Determination of Gab1 (Grb2-Associated Binder-1) Interaction with Insulin Receptor-Signaling Molecules
Stéphane Rocchi,
Sophie Tartare-Deckert,
Joseph Murdaca,
Marina Holgado-Madruga,
Albert J. Wong and
Emmanuel Van Obberghen
INSERM U145 (S.R., S.T.-D., J.M., E.V.O.) 06107 Nice
Cédex 2, France
Departments of Microbiology &
Immunology and Pharmacology (M.H.-M., A.J.W.) Jefferson Cancer
Institute Philadelphia, Pennsylvania 19107
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ABSTRACT
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The newly identified insulin receptor (IR)
substrate, Gab1 [growth factor receptor bound 2 (Grb2)-associated
binder-1] is rapidly phosphorylated on several tyrosine residues by
the activated IR. Phosphorylated Gab1 acts as a docking protein for Src
homology-2 (SH2) domain-containing proteins. These include the
regulatory subunit p85 of phosphatidylinositol 3-kinase and
phosphotyrosine phosphatase, SHP-2. In this report, using a modified
version of the yeast two-hybrid system, we localized which Gab1
phospho-tyrosine residues are required for its interaction with
phosphatidylinositol 3-kinase and with SHP-2. Our results demonstrate
that to interact with p85 or SHP-2 SH2 domains, Gab1 must be tyrosine
phosphorylated by IR. Further, we found that Gab1 tyrosine 472 is the
major site for association with p85, while tyrosines 447 and 589 are
participating in this process. Concerning Gab1/SHP-2 interaction, only
mutation of tyrosine 627 prevents binding of Gab1 to SHP-2 SH2 domains,
suggesting the occurrence of a monovalent binding event. Finally, we
examined the role of Gab1 PH (Pleckstrin homology) domain in Gab1/IR
interaction and in Gab1 tyrosine phosphorylation by IR. Using the
modified two-hybrid system and in vitro experiments, we
found that the Gab1 PH domain is not important for IR/Gab1 interaction
and for Gab1 tyrosine phosphorylation. In contrast, in intact mammalian
cells, Gab1 PH domain appears to be crucial for its tyrosine
phosphorylation and association with SHP-2 after insulin stimulation.
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INTRODUCTION
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The insulin receptor (IR) is a tyrosine kinase that becomes
activated upon hormone binding (1, 2). Known direct targets of IR
include the insulin receptor substrate (IRS) proteins (IRS-1, IRS-2,
and IRS-3) (3, 4). They serve as multidocking molecules for several
proteins, some of which have been recognized as key players in insulin
signal transduction. These molecules include Grb2 (growth factor
receptor bound 2), the regulatory subunits of phosphatidylinositol
3-kinase (PI3-K), p85 and p55, and the phosphotyrosine phosphatase
SHP-2 (SH2-containing protein tyrosine phosphatase) (4, 5, 6). PI3-K is
involved in several insulin responses, such as glucose transport, p70
S6 kinase activation, membrane ruffling, and mitogenesis (7, 8, 9, 10, 11). The
role of SHP-2 in insulin signaling is likely to be complex as the
phosphatase appears to function as a positive effector in the
insulin-stimulated mitogen-activated protein kinase pathway and
mitogenesis (12, 13, 14, 15), whereas it may also be an "attenuator" of
insulin signaling by inducing dephosphorylation of IRS-1 (5, 16).
Recently, Wong and co-workers (17) cloned a new protein called Gab1
(Grb2-associated binder-1). Gab1 is found in most human tissues except
lung, kidney, and liver. It has a molecular mass of 77 kDa, but
migrates between 115120 kDa on SDS-PAGE, which is thought to be due
to its high level of serine/threonine phosphorylation. This protein has
been identified as a substrate of the insulin and epidermal growth
factor (EGF) receptors. In addition, Weidner et al. (18)
have shown that Gab1 interacts directly with the c-Met tyrosine
kinase.
The physiological role of Gab1 is currently unknown. However, Gab1
overexpression in epithelial cells is sufficient to generate the
characteristic responses induced by c-Met tyrosine kinase receptor,
such as branching morphogenesis and cell scattering. Moreover,
overexpression of Gab1 in NIH3T3 cells enhances cell growth and
transformation stimulated by insulin and EGF. In summary, Gab1 appears
to be a key mediator in cell proliferation and transformation induced
by the EGF and c-Met receptors.
