3 min). Anti-HA immunoprecipitates of cell lysates
(400 µg total protein) were subjected to 8% SDS-PAGE and
immunoblotted with antibodies against phosphotyrosine or HA.
Representative blots are shown for experiments that were repeated
independently three times.
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We, and others, have previously shown that the catalytically inactive
substrate trapping mutant PTP1B-C215S can coimmunoprecipitate with the
phosphorylated insulin receptor (6, 35). To determine
whether interactions between PTP1B and the insulin receptor can be
altered by manipulations at Ser50, we examined
the ability of S50A/C215S and S50D/C215S mutants to coimmunoprecipitate
with the phosphorylated insulin receptor in
NIH-3T3IR cells. As expected, significant
coimmunoprecipitation of the insulin receptor and PTP1B-C215S was
observed in samples derived from insulin-stimulated cells (Fig. 9
, lanes 12). Similar results were
obtained with the S50A/C215S mutant. By contrast, the insulin receptor
did not coimmunoprecipitate to the same extent with the S50D/C215S
mutant (Fig. 9
, lanes 56). These results suggest that phosphorylation
at Ser50 may interfere with the ability of PTP1B
to interact with its substrates. Moreover, these results are also
consistent with the impaired ability of PTP1B-S50D to dephosphorylate
the insulin receptor and IRS-1.

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Figure 9. Mutations at Ser50 Affect the Ability
of PTP1B to Bind Phosphorylated Insulin Receptor
NIH-3T3IR cells transiently transfected with PTP1B-C215S,
S50A/C215S, S50D/C215S, or empty vector (control) were treated without
(-) or with (+) insulin (100 nM, 3 min). Anti-PTP1B
immunoprecipitates of cell lysates (500 µg total protein) were
subjected to 10% SDS-PAGE and immunoblotted with antibodies against
the insulin receptor or PTP1B (lanes 17). Immunoblots of cell lysates
are shown in lanes 814. Representative blots are shown for
experiments that were repeated independently four times.
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To provide further evidence that phosphorylation of PTP1B at
Ser50 by Akt is important for regulating PTP1B
activity, we coexpressed various Akt constructs along with both the
insulin receptor and either PTP1B-WT or PTP1B-S50A in Cos-7 cells.
As previously demonstrated in Fig. 7
, insulin-stimulated
phosphorylation of the insulin receptor was significantly decreased by
overexpression of either PTP1B-WT or PTP1B-S50A alone (Fig. 10
, lanes 14 and 912).
Coexpression of a dominant inhibitory Akt mutant (Akt-AAA) had no
detectable effect on the ability of either PTP1B-WT or PTP1B-S50A to
dephosphorylate the insulin receptor (Fig. 10
, lanes 78 and 1516).
Importantly, coexpression of the constitutively active Akt mutant
(Akt-myr) significantly impaired the ability of PTP1B-WT, but not
PTP1B-S50A, to dephosphorylate the insulin receptor (compare Fig. 10
.
lanes 56 with lanes 1314). Taken together with data from
experiments using PTP1B-S50D and S50D/C215S shown in
Figs. 79

, these
results are consistent with the possibility that phosphorylation of
PTP1B at Ser50 by Akt may negatively regulate
catalytic activity of PTP1B and impair its ability to dephosphorylate
the insulin receptor. To confirm the functional significance of these
results in a more physiological context, we examined insulin-stimulated
tyrosine phosphorylation of the insulin receptor in the absence and
presence of wortmannin in untransfected HepG2 cells. Consistent with an
Akt-dependent positive feedback mechanism involving regulation of PTP1B
activity, wortmannin pretreatment completely inhibited
insulin-stimulated phosphorylation of Akt (Fig. 11C
) and resulted in a small, but
statistically significant, 25% decrease in insulin receptor
autophosphorylation (Fig. 11
, A and B).

