From the Departments of Pharmacology,
Biochemistry, and ** Cellular & Structural Biology,
The University of Texas Health Science Center, San Antonio, Texas 78229 and the ¶ Joslin Diabetes Center and Department of Medicine,
Harvard Medical School, Boston, Massachusetts 02215
Received for publication, August 20, 2002, and in revised form, December 12, 2002
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ABSTRACT |
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Grb10 has been proposed to inhibit or activate
insulin signaling, depending on cellular context. We have investigated
the mechanism by which full-length hGrb10 The insulin receptor
(IR)1 transmits signals
through the actions of its intrinsic receptor tyrosine kinase. Ligand
binding results in the autophosphorylation of the IR on multiple
tyrosine residues (1, 2). Once phosphorylated, these residues serve as
docking sites for specific intracellular effectors central to the
transmission of the insulin signal. The insulin receptor substrate
(IRS) family (IRS-1 through IRS-4) is one such group of proteins that
recognizes the phosphorylated IR via their phosphotyrosine binding
domains (PTB) (3, 4). The IRS-PTB domain associates with the NPXY motif
surrounding tyrosine 972 in the juxtamembrane region of the IR, which
promotes the IR-IRS interaction (5). Once in contact with the receptor,
IRS is phosphorylated on multiple tyrosine residues by the IR tyrosine
kinase. Numerous adaptor proteins and enzymes then associate with
tyrosine-phosphorylated IRS via their Src homology 2 (SH2) domains and
convey the insulin signal downstream (6, 7).
The insulin receptor-binding protein Grb10 is of considerable interest
due to its potential to positively or negatively affect receptor-tyrosine kinase signaling. Grb10 is a member of a superfamily of adaptor proteins, which includes Grb7 and Grb14. This family of
proteins shares several structural features, including a SH2 and a
Pleckstrin homology (PH) domain (8). The C-terminal SH2 domain has been
shown to associate with the IR (9, 10), and mutation of a critical
arginine residue in this region of full-length Grb10 disrupts IR-Grb10
association in cells (11). A second domain termed the BPS (for
between the Pleckstrin and SH2) has also been suggested as a second independent IR-interacting domain (9).
Grb10 interacts with the regulatory kinase loop of the IR (10, 12, 13),
although studies have also reported a Grb10-interacting region in the
IR C terminus (14, 15). Several human Grb10 isoforms exist and most
likely arise from alternative splicing. Two isoforms of hGrb10 differ
in the PH domain. hGrb10 The functional role for Grb10 in insulin signaling remains
controversial. Grb10 has been shown to positively stimulate
insulin-induced mitogenesis (18). Microinjection of a peptide fragment
including the BPS and SH2 domain of Grb10 inhibited insulin-stimulated
DNA synthesis (13, 18), which, if functioning as a dominant negative, is consistent with Grb10 as a positive regulator for cell growth. Several studies have indicated an inhibitory role for Grb10 as well.
Overexpression of hGrb10 Stable expression of hGrb10 Insulin-stimulated tyrosine phosphorylation of IRS proteins induces the
association of the p85 regulatory subunit of PI 3-kinase and
subsequently results in an increase in PI 3-kinase activity (23). As an
IR-interacting protein, Grb10 has the potential to influence signaling
through this IRS/PI 3-kinase pathway and moderate the actions of
downstream effectors such as Akt. This study investigates the molecular
mechanisms linked to the effects of Grb10 on insulin signaling. We find
that hGrb10 Cell Lines and Reagents--
Chinese hamster ovary (CHO) cells
expressing both the human insulin receptor and hGrb10 Plasmids--
cDNAs encoding HA-tagged Grb10 Adenoviruses--
Adenoviruses encoding green fluorescence
protein (GFP, as an adenovirus control) and hGrb10 Cell Culture, Immunoprecipitation, and Western Blot--
CHO/IR
cells were maintained in Ham's F12 media supplemented with 10%
newborn calf serum and 1% penicillin/streptomycin. Cells were grown to
60-80% confluency 24 h prior to transfection using LipofectAMINE
reagent according to the manufacturer's protocol (Invitrogen). 3T3-L1
cells were cultured in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Cells were grown to confluency and then differentiated with the addition of 500 µM
isobutylmethylxanthine, 25 µM dexamethasone, and 4 µg/ml insulin for 3 days, followed by 3 days in supplemental media
with 4 µg/ml insulin added. Cells were cultured an additional 3-4
days in supplemental media to complete differentiation. For adenovirus
experiments, cells were transduced with GFP (control) or Grb10 Yeast Transformation and Interaction Assay--
The yeast
MATCHMAKER LexA two-hybrid system reagents were purchased from
Clontech. The yeast Saccharyomyces
cerevisiae strain GUSEGY48 (Mat Tri-Hybrid Disruption Assay--
GUSEGY48 was sequentially
transformed as described above with pLexA-IR, pB42AD IRS-1
1-1242aa/IRS-2 3-1321aa/IRS-2 5-591aa/IRS-2 591-1321aa (generous
gifts from Dr. Thomas A. Gustafson), and a third plasmid (pDIS)
containing various Grb10 constructs. Transformants were grown on
SD-HisTrpUra agar plates for 3 days at 30 °C. Four independent
colonies were streaked onto SD-HisTrpUra agar plates, incubated
overnight, and then replicated onto SD galactose/raffinose-HisLeuTrpUra agar plates and incubated for 5 days at 30 °C to induce the
expression of B42 and pDIS fusion proteins and promote growth.