Sequence analysis shows that Gab1 is homologous to IRS-1, IRS-2, and
IRS-3 especially in the Pleckstrin homology (PH) domain, which is
located at the N terminus of these proteins. In addition, Gab1
possesses 16 potential phosphotyrosine sites, some of which could serve
as binding sites for SH2 domains of the regulatory subunit of PI3-K,
Grb2, phospholipase C-
, Nck, and SHP-2. This suggests that Gab1
could serve as a docking protein, like the other IRS proteins. However,
in contrast to IRS proteins and Shc, Gab1 does not possess a
phosphotyrosine binding (PTB) domain, which is thought to be implicated
in direct binding to the IR phosphotyrosine 960 (19, 20, 21, 22).
In the present study, we used a modified version of the yeast
two-hybrid system and coimmunoprecipitations in intact mammalian cells
to evaluate interactions between Gab1 and PI3-K or SHP-2, and to
identify the tyrosines involved in these interactions. We demonstrate
that Gab1 must be tyrosine phosphorylated by IR to allow its
association with PI3-K and SHP-2. Finally, we show that in intact
cells, the Gab1 PH (Pleckstrin homology) domain is crucial for its
tyrosine phosphorylation and association with SHP-2 after insulin
stimulation.
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RESULTS
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In a first series of experiments, we examined, in the yeast
two-hybrid system, interaction of Gab1 with insulin-signaling molecules
such as IR ß-subunit, insulin-like growth factor-I receptor (IGF-I R)
ß-subunit, p85 regulatory subunit of PI3-K, and SH2 domains of
phosphotyrosine phosphatase SHP-2. However, we failed to detect
interaction between Gab1 and these molecules in our system (data not
shown). We hypothesized that in yeast this lack of interaction between
Gab1 and p85 or n/c SH2 SHP-2 could be due to absence of Gab1 tyrosine
phosphorylation. To test this, we engineered a novel two-hybrid vector
in which the IR ß-subunit cDNA was subcloned downstream of the
methionine-repressible promoter MET25 (pVJL-HIR-3H). The full-length
p85 cDNA or the cDNA corresponding to n/c SH2 domains of SHP-2 cDNA
were also subcloned in this vector pVJL-HIR-3H in frame with the
DNA-binding domain of lexA (they are called, respectively, pVJL-HIR-p85
and pVJL HIR n/c SH2 SHP-2) (Fig. 1
). In the presence
of methionine, expression of IR ß is repressed. Absence of methionine
allows expression of IR ß leading to phosphorylation of coexpressed
substrates in the yeast.

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Figure 1. Schematic Representation of the Modified
Two-Hybrid System
Yeast is cotransformed with the plasmid pACT-Gab1 encoding GAD-Gab1 in
combination with a new two-hybrid vector in which the IR ß cDNA was
subcloned downstream of the methionine-repressible promoter MET25 and
encoding LDBD-SHP-2 or -p85 constructs. Expression of IR ß is
prevented in the presence of methionine, while absence of methionine
allows expression of the IR ß.
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We first verified that the two vectors, pVJL-HIR-p85 and pVJL-HIR-n/c
SH2 SHP-2, were incapable by themselves, or in combination with an
unrelated GAD (Gal4 activation domain) fusion protein, of activating
expression of the two reporter genes (data not shown). We then tested
interaction of full-length p85 with Gab1 in the presence or in the
absence of IR ß-subunit (Fig. 2A
). We observed that
coexpression of lexA DNA-binding domain (LDBD)-p85 and GAD-Gab1
produces a detectable level of ß-galactosidase activity only if the
IR is expressed (without methionine in the medium). In the absence of
IR ß, interaction was undetectable. These results suggest that IR ß
phosphorylates Gab1 on tyrosine residues, resulting in interaction of
Gab1 with p85. To further address this issue, lysine 1018 of the IR
ATP-binding site was mutated to obtain a kinase-deficient IR (pVJL HIR
K1018A p85). No interaction between Gab1 and p85 was detected using
mutant IR ß K1018A, suggesting that the interaction depends on
receptor tyrosine kinase activity. Similar results were obtained with
LDBD-n/c SH2 SHP-2 (data not shown). Concurrently, we verified
expression and tyrosine phosphorylation of IR. Yeast coexpressing wild
type (WT) or K1018A IR constructs were incubated in the presence or
absence of methionine, after which yeast lysates were prepared, and IR
constructs were immunoprecipitated. Phosphorylated IR was revealed by
immunoblotting with an antibody to phosphotyrosine. Expression was
measured by immunoblotting using an antibody to the hemagglutinin (HA)
epitope, which is present in the fusion protein (Fig. 2B
). As shown,
expression of IR WT and IR K1018A was observed in absence of
methionine, whereas expression was repressed in the presence of
methionine (bottom panel). Moreover, as expected, IR WT is
tyrosine phosphorylated in yeast in contrast to kinase-deficient IR (IR
K1018A).