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Figure 10. Action of Activated Akt to Inhibit
Dephosphorylation of the Insulin Receptor by PTP1B Requires
Ser50
Cos-7 cells transiently cotransfected with human insulin receptor, a
PTP1B construct, and either empty vector (control) or the indicated Akt
constructs were treated without (-) or with (+) insulin (100
nM, 3 min). Whole-cell lysates (30 µg total protein) were
subjected to 10% SDS-PAGE and immunoblotted with antibodies against
phosphotyrosine, insulin receptor, PTP1B, or Akt. Note that
coexpression of the constitutively active Akt-myr significantly
inhibits the ability of PTP1B-WT to dephosphorylate the insulin
receptor (lanes 5 and 6) but has no effect on the ability of PTP1B-S50A
to dephosphorylate the insulin receptor (lanes 13 and 14).
Representative blots are shown from experiments that were repeated
independently four times.
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Figure 11. Insulin-Stimulated Autophosphorylation of the
Insulin Receptor Is Impaired by Wortmannin Pretreatment in
Untransfected HepG2 Cells
Cells were pretreated without or with wortmannin and then stimulated
without or with insulin (100 nM, 10 min). A, Insulin
receptor immunoprecipitates of cell lysates were subjected to 10%
SDS-PAGE followed by immunoblotting with antiphosphotyrosine or
antiinsulin receptor antibodies. A representative immunoblot is shown
from experiments that were repeated independently four times. B,
Quantification of antiphosphotyrosine immunoblots (mean ±
SEM of four independent experiments normalized for insulin
receptor recovery). C, Akt and phospho-Akt immunoblot of cell lysates
demonstrating inhibition of Akt phosphorylation by wortmannin
pretreatment.
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DISCUSSION
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The importance of PTP1B in negatively regulating metabolic actions
of insulin has been unequivocally demonstrated by the presence of
increased insulin sensitivity, enhanced insulin receptor
phosphorylation, and resistance to obesity in PTP1B knockout mice
(1, 2). Thus, it is of interest to identify novel
mechanisms for regulating catalytic activity and function of PTP1B,
such as phosphorylation of critical residues by known kinases. Akt is a
ser/thr kinase downstream from PI3K that plays essential roles in cell
growth, differentiation, proliferation, and survival (30).
With respect to insulin signaling, Akt has been implicated in both
metabolic actions of insulin to promote glucose transport (27, 28, 36) as well as vasodilator actions of insulin mediated by
nitric oxide (37). Known substrates of Akt that
participate in insulin signaling pathways and whose functions are
regulated by Akt phosphorylation include glycogen synthase kinase-3
(GSK-3) (25), phosphofructokinase-2 (38),
mammalian target of rapamycin (39, 40), forkhead
transcription factor FHKR (41, 42), insulin receptor
substrate-1 (IRS-1) (43, 44), cyclic nucleotide
phosphodiesterase 3B (45, 46), and endothelial nitric
oxide synthase (47, 48). The identification of many of
these Akt substrates has been facilitated by the existence of a robust
consensus phosphorylation motif (30). We found that PTP1B
contains this motif [amino acids (a.a.) 4550] in a region that
forms important stabilizing contacts with amino acids immediately
upstream from the phosphotyrosine residue of PTP1B substrates. Thus,
phosphorylation of Ser50 in this region might be
predicted to have functionally important consequences. Interestingly,
only two other PTPases (TC-PTP and PTP-MEG1) contain this exact Akt
phosphorylation motif despite high homology of this region in general
among many PTPases. The absolute conservation of this region in PTP1B
among different species is also consistent with its potential role as
an Akt phosphorylation site.
PTP1B Is a Novel Substrate for Akt Both in Vitro and
in Intact Cells
We demonstrated that PTP1B can function as a direct substrate for
Akt under in vitro conditions using both a commercially
available purified truncated PTP1B (a.a. 1322) as well as full-length
PTP1B immunoprecipitated from transfected cells as a substrate. The
phosphorylation of PTP1B by Akt when Ser50 was
present (PTP1B-WT or PTP1B-C215S) was significantly greater
than when Ser50 was replaced by either Ala
or Asp. The small amount of phosphorylation observed with PTP1B-S50A
and PTP1B-S50D may be due to nonspecific incorporation of
[
-32P]-ATP or the presence of other cryptic
Akt phosphorylation sites. Alternatively, it is possible that known
sites of phosphorylation on PTP1B such as Tyr66,
Tyr152, Tyr153
(35), Ser352,
Ser378, Ser386
(49), or other unknown sites may undergo weak
phosphorylation by kinases that coimmunoprecipitate with PTP1B.