Grb10
Previous reports indicated that insulin rapidly induces phosphorylation
of AktT308 and that this is stable during extended
stimulation (31, 32). Furthermore, limited AktS473
phosphorylation in the basal state rapidly increases following growth
factor stimulation (33). Since hGrb10 Full-length Grb10 Inhibits Insulin-stimulated Tyrosine
Phosphorylation of IRS-1 and IRS-2--
The IRS proteins are rapidly
phosphorylated in response to insulin stimulation. Since
tyrosine-phosphorylated IRS-1/2 increases PI 3-kinase activity in the
cell by binding the regulatory subunit of PI 3-kinase (7), we
considered whether Grb10-mediated inhibition of Akt phosphorylation
might arise from decreased signaling through IRS-1/2. A partial
inhibition of insulin-stimulated tyrosine phosphorylation of IRS has
been observed with hGrb10 hGrb10 Grb10 Binding to IR Correlates with Inhibited IRS-1
Phosphorylation--
Because hGrb10 Autophosphorylation of IR Is Not Affected by Grb10
Expression--
IRS interacts directly with the
tyrosine-phosphorylated NPEY972 (34) motif in the IR
juxtamembrane (4). Studies have indicated that autophosphorylation of
the IR initiates at tyrosines 1158, 1162, and 1163 in the core
catalytic domain (35), and phosphorylation in the C-terminal and
juxtamembrane domains rapidly follows. Since Grb10 associates with the
regulatory kinase domain of the IR (10, 12, 13), we considered that the
effects of Grb10 on IRS tyrosine phosphorylation might arise from a
decrease or loss in receptor autophosphorylation. The insulin receptor
was immunoprecipitated from cell lysates of either CHO/IR or CHO/IR
stably expressing hGrb10 Grb10 Disrupts the Association of IRS-1/2 with the
Insulin Receptor--
To determine the mechanism behind Grb10-mediated
inhibition of IRS-1/2 tyrosine phosphorylation, a yeast tri-hybrid
disruption assay was performed using IR as bait, IRS-1 or IRS-2 as prey
and various Grb10 constructs (Fig.
6A) as disrupters. The
interaction between IR and IRS-1 (36) and IRS-2 (37, 38) in yeast, as determined by growth on selective media, has been well characterized. Co-expression of full-length hGrb10 Complete Inhibition of IRS Phosphorylation Requires an Intact Grb10
SH2 Domain--
Results of the yeast tri-hybrid system indicated that
disruption of IR/IRS interactions required both the BPS and SH2
domains. Furthermore, the Grb10 SH2 domain appeared to be essential and disruption occurred regardless of whether the SH2 domain was
binding-competent. To determine if the BPS/SH2 region was necessary for
inhibition of insulin-stimulated IRS phosphorylation in mammalian
cells, we co-expressed full-length or BPS/SH2 fragments of wild-type or
mutated forms of hGrb10 Grb10 has been shown to play both a positive and negative role in
insulin signaling, and these differences appear to depend both on cell
type and on the Grb10 splice variant being studied. To understand these
functional differences, we investigated the molecular mechanism of
Grb10 action at the insulin receptor. Here, we show that full-length
human Grb10 has an inhibitory effect on insulin-stimulated signaling
through the IRS/PI 3-kinase pathway to Akt. Using a yeast tri-hybrid
system, we found Grb10 can disrupt the association of IRS proteins with
IR. The Grb10 SH2 domain is essential for this disruption and for
complete inhibition of IRS tyrosine phosphorylation in cells. Our
findings support a mechanism for Grb10-mediated inhibition of IRS/PI
3-kinase/Akt signaling, in which association of IRS proteins with the
IR kinase domain and/or the juxtamembrane NPXY motif
(Tyr972) is sterically hindered by Grb10.
Previous studies have concluded that blocking IR catalytic activity by
either full-length Grb10 or domain fragments results in decreased
substrate phosphorylation (19, 20). In agreement with these studies, we
have observed a Grb10-mediated decrease in phosphorylation of IR
substrates, including IRS-1/2 (this study) (16), p62dok
(16, 39), and Shc (data not shown). However, Mournier et al.
(21) reported that overexpression of hGrb10 did not affect IRS tyrosine
phosphorylation, PI 3-kinase activity or Akt activity in rat
hepatocytes, although IR kinase activity and IR autophosphorylation were reportedly reduced. Our results in CHO/IR cells show that the
catalytic activity of the RTK toward itself is not significantly impaired by Grb10. To a degree this is intuitive since tyrosine phosphorylation of the IR is necessary for Grb10 association. The rapid
association of Grb10 with the IR could suggest that if Grb10 blocked
the enzymatic activity of the RTK, autophosphorylation of the IR would
be reduced in CHO/IR/hGrb10 Although both the BPS and SH2 domains can independently interact with
the IR, the two regions together are necessary for disruption of
interactions between IRS-1/2 and IR. Our results with the yeast tri-hybrid system indicate that the BPS domain with either an active or
inactive SH2 domain is sufficient for disruption. This would suggest
that the BPS domain mediates the interaction with the IR and the SH2
domain facilitates a physical disruption of IR/IRS interaction. When
the Grb10 SH2 domain is deleted or replaced with the Shc SH2 domain,
the IR/IRS interaction is not disrupted although both constructs
interact with the IR in yeast two-hybrid assay. These findings support
a structural role for the Grb10 SH2 domain in hindering IR/IRS
interactions. However, Grb10 molecules lacking an intact SH2 domain
cannot support complete inhibition of IRS tyrosine phosphorylation in
mammalian cells (Fig. 7B). Interestingly, while the BPS
domain with either an active or inactive SH2 domain disrupted IR/IRS
interactions in the yeast tri-hybrid studies, only Grb10 (full-length
and BPS/SH2 fragment) with an active SH2 domain inhibited
insulin-stimulated IRS-1 tyrosine phosphorylation in mammalian cells.