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Figure 2. Gab-1 Binding to PI3-Kinase in the Yeast
Two-Hybrid System Depends on the IR Kinase
A, The yeast strain L40 was cotransformed with a plasmid encoding
GAD-Gab1 and with a plasmid encoding LDBD-p85 and IR ß. Transformants
were isolated on selective plates in the absence or in the presence of
methionine. Activation of ß-galactosidase was measured using the
substrate CPRG, and indicated activities were calculated according to
Miller. Values represent the average ± SE of six
independent transformants. Similar results were obtained using the
filter color assay or growth on SC plates lacking histidine. K1018A
corresponds to IR ß in which lysine 1018 is replaced by alanine and
is kinase-dead. B, Yeasts cotransformed with different IR ß
constructs were grown overnight in 100 ml yeast medium. The yeasts were
solubilized, and IR ß was immunoprecipitated with an antibody to
hemagglutinin. The immunoprecipitated proteins were separated by
SDS-PAGE under reducing conditions and transferred to an Immobilon P
membrane. The membrane was probed with antibodies to hemagglutinin
(upper part of the figure) or antibodies to
phosphotyrosine (lower part of the membrane). A representative
experiment is shown.
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As a whole, these results validate our hypothesis that the IR kinase
activity is required for tyrosine phosphorylation of GAB1 and for its
association with p85 subunit of PI3-K and with phosphotyrosine
phosphatase SHP-2.
Using this system, we identified the phosphorylated tyrosine residue(s)
of Gab1 which is (are) involved in interaction with p85.
Phosphotyrosines in YXXM motifs are potential binding sites for p85 SH2
domains. Sequence analysis has shown that Gab1 contains at least 16
potential tyrosine phosphorylation sites, including 3 in YXXM motifs,
Y447, Y472, and Y589. We replaced tyrosine residues 447, 472, and 589
in GAD-Gab1 individually or in combination, and analyzed the ability of
the different constructs to interact with LDBD-p85 in the presence of
IR ß (Fig. 3A
). As shown in Fig. 3A
, mutation of
tyrosines 447 and 589 (Y447F and Y589F constructs) did not
significantly alter interaction with Gab1. Mutation of tyrosine 472, or
mutation at both tyrosines 447 and 589, decreased by approximately 50%
the interaction of Gab1 with p85. However, replacement of tyrosines 447
and 472 (GAD-Gab1 Y447F/Y472F) and tyrosines 472 and 589 (GAD-Gab1
Y472F/Y589F) completely abolished interaction between Gab1 and p85.
Lack of interaction was also obtained with a construct containing the
three mutated tyrosines (GAD-Gab1 Y447F/Y472F/Y589F). To confirm the
yeast two-hybrid results, the interaction between Gab1 and the p85 of
PI3-K was analyzed by measurement of PI3-K activity associated with
Gab1 (WT and mutants) after insulin stimulation in intact cells (Fig. 3B
). After transfection, the cells were stimulated with insulin, and
Gab1 was immunoprecipitated from the cell lysates. As a control of
expression, one-tenth of the cell lysates was analyzed by SDS-PAGE and
immunoblotted with antibodies to Gab1. In all experiments, the
expression levels of Gab1 WT and mutant proteins were comparable (data
not shown). The PI3-K activity associated with Gab1 was measured as
described in Materials and Methods. In 293 EBNA cells
transfected with Gab1 WT, insulin induced a 3-fold increase in PI3-K,
which was chosen to represent 100% (Fig. 3B
). In cells transfected
with Gab1 Y447F/Y589F, the insulin-induced PI3-K activity associated to
GAB was approximately increased 50% of that observed for the Gab1 WT.
In cells transfected with the other Gab1 mutants (Y447F/Y472F,
Y472F/Y589F, and Y447F/Y472F/Y589F), the insulin-stimulated PI3-K
activity associated with Gab1 was completely abolished compared with
that found with the Gab1 WT. The absence of PI3-K activity associated
with Gab1 Y447F/Y472F, Y472F/Y589F, and Y447F/Y472F/Y589F is not due to
a lack of tyrosine phosphorylation, since immunoblotting using
phosphotyrosine antibodies revealed that all Gab1 mutants are
phosphorylated on tyrosine residues (data not shown). Taken together
these results indicate that tyrosines 447, 472, and 589 of Gab1 are
important for binding p85.