Nevertheless, substantial phosphorylation of PTP1B by Akt occurs only
when Ser50 is intact. This strongly suggests that
Ser50 is a bona fide target for direct
phosphorylation by Akt in vitro.
Results from our in vivo labeling experiments were
also consistent with the presence of an Akt phosphorylation site in
PTP1B at Ser50 and suggest that PTP1B can
function as a substrate for Akt in intact cells. Insulin treatment of
NIH-3T3IR cells cotransfected with PTP1B and Akt
caused a significant increase in phosphorylation of PTP1B-WT, but not
PTP1B-S50A. Furthermore, this in vivo phosphorylation
of PTP1B-WT was blocked by pretreatment of cells with the PI3K
inhibitor wortmannin and did not require coexpression of Akt.
Importantly, we also observed similar results in untransfected
HepG2 cells, suggesting that endogenous PTP1B can be phosphorylated by
Akt in response to insulin stimulation in a more physiologically
relevant cell type. Moreover, a dominant inhibitory Akt mutant blocked
the phosphorylation of PTP1B in response to insulin. We, and others,
have previously documented that the mutant Akt used in this assay
inhibits wild-type Akt activity (46, 50). Finally,
coexpression of a constitutively active Akt mutant resulted in robust
phosphorylation of PTP1B. Interestingly, the phosphorylation of PTP1B
appeared to be much greater in response to cotransfection of Akt-myr
than in response to insulin stimulation. This suggests that not all
PTP1B molecules are phosphorylated in response to insulin stimulation.
One possible explanation for these observations is that the amount of
label incorporated into PTP1B during the 10-min insulin stimulation is
likely to be much less than during the lengthy period of time
transfected cells are exposed to elevated levels of Akt-myr. It is also
possible that Akt-myr is a potent kinase that can phosphorylate PTP1B
at additional sites. When we directly assessed PTP1B activity using an
in vitro phosphatase assay, we observed a significant
inhibition of PTP1B activity in both PTP1B immunoprecipitates derived
from insulin-treated cells or cells cotransfected with Akt-myr. Thus,
it is plausible that insulin stimulation may result in impaired PTP1B
activity under physiological conditions. It seems unlikely that the
phosphorylation of PTP1B we observed in response to insulin
stimulation represents tyrosine phosphorylation because we did
not observe any significant increase in
tyrosine-phosphorylated PTP1B by immunoblotting under our experimental
conditions. Taken together, our results suggest that
Ser50 is an insulin- and PI3K-dependent Akt
phosphorylation site on PTP1B in vivo, and it is possible
that phosphorylation of PTP1B on Ser50 by Akt may
contribute to feedback regulation of insulin signaling.
Role of Phosphorylation at Ser50 in Regulating Function
of PTP1B
We hypothesized that phosphorylation at
Ser50 by Akt impairs the ability of PTP1B to
dephosphorylate its substrates. We evaluated this possibility by
comparing the ability of wild-type and mutant forms of PTP1B to
dephosphorylate physiological substrates such as the insulin receptor
and IRS-1 in intact cells. Insulin-stimulated receptor
autophosphorylation was greatly decreased (compared with results from
control cells) in cells overexpressing wild-type PTP1B. Since this was
not observed with the catalytically inactive PTP1B-C215S, we conclude
that wild-type PTP1B is effective at dephosphorylating the insulin
receptor as previously described (3). Overexpression of
PTP1B-S50A (mutant unable to undergo phosphorylation at
Ser50 resulted in dephosphorylation of the
insulin receptor to a similar extent as wild-type PTP1B. By contrast,
PTP1B-S50D (mutant designed to mimic phosphorylation at
Ser50 had an impaired ability to dephosphorylate
the insulin receptor. We observed similar results with respect to the
effects of our PTP1B constructs on insulin-stimulated phosphorylation
of IRS-1. Since IRS-1 is a substrate for PTP1B (4),
decreased levels of insulin-stimulated IRS-1 phosphorylation in the
presence of PTP1B-WT or PTP1B-S50A may represent both direct effects of
PTP1B on IRS-1 as well as secondary effects of PTP1B mediated by
dephosphorylation of the insulin receptor. Although we cannot entirely
rule out the possibility that our results with IRS-1 are completely
secondary to dephosphorylation of the insulin receptor, this seems less
likely because the relative impairment of PTP1B-S50D with respect to
IRS-1 dephosphorylation is less than that observed for insulin receptor
dephosphorylation (compare Fig. 7
, lanes 2 and 6 with Fig. 8
, lanes 2
and 6). Thus, data regarding the ability of our mutant PTP1B constructs
to dephosphorylate the insulin receptor and IRS-1 in intact cells are
consistent with the idea that phosphorylation of PTP1B at
Ser50 negatively regulates its function. In
addition, data from coimmunoprecipitation experiments suggest that this
impairment in PTP1B function may be the result of decreased binding of
PTP1B to its substrates when Ser50 is
phosphorylated (mimicked by the S50D mutant).