Complete inhibition appeared to correlate with the ability of Grb10 to
co-immunoprecipitate the IR. In contrast with results using yeast
two-hybrid, in which the BPS domain is sufficient for interaction with
the IR, regardless of an active SH2 domain (Ref. 9 and Fig. 6), only
Grb10 with an active SH2 domain could co-immunoprecipitate the IR in
cells. The high sensitivity of the yeast system may allow for the
detection of the BPS-mediated IR interaction and may override the need
for an active SH2 domain. In mammalian cells, the SH2 domain may be needed to facilitate or strengthen BPS-mediated association with the
IR. For this reason, we could not fully assess the structural requirement of the SH2 domain in physically blocking IRS-1/2 access to
the IR in cells. In addition, we were unable to express fragments encoding only the BPS or SH2 domains, most likely due to their instability in mammalian cells. While we were unable to detect Grb10
association with the IR when the SH2 was inactivated or deleted,
partial inhibition of IRS tyrosine phosphorylation with overexpression
of these mutants suggests that some interaction via the BPS domain is likely.
Full-length hGrb10 In this study, the mechanism for Grb10-mediated inhibition of insulin
signaling was investigated. We found that full-length hGrb10 could
significantly inhibit insulin-stimulated tyrosine phosphorylation of
IRS-1 and IRS-2 and subsequently delay signaling to the downstream
effector, Akt. Autophosphorylation of the IR is not impaired by the
binding of Grb10 to the receptor, which indicates functional activity
of the RTK in cells. hGrb10 inhibits signaling through the insulin receptor substrate (IRS) proteins. Overexpression of
hGrb10
in CHO/IR cells and in differentiated adipocytes
significantly reduced insulin-stimulated tyrosine phosphorylation of
IRS-1 and IRS-2. Inhibition occurred rapidly and was sustained for 60 min during insulin stimulation. In agreement with inhibited signaling through the IRS/PI 3-kinase pathway, we found hGrb10
to both delay
and reduce phosphorylation of Akt at Thr308 and
Ser473 in response to insulin stimulation. Decreased
phosphorylation of IRS-1/2 may arise from impaired catalytic activity
of the receptor, since hGrb10
directly associates with the IR kinase
regulatory loop. However, yeast tri-hybrid studies indicated that
full-length Grb10 blocks association between IRS proteins and IR, and
that this requires the SH2 domain of Grb10. In cells, hGrb10
inhibited insulin-stimulated IRS-1 tyrosine phosphorylation in a
dose-dependent manner, but did not affect IR catalytic
activity toward Tyr972 in the juxtamembrane region and
Tyr1158/1162/1163 in the regulatory domain. We
conclude that binding of hGrb10
to IR decreases signaling through
the IRS/PI 3-kinase/AKT pathway by physically blocking IRS access to
IR.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
encodes for the full-length protein, while
Grb10
(previously named Grb-IR) lacks an intact PH domain as it
contains a 46-amino acid deletion in this region (16, 17). A third
isoform, hGrb10
is identical to hGrb10
with the exception of 58 amino acids at the extreme N terminus of the
isoform (12, 17).
negatively regulates insulin
receptor-mediated tyrosine phosphorylation of GTPase-activated protein
(GAP)-associated protein p60 and IRS-1 (16). Furthermore, binding of
Grb10 via its BPS/SH2 domains has been shown to inhibit IR catalytic
activity in vitro (19-21).
in CHO/IR reduces
insulin-dependent phosphorylation of IRS-1 (16), although a
direct effect of full-length Grb10 on IRS-1/2 and downstream effectors
of the insulin signal, such as Akt, has not been reported. However,
several studies indicate that Grb10 may regulate downstream events in various signaling pathways. Overexpression of hGrb10
(Grb10
) in
rat hepatocytes inhibits insulin-stimulated glycogen synthase activity,
through a proposed novel pathway outside of the classical PI 3-kinase
to Akt/glycogen synthase kinase-3 signaling (21). Grb10 has also been
found to associate with tyrosine-phosphorylated c-kit receptor, and
synergistically promote Akt activation. This study indicated Grb10
forms a constitutive complex with Akt and proposed that Grb10
positively influenced Akt activity by promoting its translocation to
the cell membrane leading to the phosphorylation and activation of this
kinase (22).
both delays and decreases Akt phosphorylation. This
inhibition stems from a rapid and sustained inhibition of
insulin-stimulated tyrosine phosphorylation of IRS-1 and IRS-2. Our
studies suggest that the mechanism for Grb10-mediated inhibition of
insulin signaling results from a physical disruption of IRS association
with phosphorylated residues of the IR kinase domain and/or the
NPXY motif (Tyr972). The SH2 domain of Grb10 is
essential for IR/IRS disruption. In addition, this region is required
to facilitate Grb10 association with the IR and inhibition of IRS
tyrosine phosphorylation in cells. This physical disruption of IR/IRS
interaction by Grb10 functions to slow signaling relayed through
tyrosine-phosphorylated IRS proteins.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
have been
previously described (16, 17). 3T3-L1 cells were from ATCC. Anti-HA
antibody was obtained from BABCO. Monoclonal anti-Myc antibody was from
Santa Cruz Biotechnology, Inc. Antibodies recognizing IRS-1 and IRS-2
were from Upstate Biotechnology. Akt and phospho-Akt
(Thr308 and Ser473) antibodies were from New
England Biolabs. The anti-phosphotyrosine (RC-20) antibody was from
Transduction Laboratories. Phosphospecific antibodies to
Tyr972, Tyr1158, and Tyr1162/1163
of the insulin receptor were obtained from
BIOSOURCE International. Secondary antibodies
conjugated to alkaline phosphatase and horseradish peroxidase were from
Promega.