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Figure 3. Identification of Gab1 Tyrosine Residues Are
Implicated in Interaction with p85
A, Yeast was cotransformed with plasmids encoding the indicated
GAD-Gab1 mutants and a plasmid encoding LDBD-p85 and IR ß.
Transformants were isolated on selective plates. Activation of
ß-galactosidase was measured using CPRG, and indicated activities
were calculated according to Miller. Values represent the average
± SE of six independent transformants. Similar results
were obtained by the filter color assay and growth on SC plates lacking
histidine. Y447F, Y472F, and Y589F are mutated forms of GAD-Gab1 in
which tyrosines 447, 472, and 589 were replaced by phenylalanine. B,
Insulin-stimulated PI3-K activity associated with Gab1 mutants
expressed in 293 EBNA cells. 293 EBNA cells transfected with plasmids
expressing the various Gab1 forms were incubated for 5 min at 37 C in
the absence or presence of 10-7 M insulin. The
cells were solubilized, and Gab1 was immunoprecipitated with
antibodies to Gab1. The PI3-K activity associated with Gab1 was
measured as described in Materials and Methods and
analyzed by TLC and autoradiography. [32P]Phosphate
incorporation into PI3-P was quantified using a Molecular Imager
(Bio-Rad). Results are presented as a percentage of the
insulin-dependent increase in PI3-K activity associated with the Gab1
WT after subtraction of the basal activity. A representative experiment
is shown.
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We next studied interaction between Gab1 and phosphotyrosine
phosphatase SHP-2. For several proteins, it has been shown that YXXL or
YXXI motifs are potential binding sites for SHP-2 SH2 domains. In Gab1,
two tyrosines are contained in a YXXL motif, i.e. tyrosines
183 and 627. Hence we constructed mutants of the yeast two-hybrid
construct, GAD-Gab1, in which tyrosines 183 and 627 were replaced by
phenylalanine (GAD-Gab1 Y183F and GAD-Gab1 Y627F). Using a modified
yeast two-hybrid system, we tested their ability to interact with
LDBD-n/c SH2 SHP-2 in the presence of IR ß (Fig. 4A
). Mutation of tyrosine 183 did not affect the
interaction of Gab1 with SHP-2 SH2 domains. In contrast, mutation of
tyrosine 627 completely abolished the interaction. Next, we examined
whether the Gab1 tyrosine 627 is involved in the interaction between
Gab1 and SHP-2 in intact cells, by mutation of this residue to
phenylalanine. We immunoprecipitated Gab1 from the transfected 293 EBNA
cells and looked for the association with SHP-2 by immunoblotting with
antibodies to SHP-2 (Fig. 4B
). As a control of expression, anti-Gab1
immunoprecipitates were also analyzed by immunoblotting with antibodies
to Gab1. Gab1 was not detected in anti-Gab1 immunoprecipitates from
vector-only transfected cells (MOCK condition), and Gab1
Y627F-transfected cells expressed slightly more protein than Gab1
WT-transfected cells. In cells transfected with Gab1 WT, association of
Gab1 WT with SHP-2 was detected in unstimulated cells, and incubation
with insulin increased this association. In contrast to the findings
with Gab1 WT, SHP-2 was not bound to Gab1 Y627F. Therefore, we conclude
that tyrosine 627 of Gab1 is the major interaction site of Gab1 with
SH2 domains SHP-2 demonstrated using both two-hybrid system and intact
cells.

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Figure 4. Identification of Gab1 Tyrosine Residues Are
Implicated in Interaction with SHP-2
A, Yeast was cotransformed with plasmids encoding indicated GAD-Gab1
mutated forms in combination with a plasmid encoding LDBD-n/c SH2 SHP-2
and IR ß. Transformants were isolated on selective plates. Activation
of ß-galactosidase was measured as described in legend to Fig. 3 .
Y183F and Y627F are mutated forms of GAD-Gab1 in which tyrosines 183
and 627 were replaced by phenylalanine. B, 293 EBNA cells were
transfected with plasmids expressing Gab1 WT or Y627F. Then, cells were
incubated with 10 mM sodium orthovanadate for 10 min at 37
C and thereafter for 5 min at 37 C in the absence or presence of
10-7 M insulin. The cells were solubilized,
and the extracted proteins were subjected to immunoprecipitation with
antibodies to Gab1. The immunoprecipitated proteins were separated by
SDS-PAGE under reducing conditions and transferred to an Immobilon P
membrane. The membrane was probed with antibodies to Gab1 (upper
part of the membrane) and antibodies to SHP-2 (lower
part of the membrane). A representative experiment is shown.