Our experiments examining insulin receptor autophosphorylation in cells
coexpressing constitutively active Akt (Akt-myr) with PTP1B further
support the idea that phosphorylation of Ser50 in
PTP1B by Akt is functionally relevant. Results from cells coexpressing
Akt-myr and PTP1B-WT were similar to results from cells overexpressing
PTP1B-S50D, suggesting that phosphorylation of PTP1B by Akt impairs
PTP1B function. In addition, these results argue against the
possibility that the impaired function of PTP1B-S50D is due to
misfolding of the mutant protein. More importantly, the impairment of
PTP1B function caused by coexpression of Akt-myr was not observed with
PTP1B-S50A, strongly suggesting that it is the ability of Akt to
phosphorylate Ser50 that results in impaired
PTP1B function. Finally, the impairment of insulin receptor
autophosphorylation caused by wortmannin pretreatment of untransfected
HepG2 cells is consistent with the idea that endogenous Akt may be
phosphorylating endogenous PTP1B and impairing its ability to
dephosphorylate the insulin receptor under more physiological
conditions. This may represent a novel mechanism for negatively
regulating PTP1B activity.
Implications for Insulin Signaling
Previously identified sites on PTP1B in the noncatalytic region at
Ser352, Ser378, and
Ser386 undergo phosphorylation in response to
12-O-tetradecanoyl phorbol-13-acetate stimulation or
changes in cell cycle (49). Phosphorylation at these
sites has been suggested to alter subcellular targeting of PTP1B and is
associated with modest decreases in PTPase activity. Bandyopadhyay
et al. have previously identified
Tyr66, Tyr152, and
Tyr153 as sites on PTP1B that can be
phosphorylated by the insulin receptor and enhance interactions between
PTP1B and the insulin receptor (35). It is possible that
differences in cell type may account for our inability to detect
tyrosine phosphorylation of PTP1B in our experiments. We have now
identified a novel Akt phosphorylation site on PTP1B at
Ser50. Our data suggest that phosphorylation of
PTP1B at Ser50 by Akt negatively modulates the
ability of PTP1B to dephosphorylate important physiological targets of
PTP1B. There are multiple phosphorylated tyrosine residues on both the
insulin receptor and IRS proteins. The exact sites on the insulin
receptor that are targets for PTP1B are not known, but it seems likely
that a majority of the sites are dephosphorylated by PTP1B since
significant effects can be detected by antiphosphotyrosine
immunoblotting. The observed decreases in IRS-1 tyrosine
phosphorylation may represent both direct effects of PTP1B and
secondary effects of reduced insulin receptor activity.
Since Akt is a downstream effector of insulin action and PTP1B acts at
upstream sites to inhibit insulin action, the ability of Akt to impair
PTP1B function may represent a positive feedback mechanism. This is not
unprecedented since Akt participates in positive feedback mechanisms
for insulin signaling at the level of IRS-1 (43). In
addition to the putative pathophysiological role of PTP1B in
insulin-resistant states such as diabetes and obesity, the recent
identification of PTP1B as the major PTPase that dephosphorylates and
activates c-src in human breast cancer cell lines suggests
that excessive PTP1B activity may also play a role in breast cancer and
other malignancies (51). Identification of novel
mechanisms for negatively regulating PTP1B activity and function, such
as phosphorylation by Akt, may lead to critical insights for the
development of therapies for a variety of important public health
problems.