and
Myc-tagged Akt have been previously described (17, 24). HA-tagged
Grb10
R520G was generated by single-stranded
site-directed mutagenesis according to the protocol described by Kunkel
et al. (25). The forward PCR primer 5'-
GCTCTAGATGGCCTTGCTGTCCCCGTTC-3' and the reverse PCR primer
5'-CGGAATTCCACTCGGATGCAGTGGTG-3' were used to amplify the BPS/SH2
fragment using pBex/Grb10
or pBex/Grb10
R520G as
template. The reverse PCR primer 5'-GCGAATTCGTGCTGTGTCCTGTGAATC-3' was
used to amplify the
SH2 fragment. The PCR products were inserted in-frame with the HA tag using the XbaI and EcoRI
restriction sites of the mammalian expression vector pBex (16). The
forward PCR primer 5'-TGCTCTAGACAGTGCTACAGAGCCAAC-3' and reverse PCR
primer 5'-GATCGGTCGACGTGCTGTGTCCTGTG-3' were used to amplify a 1.5-kb fragment from hGrb10
(region encoding for amino acids 1-491). The
forward PCR primer 5'-GATCGGTCGACCCCCCAAATCCTTCTCTT-3' and the reverse
primer 5'-CGGAATTCCACTTTCCGATCCACGGG-3' were used to amplify a 0.4-kb
(SH2 domain) fragment from pRK/Shc-myc (plasmid was a generous gift
from Dr. Ben Margolis). The individual PCR products were separately
subcloned into an intermediate vector using the
XbaI/SalI and SalI/EcoRI
restriction sites. The resulting chimeric fragment was inserted
in-frame with the HA tag using the XbaI/EcoRI
restriction sites of pBex. A plasmid encoding Myc-tagged IRS-1 was a
generous gift from Dr. Richard Roth (26). A plasmid encoding
full-length human IRS-2 was a generous gift from Dr. Klein-Hitpass
(27).
were produced by
using the AdEasy system (The AdEasy system is a generous gift of Drs.
Tong-Chuan He and Bert Vogelstein, Ref. 28). The cDNA encoding
HA-tagged Grb10
was cloned into the pAdTrack-CMV transfer vector
using EcoRV and XbaI restriction sites. The
hGrb10
-HA encoding sequence was then transferred into the pAdEasy
viral DNA plasmid by homologous recombination in the BJ5183
Escherichia coli strain. The recombinant adenoviral
construct was transfected into HEK293 cells to produce viral particles.
Adenoviruses were purified by CsCl gradient centrifugation. The
infection efficiency was estimated for GFP expression using an Olympus
IX70 immunofluorescence microscope.
viral
stocks at a multiplicity of infection of 20. Twenty-four hours
post-transfection/post-transduction cells were serum-starved for 4-18
h, treated with 10 nM (CHO/IR) or 100 nM
(adipocytes) insulin, and lysed in ice-cold Buffer A (50 mM
HEPES, pH 7.6, 150 mM NaCl, 1% Triton-X, 10 mM
NaF, 20 mM sodium pyrophosphate, 20 mM
-glycerophosphate, 1 mM sodium orthovanadate, 10 µg/ml
aprotinin, 10 µg/ml leupeptin, 1 µM microcystin-LR, and
1 mM phenylmethylsulfonyl fluoride). The homogenate was
centrifuged (10,000 × g, 4 °C, 10 min), and the
supernatants used for immunoprecipitation or Western blot experiments.
For immunoprecipitation, cell lysates were incubated with specific
antibodies bound to either protein A or protein G-Sepharose beads
(Amersham Biosciences) for 6-18 h at 4 °C with gentle rotation.
Immunoprecipitates were washed extensively with ice-cold Buffer B (50 mM HEPES, pH 7.6, 150 mM NaCl, 0.1% Triton X).
Proteins bound to beads were eluted by heating at 95 °C for 4 min in
SDS sample loading buffer. Eluted proteins were separated by SDS-PAGE,
transferred to a nitrocellulose membrane, and detected with specific
antibodies. Quantitation of the relative increase in Akt
phosphorylation was performed by analyzing Western blots using the
Scion Image program and normalized to the level of Akt expression in
each experiment.
, trp1, his3, urs3,
6LexAop-LEU2, LYS2) and the pLexA-IR, and various pB42AD Grb10
constructs were generous gifts from Dr. Thomas A. Gustafson. GUSEGY48
was sequentially transformed with plasmid constructs by the
polyethylene glycol/lithium acetate method according to the
Clontech protocol. Transformants were grown on
SD-HisTrp agar plates for 3 days at 30 °C. Four independent colonies
were streaked onto SD-HisTrp agar plates, grown overnight, replica-plated onto SD galactose/raffinose -HisLeuTrp agar plates and
incubated for 5 days at 30 °C to induce expression of B42 fusion
proteins and to determine interacting partners.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Inhibits Akt Phosphorylation in Response to Insulin
Stimulation--
In the insulin signaling cascade, elevated
phospholipid levels result from PI 3-kinase activity and provide for
the activation of downstream mediators, such as Akt (29, 30). To test
the effects of hGrb10
on Akt activation, Myc-tagged Akt was
expressed in CHO/IR cells together with HA-tagged hGrb10
.
Insulin-stimulated Akt phosphorylation was detected using
phosphospecific antibodies to AktT308 and
AktS473. Significant phosphorylation on both
AktT308 and AktS473 was seen with a 5-min
insulin stimulation (Fig. 1A,
lanes 1 and 2, first and third
panel), which is consistent with published results (31). The
presence of hGrb10
greatly reduced phosphorylation on
AktT308 (Fig. 1A, lanes 3 and
4, top panel). Phosphorylation at
AktS473 was also decreased with hGrb10
overexpression
(Fig. 1A, lanes 3 and 4, third
panel). Quantitative analyses indicated that hGrb10
reduced
AktS473 phosphorylation by ~50-60% and virtually
abolished AktT308 phosphorylation (Fig. 1B).
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Fig. 1.
Effects of Grb10 on
insulin-stimulated phosphorylation of Akt. A,
full-length Grb10 decreases insulin-stimulated phosphorylation of Akt.
CHO/IR cells were transiently transfected with Myc-tagged Akt and
either the empty vector or an expression construct encoding full-length
Grb10. Cells were serum-starved and stimulated with 10 nM
insulin for 5 min. Cell lysates were run on a 10% SDS-PAGE gel and
insulin-stimulated phosphorylation of Akt at Thr308 and
Ser473 was detected using phosphospecific antibodies. The
expression level of Akt was detected by antibody to the Myc tag and
Grb10
expression detected by antibody recognizing the HA tag.
Experiments were performed at least three times with similar results.
B, quantitative analysis of Akt phosphorylation. Akt
phosphorylation was quantified using Scion Image and normalized to Akt
expression levels. The mean fold stimulation expressed as a percentage
of the maximum ± S.E. from three independent experiments is
shown. C, insulin-stimulated phosphorylation of Akt is
delayed by the presence of Grb10
. CHO/IR cells were transfected and
blotted as described in A, except cells were stimulated with
10 nM insulin for the indicated time periods.
significantly inhibits
AktT308 and partially inhibits insulin-stimulated
AktS473 phosphorylation, we hypothesized that hGrb10
might have a temporal effect on Thr308 and
Ser473 phosphorylation. Akt was overexpressed in CHO/IR
cells in the absence or presence of hGrb10
. Cells were treated with
10 nM insulin for increasing times, and phosphorylation was
detected in whole cell lysates using phosphospecific antibodies.
hGrb10
delayed Akt phosphorylation at both sites, while concurrently decreasing the overall extent of phosphorylation for up to 1 h of
insulin treatment (Fig. 1C).
(the isoform that lacks a PH-domain) (16),
although the effects of full-length hGrb10
have not been
investigated. hGrb10
and IRS-1 were co-expressed in CHO/IR cells and
insulin-stimulated IRS-1 tyrosine phosphorylation was measured. In the
absence of Grb10, IRS-1 was tyrosine-phosphorylated after 5 min of
insulin stimulation (Fig. 2A,
lanes 1 and 2). Insulin-stimulated tyrosine
phosphorylation of IRS-1 was substantially inhibited when hGrb10
was
co-expressed (Fig. 2A, lanes 3 and 4).
IRS-1 tyrosine phosphorylation was inhibited in a
dose-dependent manner (Fig. 2B, lane
2 versus lanes 3-6). We also examined the
effects of hGrb10
on IRS-2 phosphorylation. Since little endogenous
IRS-2 was detected in CHO/IR cells (data not shown), we co-expressed IRS-2 and hGrb10
in CHO/IR cells. We found that treatment of cells
with insulin led to a significant increase in IRS-2 tyrosine phosphorylation (Fig. 2C, lane 2 versus lane 1). The insulin-stimulated IRS-2
tyrosine phosphorylation was greatly inhibited by overexpressing hGrb10
(Fig. 2C, lane 4 versus lane 2). These
findings demonstrate that the Grb10-mediated inhibition is not
specific for IRS-1.
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Fig. 2.
Full-length Grb10 inhibits
insulin-stimulated tyrosine phosphorylation of IRS. A,
Grb10 inhibits insulin-stimulated tyrosine phosphorylation of
overexpressed IRS-1 in CHO/IR cells. CHO/IR cells were transiently
transfected with mammalian expression constructs encoding Myc-tagged
IRS-1 and HA-tagged Grb10
. Cells were serum-starved, stimulated with
10 nM insulin for 10 min, lysed, and IRS-1
immunoprecipitated with antibody to the Myc tag. Insulin-stimulated
tyrosine phosphorylation of IRS-1 was detected by Western blot with a
phosphotyrosine-specific antibody. Membranes were then stripped and
reblotted for IRS-1 using an antibody to the Myc tag. Grb10
expression was detected in cell lysates using an antibody to the HA
tag. B, Grb10 inhibits insulin-stimulated IRS tyrosine
phosphorylation in cells in a dose-dependent manner.
Myc-tagged IRS-1 was co-transfected with increasing amounts (1, 2, 4, or 8 µg) of HA-tagged Grb10
into CHO/IR cells. Cells were
serum-starved, stimulated for 5 min with 10 nM insulin and
lysed. Tyrosine phosphorylation of IRS-1 in cell lysates was determined
as in A. C, full-length Grb10 inhibits
IR-mediated tyrosine phosphorylation of IRS-2 overexpressed in CHO/IR
cells. CHO/IR cells were transiently transfected with an expression
construct encoding IRS-2, and either co-transfected with empty vector
or an expression construct for Grb10
. Twenty-four hours
post-transfection cells were serum-starved and stimulated with 10 nM insulin for 10 min. Cell lysates were run on a 7.5%
SDS-PAGE gel and tyrosine phosphorylation of overexpressed IRS proteins
detected with a phosphotyrosine-specific antibody. Membranes were
stripped and IRS-2 was detected with an antibody specific to the
protein. The presence of Grb10 in the lysate was determined by probing
with an antibody to the HA tag.