 |
MATERIALS AND METHODS
|
---|
Reagents
Reagents were obtained from the following sources:
monoclonal anti-PTP1B antibody from Oncogene Science, Inc. (Boston, MA); recombinant PTP1B protein,
phosphorylated insulin receptor peptide substrate (IR 5, 9, 10),
and Biomol Green reagent from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA); rabbit polyclonal anti-PTP1B
antibody, purified recombinant activated Akt, and GSK peptide from
Upstate Biotechnology, Inc. (Lake Placid, NY);
antiinsulin receptor and antiphosphotyrosine antibodies (PY20) from
Transduction Laboratories, Inc. (Lexington, KY);
anti-HA antibody (HA-11) from BabCO (Richmond, CA); wortmannin from
Sigma (St. Louis, MO); protein G agarose and LipofectAMINE
PLUS from Life Technologies, Inc. (Gaithersburg, MD);
rabbit polyclonal anti-phospho-Akt (Ser473) from
New England Biolabs, Inc. (Beverly, MA); and
[
-32P]-ATP and
[32P]-orthophosphate from ICN Biomedicals, Inc. (Irvine, CA).
Expression Plasmids
pCIS2.
pC152 is a parental expression vector with a CMV promoter/enhancer
(52, 53).
PTP1B-WT.
cDNA for human PTP1B was ligated into the multiple cloning region of
pCIS2 as described (5).
PTP1B-C215S.
cDNA for a catalytically inactive mutant PTP1B with substitution of Ser
for Cys215 was ligated into the multiple cloning
region of pCIS2 as described (5).
PTP1B-S50D.
A point mutant derived from PTP1B-WT with Asp substituted for
Ser50 was constructed using the mutagenic
oligonucleotide 5'-GG TAC AGA GAC GTG GAT CCC
TTT GAC CAT AG-3' and the Morph Mutagenesis kit (5 Prime to 3
Prime, Boulder, CO). This mutagenesis also introduced a silent mutation
at the position encoding Val49 creating a new
BamHI site.
PTP1B-S50A.
A point mutant derived from PTP1B-S50D with Ala substituted at position
50 was constructed using the mutagenic oligonucleotide 5'-GG TAC AGA
GAC GTG GCT CCC TTT GAC CAT AG-3' and the Morph
kit.
S50D/C215S.
A mutant PTP1B containing both substitutions of Asp for
Ser50 and Ser for Cys215
was derived from PTP1B-C215S using the mutagenic oligonucleotide pairs
5'-CGA AAT AGG TAC AGA GAC GTG GAT CCC TTT GAC
CAT AGT CGG-3' and 5'-CCG ACT ATG GTC AAA GGG ATC
CAC GTC TCT GTA CCT ATT TCG-3' with the QuikChange
mutagenesis kit (Stratagene, La Jolla, CA). This also
introduced a silent mutation at the position encoding
Val49, creating a new BamHI site.
S50A/C215S.
A mutant PTP1B containing both substitutions of Ala for
Ser50 and Ser for Cys215
was derived from PTP1B-C215S using the mutagenic oligonucleotide pairs
5'-CGA AAT AGG TAC AGA GAC GTG GCT CCC TTT GAC
CAT AGT CGG-3' and 5'-CCG ACT ATG GTC AAA GGG
AGC CAC GTC TCT GTA CCT ATT TCG-3' with the
QuikChange kit. The presence of the desired mutations in all PTP1B
constructs was verified by direct sequencing.
Akt-WT.
cDNA for mouse Akt-1 was ligated into multiple cloning region of pCIS2
as described previously (28).
Akt-AAA.
A dominant inhibitory mutant of Akt with substitutions of Ala for
Lys179 in the ATP binding domain, as well as for
the regulatory phosphorylation sites, Thr308 and
Ser473, was created and subcloned into pCIS2 as described
(46).
Akt-myr.
cDNA for mouse Akt-1 with a myristoylation sequence from pp60 c-src
(54) fused in-frame with the N terminus of Akt
(generous gift from Drs. P. N. Tsichlis and K. Datta) was
ligated into the multiple cloning region of pCIS2 as described
(28).
pCIS-hIR.
cDNA for the human insulin receptor was ligated into the multiple
cloning site of pCIS2 as described (55).