Inhibits Endogenous IRS-1 and Akt Phosphorylation in
Differentiated Adipocytes--
To determine if hGrb10
inhibits
phosphorylation of endogenous IRS and Akt in physiologically relevant
cells, we used an adenovirus system to express hGrb10
in
differentiated 3T3-L1 adipocytes. Infection efficiency was 85-90% as
assessed by visualization of green fluorescence emitted from
co-expressed GFP (data not shown). Insulin-stimulated phosphorylation
of endogenous IRS-1 was inhibited (Fig.
3A), and Akt
Thr308 and Ser473 phosphorylation significantly
reduced in adipocytes expressing hGrb10
(Fig. 3B).
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Fig. 3.
Grb10 inhibits
endogenous IRS-1 and Akt phosphorylation in differentiated
adipocytes. A, Grb10
inhibits insulin-stimulated
tyrosine phosphorylation of endogenous IRS-1. 3T3-L1 adipocytes were
infected with either GFP or Grb10 adenovirus. Post-infection, cells
were serum-starved and stimulated with 100 nM insulin for 5 min. Insulin stimulated tyrosine phosphorylation of endogenous IRS-1
was detected in the lysates with a phosphotyrosine-specific antibody.
Membranes were stripped and endogenous IRS-1 detected using an antibody
specific to the protein. The amount of adenovirally expressed HA-tagged
Grb10
was detected in the lysates using an antibody to the tag.
B, Grb10
inhibits phosphorylation of endogenous Akt.
3T3-L1 adipocytes were treated and lysates prepared as in A.
Insulin-stimulated phosphorylation of Akt at Thr308 and
Ser473 was detected using phosphospecific antibodies.
Endogenous Akt was detected using an antibody specific to the protein.
Data is representative of three independent experiments.
delayed and sustained reduced
levels of insulin-induced Akt phosphorylation (Fig. 1C), we
investigated the temporal effects of hGrb10
on IRS-1 tyrosine
phosphorylation. Time course studies indicated that hGrb10
inhibited
IRS-1 tyrosine phosphorylation as early as 30 s after insulin
stimulation, and that hGrb10
-mediated inhibition continued through
an hour of insulin stimulation (Fig.
4A, top panel).
Since hGrb10
inhibits insulin-stimulated IRS-1 tyrosine
phosphorylation over an extended period, we studied the kinetics of
hGrb10
binding to the IR. CHO/IR cells transiently expressing
HA-tagged hGrb10
were stimulated with insulin for varying times, and
hGrb10
was immunoprecipitated from the cell lysates with antibody to
the HA tag. Bound IR was detected using an antibody specific to the
-subunit of IR. hGrb10
rapidly associated with the IR within
30 s of insulin stimulation, and the association persisted through
60 min of hormonal treatment (Fig. 4B), which mirrors the
speed and duration of hGrb10
inhibition of insulin stimulated IRS-1
tyrosine phosphorylation (Fig. 4A). We observed a decrease
in Grb10
association following a 10-min insulin stimulation, which
may result from subsequent dephosphorylation of the IR by activated
tyrosine phosphatases.
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Fig. 4.
Grb10 binding to IR correlates with inhibited
IRS-1 phosphorylation. A, IRS-1 tyrosine
phosphorylation is rapidly and stably inhibited by Grb10 . CHO/IR
cells were co-transfected with Myc-tagged IRS-1 and empty vector or
HA-tagged Grb10
. Cells were serum-starved and stimulated with 10 nM insulin for the indicated times prior to cell lysis.
Lysates were separated by SDS-PAGE and insulin-stimulated tyrosine
phosphorylation detected using a phosphotyrosine-specific antibody.
Blots were stripped and IRS-1 expression determined by blotting with
-Myc antibody. Grb10
expression was detected by Western blot with
antibody to the HA tag. B, Grb10
rapidly associates with
IR. CHO/IR cells were transiently transfected with an expression
construct for Grb10
. Cells were serum-starved and stimulated with 10 nM insulin for the indicated times. Grb10 was
immunoprecipitated from the cell lysates using an antibody to the HA
tag and samples separated by SDS-PAGE. Co-immunoprecipitated IR was
detected by Western blot using an antibody specific to the IR
subunit. hGrb10
expression was detected by Western blot with an
antibody to the HA tag. Data are representative of three
independent experiments.
and the extent of insulin-stimulated
phosphorylation at Tyr972, Tyr1158, and
Tyr1162/1163 was determined using phosphospecific
antibodies. Phosphorylation at Tyr972 was not significantly
reduced in cells that stably expressed hGrb10
(Fig.
5, lanes 2 versus 4,
first panel), suggesting that the reduction in IRS tyrosine
phosphorylation is not a direct consequence of an impaired IRS docking
site on the IR. Phosphorylation of the IR on the three critical
tyrosine residues in the regulatory loop were similarly unaffected by
expression of hGrb10
(Fig. 5, lanes 2 versus 4,
second and third panels). While the presence of
Grb10 has been shown to inhibit tyrosine phosphorylation of IR
substrates in vitro (19-21), it is unclear whether this is
directly related to impaired catalytic activity. We considered that if Grb10
does not significantly impair IR catalytic activity in cells,
then an alternative mechanism for Grb10-mediated inhibition of
substrate phosphorylation may exist. The results in Fig. 5 illustrate
that the catalytic activity of IR, at least toward itself, is not
significantly hindered by Grb10
overexpression in cells.
View larger version (29K):
[in a new window]
Fig. 5.