IRS1-HA.
cDNA for human IRS-1 was subcloned into pCIS2 with a sequence coding
for an HA-epitope tag fused to the C terminus as described
(56).
Cell Culture and Transfection
NIH-3T3 fibroblasts overexpressing human insulin receptors
(NIH-3T3IR) or Cos-7 cells were maintained in
DMEM containing 10% FBS, L-glutamine (2 mM),
penicillin (100 U/ml), and streptomycin (100 µg/ml), in a humidified
atmosphere with 5% CO2 at 37 C. Cells were
transiently transfected with various constructs using LipofectAMINE
PLUS according to the manufacturers instructions. HepG2 cells were
maintained in DMEM containing 100 mM glucose, 10% FBS,
L-glutamine (2 mM), penicillin (100 U/ml),
and streptomycin (100 µg/ml).
In Vitro Akt Kinase Assays
In vitro assays using purified activated Akt as the
kinase and PTP1B as substrate were carried out at 30 C for 30 min in
kinase assay buffer containing 50 mM Tris-HCl, pH
7.4, 10 mM MgCl2, 1
mM dithiothreitol, 50 µM
ATP, and 2.5 µCi [
-32P]-ATP/assay.
Reactions were stopped by adding Laemmli sample buffer and boiling for
10 min. Samples were subjected to 10% SDS-PAGE and a PhosphorImager
(Molecular Dyamics, Inc., Sunnyvale, CA) was used to
detect phosphorylated PTP1B and autophosphorylated Akt. In
addition, gel contents were transferred to nitrocellulose and
immunoblotted with anti-PTP1B antibody. Finally, activity of Akt in
each assay was independently verified using a peptide substrate derived
from GSK-3 (RPRAATF) as described (29, 57). For assays
using purified PTP1B (truncated protein containing amino acids 1322),
2.5 µg of PTP1B, and approximately 0.3 µg of Akt (specific
activity,154 nmol phosphate transferred to GSK-3 peptide/min/mg
protein) were used. In some experiments, full-length recombinant
wild-type and mutant PTP1B proteins immunoprecipitated from lysates of
transfected NIH-3T3IR cells were used as
substrate. One day after transfection, cell lysates were prepared using
lysis buffer A (50 mM Tris-HCl, pH 7.4, 125
mM NaCl, 1% Triton X-100, 0.5% NP-40, 1
mM
Na3VO4, 50
mM NaF, 0.1 mM okadaic
acid, and complete protease inhibitor cocktail (Roche Molecular Biochemicals, Indianapolis, IN). Lysates (500 µg total
protein) were precleared with protein G agarose beads for 1 h at 4
C and then incubated with anti-PTP1B antibody (2.5 µg) and
protein G agarose beads for 2 h at 4 C. The immunocomplexes
were washed four times with kinase assay buffer and used in the
in vitro kinase assays as described above.
In Vivo Phosphorylation Experiments
Untransfected HepG2 cells or
NIH-3T3IR cells transiently transfected with
various PTP1B and Akt constructs were serum starved overnight and then
labeled for 4 h with [32P]-orthophosphate
(75 µCi/ml in KRB buffer, pH 7.4, containing 1% BSA) as described
previously (56). After labeling, cells were treated
without or with insulin (100 nM, 10 min) and washed four
times with PBS, after which whole-cell lysates were prepared as
described above. Lysates were immunoprecipitated with anti-PTP1B
antibody as described above, and samples were separated by 10%
SDS-PAGE. Phosphorylated PTP1B was detected and quantified using a
PhosphorImager. In addition, gel contents were transferred to
nitrocellulose for immunoblotting with anti-PTP1B antibody. In some
experiments, transfected cells were pretreated with wortmannin (100
nM) 2.5 h after initiation of labeling with
[32P]-orthophosphate (90 min before insulin
treatment).
In Vitro PTP1B Phosphatase Assay
NIH-3T3IR cells transiently
cotransfected with PTP1B-WT and either a control vector or Akt-myr were
serum starved overnight and then treated without or with insulin (100
nM, 5 min) and washed with PBS, and whole-cell lysates
were prepared as described above using Buffer A without
Na3VO4 and NaF. Lysates
were immunoprecipitated with anti-PTP1B antibody and Protein G as
described above, and the immunoprecipitates were washed four
times with phosphatase assay buffer (50 mM HEPES, pH 7.2, 1
mM EDTA, 1 mM dithiothreitol, 0.05% NP-40).
The Protein G beads were then suspended in 55 µl of assay buffer and
5 µl of 1.5 mM phosphorylated insulin receptor peptide
(residues 11421153 of the insulin receptor phosphorylated at Tyr
1146, 1150, and 1151) were added as the substrate. After incubation at
30 C for 30 min, 25 µl of the clear supernatant were transferred to
half-size 96-well plates, and 100 µl of Biomol Green reagent were
added to each well. Samples were gently agitated for 2030 min, and
the free phosphate liberated was determined by absorbance at 620
nM using a microtiter-plate spectrophotometer. A standard
curve was generated for each experiment to quantify results.
Dephosphorylation of Insulin Receptor and IRS-1 by PTP1B
The ability of wild-type and mutant PTP1B to
dephosphorylate the insulin receptor was evaluated by
immunoblotting whole-cell lysates derived from Cos-7 cells transiently
cotransfected with human insulin receptor and either pCIS2 (empty
vector) or PTP1B constructs. The day after transfection, cells were
serum-starved overnight and then treated without or with insulin (100
nM) for 3 min. Whole-cell lysates were made as previously
described (58) using lysis buffer B (50 mM
Tris, pH 7.4, 300 mM NaCl, 1% Triton X-100, 1
mM Na3VO4, and
complete protease inhibitor cocktail) and centrifuged at 6,000 x
g for 3 min at 4 C to pellet the remaining cellular debris.
An aliquot from each group was resolved by SDS-PAGE, transferred to
nitrocellulose, and immunoblotted with antibodies against
phosphotyrosine, insulin receptor, or PTP1B. In some experiments cells
were also cotransfected with various Akt constructs. In addition,
similar experiments were performed on anti-HA immunoprecipitates of
cell lysates derived from cells also cotransfected with IRS1-HA.
For immunoprecipitation, an aliquot of each lysate was incubated with 2
µg of HA-11 antibody overnight at 4 C followed by incubation with
washed protein G-conjugated agarose beads for 2 h at 4 C on a
rotating wheel. Immunocomplexes were washed twice with lysis buffer
B (without protease inhibitors), once with buffer C (20
mM Tris, pH 7.4, 150 mM
NaCl), and then subjected to SDS-PAGE and immunoblotting.
Coimmunoprecipitation of insulin receptor with PTP1B was assessed in
cell lysates derived from NIH-3T3IR cells
transiently transfected with PTP1B mutants C215S, S50A/C215S, or
S50D/C215S that were treated without or with insulin (100
nM) for 3 min. Samples were
immunoprecipitated with anti-PTP1B antibody followed by
immunoblotting with antiinsulin receptor antibody.
Assessment of Insulin Receptor Autophosphorylation and Akt
Phosphorylation in HepG2 Cells
Confluent HepG2 cells were serum starved overnight,
pretreated without or with wortmannin (100 nM) for 90 min,
and then stimulated without or with insulin (100 nM, 10
min). Insulin receptor and PTP1B were immunoprecipitated from
whole-cell lysates (500 µg total protein) using antiinsulin receptor
and anti-PTP1B antibodies, respectively, and samples were immunoblotted
with antiphosphotyrosine antibody (PY20), antiinsulin receptor
antibody, or anti-PTP1B antibodies. Akt and phospho-Akt immunoblots
were also performed using whole-cell lysates (100 µg total
protein).
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. Monica Montagnani and Fredly Bataille for technical
assistance with some experiments.
 |
FOOTNOTES
|
---|
1 These authors made equal contributions to this work. 
Abbreviations: a.a., Amino acids; GSK-3, glycogen synthase
kinase-3, HA, hemagglutinin; IRS-1, insulin receptor substrate 1;
PTPase, protein tyrosine phosphatase; PTP1B-WT, wild-type PTP1B.
Received for publication March 13, 2001.
Accepted for publication June 19, 2001.
 |
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