Insulin receptor autophosphorylation is
unaffected by Grb10 . CHO/IR cells alone or stably
expressing Grb10
(HA-tagged) were serum-starved and stimulated with
10 nM insulin for 5 min. The IR was immunoprecipitated from
the cell lysates, and samples were separated by SDS-PAGE. IR tyrosine
phosphorylation was detected using phosphospecific antibodies to
Tyr972, Tyr1158, and Tyr1162/1163.
Grb10
and IR expression levels were detected in whole cell lysates
using antibodies specific for the HA tag and the IR
subunit,
respectively.
in the yeast tri-hybrid system prevented growth on selective media, indicating that IR/IRS-1/2 interactions were inhibited (Fig. 6B). Since interactions
between Grb10 and IR are mediated by two separate "domains," the
BPS and SH2 (9), we set out to determine which is responsible for the disruption of IR/IRS-1/2 interactions. A truncated construct containing the BPS and SH2 domains (342-536) blocked growth on selective media
(Fig. 6B), indicating that the N terminus and PH domain are
not required for inhibition of IR/IRS-1/2 binding. To block SH2 domain
function, the critical arginine residue at 462 was replaced with
alanine (R462A) in the full-length and truncated (BPS/SH2) hGrb10
proteins. Both constructs interacted with IR (Fig. 6A and
Ref. 9) and both also disrupted the IR/IRS-1/2 interactions similar to
wild-type Grb10 (Fig. 6B). These data suggest that the BPS
domain may be responsible for inhibiting IR/IRS-1/2 binding. However,
the BPS (358-412) or SH2 (413-536) domains alone, which interact with
IR in yeast two-hybrid experiments (Fig. 6A and Ref. 9), do
not prevent yeast growth on selective media, indicating that the
domains by themselves are unable to disrupt IR/IRS-1/2 interactions
(Fig. 6B). Deleting the SH2 domain (Grb10
1-412 aa) or
replacing it with the SH2 domain of Shc(p52) (Grb10
1-491aa:ShcSH2) does not inhibit association with the IR in yeast two hybrid experiments (Fig. 6A); however,
removing or swapping the SH2 domain of Grb10 blocks the ability of
Grb10 to inhibit the binding of IR/IRS-1/2 (Fig. 6B).
Furthermore, an inactive hGrb10
SH2 domain (413-536, R462A), which
cannot associate with the IR in yeast two hybrid experiments (Fig.
6A and Ref. 9), can support interaction and disruption when
combined with the BPS domain (Fig. 6, A and B).
Therefore, BPS directed disruption of IR/IRS-1/IRS-2 requires the
structural presence of an active or inactive (R462A) Grb10 SH2 domain,
which may physically block binding of IRS-1/2 to the
NPXY972 motif.
View larger version (43K):
[in a new window]
Fig. 6.
Grb10 disrupts the interaction between IRS
proteins and the insulin receptor. A, yeast two-hybrid:
interaction between the insulin receptor (bait) and various Grb10
constructs (prey) was determined by the ability to induce growth on
selective media (+). Grb10 constructs that do not interact with the
insulin receptor do not induce growth on selective media ( ).
B, Yeast tri-hybrid: disruption of the insulin receptor
(bait) and IRS-1 or IRS-2 (prey) interaction by various Grb10
constructs (disruptants) was determined by the inhibition of
yeast growth on selective media.
along with IRS-1 in CHO/IR cells. The
hGrb10
(yeast tri-hybrid studies) and hGrb10
(mammalian cell
studies) isoforms are identical outside of a 58-amino acid extension at
the protein's N terminus (12, 17). hGrb10
R462
corresponds to hGrb10
R520 (Fig.
7A). As previously observed,
insulin-stimulated IRS-1 phosphorylation was inhibited in the presence
of full-length wild-type hGrb10
(Fig. 7B). Consistent
with our yeast tri-hybrid findings, co-expression of the BPS/SH2
fragment in cells similarly decreased insulin-stimulated IRS-1
phosphorylation (Fig. 7B, first panel, lane
4). Inhibition of IRS-1 tyrosine phosphorylation correlated with
Grb10 association with the IR, and the BPS/SH2 fragment was sufficient
to maintain this interaction (Fig. 7B, third
panel, lanes 3 and 4). Interestingly, IRS-1
tyrosine phosphorylation was partially restored with either the
BPS/SH2R520G fragment or full-length
Grb10
R520G (Fig. 7B, first
panel, lanes 5 and 8). These findings
suggest that an active SH2 domain is necessary for complete inhibition of IRS tyrosine phosphorylation in cells. Studies have indicated that
the BPS and SH2 domains may act cooperatively to facilitate the
interaction of Grb10 with the IR (9). Full-length Grb10
with an
inactive SH2 domain is unable to co-immunoprecipitate detectable levels
of the IR (Fig. 7B, lane 8, third
panel and Ref. 11). Similarly, the BPS/SH2R520G
fragment was unable to co-immunoprecipitate detectable amounts of the
IR (Fig. 7B, lane 5, third panel). Our
results suggest that the BPS domain is necessary, but not sufficient to
mediate a tight association with the IR in cells. In the absence of an intact SH2 domain, the interaction between Grb10 and the IR may be
transient and therefore undetectable by co-immunoprecipitation (Fig.
7B, third panel, lanes 5 and 8 versus 3 and 4). However, this transient association is
sufficient to affect IR/IRS-1 interactions as indicated by the partial
inhibition of IRS-1 tyrosine phosphorylation (Fig. 7B). To
confirm the necessity of the SH2 domain for Grb10-IR association and
its effect on the inhibition of insulin-stimulated IRS tyrosine, we
tested two additional Grb10
constructs. By either replacing the
Grb10 SH2 domain with the SH2 domain of Shc(p52) or by deleting this
domain altogether, IRS-1 tyrosine phosphorylation was restored, 41 and
30%, respectively (Fig. 7B, first panel, lanes 6 and 7). Immunoprecipitation with either the
Grb10
1-491:ShcSH2 chimera or Grb10
SH2 revealed
that the BPS domain alone can not maintain a sufficient interaction to
co-immunoprecipitate the IR (Fig. 7B, lanes 6 and
7, third panel). Taken together with the yeast
tri-hybrid results, the SH2 domain makes dual contributions to
Grb10-mediated inhibition of IRS phosphorylation: in cells, the SH2
domain stabilizes the interaction of Grb10 with the IR, and with a
stable Grb10/IR interaction, the SH2 domain sterically hinders IRS
access to its binding motif on the IR.
View larger version (24K):
[in a new window]
Fig. 7.
Complete inhibition of IRS-1 tyrosine
phosphorylation requires an intact SH2 domain. A,
schematic diagram of Grb10 constructs used in B. Full-length
Grb10 is shown for a comparison. B, Myc-tagged IRS-1 was
co-expressed in CHO/IR cells with the indicated Grb10
construct.
Twenty-four hours post-transfection cells were serum-starved and
stimulated with 10 nM insulin for 5 min. Cells were lysed
and the lysates split for IRS-1 immunoprecipitation using an antibody
to the Myc tag and IR co-immunoprecipitation with Grb10 using an
antibody to the HA tag. Insulin-stimulated tyrosine phosphorylation of
IRS-1 was detected with a phosphotyrosine-specific antibody, and the
level of IRS-1 determined by reblotting the stripped membrane with Myc
antibody. IR was detected with antibody to the
-subunit. Grb10
expression was detected in cell lysates using an antibody to the HA
tag. Data are representative of three independent
experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
stable cells. However, we found that the
overall tyrosine phosphorylation of the IR was also unaffected by the
presence of hGrb10
(data not shown). It is possible that the effects
of Grb10 on catalytic activity arise from conformational changes that
sterically hinder access of the kinase with its substrates. While we
can not exclude inhibition of enzymatic activity of the insulin
receptor for IRS-1/2 as a cause for decreased substrate
phosphorylation, the observation that hGrb10 physically disrupts the
interaction between IR and IRS proteins using the yeast tri-hybrid
system supports structural interference as a mechanism for
Grb10-mediated inhibition. The interaction of IRS proteins with the IR
is transient in nature, and several negative feedback mechanisms exist
in cells to prompt rapid dissociation of tyrosine-phosphorylated IRS
proteins with the receptor (40-42). As a result, we were unable
to detect an interaction between IR and IRS proteins by
co-immunoprecipitation (data not shown), and cannot completely assess
physical disruption as an alternative mechanism for Grb10-mediated
inhibition in mammalian cells.
significantly reduced phosphorylation of Akt on
both Thr308 and Ser473 in CHO/IR cells. A
similar inhibitory effect on phosphorylation of endogenous Akt was seen
with adenoviral expression of hGrb10
in differentiated adipocytes.
Our findings differ from a recent study reporting mGrb10 functioned as
a positive regulator of Akt in the c-kit signaling pathway by promoting
relocalization to the membrane (22). We do not find hGrb10 to
co-immunoprecipitate with Akt in CHO/IR cells (data not shown). Protein
interactions with Grb10 may be species-specific. Receptor-associated
variations in recruited signaling pathways may also account for the
observed differences in Grb10 function. IRS signaling is directly
impaired by Grb10 interactions with IR and inhibition of Akt activity
by hGrb10
may stem from a specific requirement for IRS proteins and
their supporting role in the activation of PI 3-kinase in response to
insulin stimulation.
inhibits insulin-stimulated tyrosine
phosphorylation of IRS in a dose-dependent manner, and the
interaction between IRS and the IR is disrupted when full-length Grb10
is used in the yeast tri-hybrid system. These studies describe a new
molecular mechanism for Grb10-mediated inhibition. By structurally
hindering access to the IR kinase domain and/or the NPXY
motif (Tyr972), Grb10 inhibits tyrosine phosphorylation of
IRS-1 and IRS-2 and functions to decrease intracellular insulin
signaling through the IRS/PI 3-kinase pathway.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Richard Roth for the Myc-tagged IRS-1 construct, Dr. Klein-Hitpass for the IRS-2 construct, Dr. Thomas A. Gustafson for the yeast strain GUSEGY48, pLexA IR pB42AD Grb10 constructs and pB42AD IRS-1/2 constructs, and Dr. Ben Margolis for the mouse Shc (p52) cDNA. We acknowledge Drs. Tong-Chuan He and Bert Vogelstein for the pAdEasy system.
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FOOTNOTES |
---|
* This research was supported by National Institutes of Health RO1 Grants DK52933 (to F. L. and L. Q. D.) and DK43123 (to S. E. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by National Institutes of Health Postdoctoral Training Grant 2T32AG00205-11.
To whom correspondence should be addressed: Dept. of
Pharmacology, The University of Texas Health Science Center, San
Antonio, TX 78229. Tel.: 210-567-3097; E-mail: liuf@uthscsa.edu.
Published, JBC Papers in Press, December 18, 2002, DOI 10.1074/jbc.M208518200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: IR, insulin receptor; BPS, between the Pleckstrin and SH2; CHO, Chinese hamster ovary; GFP, green fluorescence protein; IRS, IR substrate; PH, Pleckstrin homology; PI 3-kinase, phosphatidylinositol 3-kinase; RTK, receptor-tyrosine kinase; SH2, Src-homology 2; HA, hemagglutinin; aa, amino acid.
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