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Finally, we investigated the role of the Gab1 PH domain in tyrosine
phosphorylation of Gab1 by IR. It has been shown previously that the
IRS-1 PH domain is essential for insulin-stimulated tyrosine
phosphorylation of IRS-1, IRS-1-associated PI3-K activity, and
subsequent p70 S6 kinase phosphorylation (23, 24). By analogy, we
hypothesized that the Gab1 PH domain could play a role in the Gab1/IR
interaction. The Gab1 PH domain was deleted, and the ability of
GAD-Gab1
PH to interact with p85 in the presence or absence of IR
was tested using the modified yeast two-hybrid system. In the absence
of IR, we did not see binding of Gab1 to p85 (Fig. 5A
). In contrast, in the presence of IR, interaction
was detectable with GAD-Gab1 as well as with GAD-Gab1
PH. No
significant difference was observed between GAD-Gab1
PH and GAD-Gab1
WT. These results suggest that, in this system, the Gab1 PH domain is
not involved in Gab1 phosphorylation by IR.

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Figure 5. Analysis of the Role of the Gab1 PH Domain in
Interaction of Gab1 with the IR Using a Modified Yeast Two-Hybrid
System and in Vitro Phosphorylation
A, The yeast strain L40 was cotransformed with a plasmid encoding
GAD-Gab1 or Gab1 PH and with a plasmid encoding LDBD-p85 and the IR
ß. Transformants were isolated on selective plates in the absence or
in the presence of methionine. Activation of ß-galactosidase was
measured using CPRG, and indicated activities were calculated according
to Miller. Values represent the average ± SE of six
independent transformants. B, 293 cells were transfected with plasmid
expressing various HA-tagged forms of Gab1. After cell lysis, HA-Gab1
was immunoprecipitated with a specific antibody to hemagglutinin. In
parallel, WGA-purified IRs were incubated in the absence or presence of
insulin. The receptors were added to pellets containing
immunoprecipitated Gab1. Tyrosine phosphorylation reaction was
initiated by addition of phosphorylation buffer and was stopped after
30 min. Phosphorylated proteins were separated by SDS-PAGE under
reducing conditions and transferred to an Immobilon P membrane. The
membrane was probed with antibodies to phosphotyrosine. A
representative experiment is shown.
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To confirm this, we examined the role of the PH domain in Gab1 in
vitro tyrosine phosphorylation by partially purified IR. To do
this, WT and
PH HA-Gab1 were immunoprecipitated with antibodies to
HA from lysates of 293 EBNA cells overexpressing the respective
constructs. Purified receptors activated by addition of insulin were
added to pellets containing Gab1 WT or Gab1
PH. Phosphorylation was
initiated by the addition of 30 µM ATP, 8 mM
MgCl2, and 4 mM MnCl2, and the
reaction was stopped after 30 min. Phosphorylated Gab1 was revealed by
Western blotting using an antibody to phosphotyrosine. Figure 5B
shows
that Gab1 WT and Gab1
PH are phosphorylated in vitro by
activated IRs. No tyrosine phosphorylation was detected in the absence
of receptor or in the absence of insulin. These results indicate that
in vitro, Gab1 is a direct substrate of the purified IR, and
that deletion of the Gab1 PH domain does not prevent Gab1 tyrosine
phosphorylation.
Finally, we studied in 293 EBNA cells insulin-induced
phosphorylation of WT Gab1 and Gab1
PH and their hormone-induced
association with SHP-2. Transfected cells expressing WT or
PH
HA-Gab1 constructs were incubated with insulin or buffer, after which
cell lysates were prepared and HA-Gab1 was immunoprecipitated with an
antibody to HA. Tyrosine- phosphorylated Gab1 was revealed by Western
blotting with an antibody to phosphotyrosine, and its association with
SHP-2 was monitored by immunoblotting with antibodies to SHP-2. As a
control of expression and immunoprecipitation, one third of the HA-Gab1
immunoprecipitates was analyzed by immunoblotting with antibodies to
HA. In all experiments, the expression levels of WT and
PH Gab1 were
similar (data not shown). As shown in Fig. 6
, basal
phosphorylation of Gab1 WT and association with SHP-2 were detected in
unstimulated cells. Insulin stimulation enhanced tyrosine
phosphorylation of Gab1 WT by approximately 5-fold and concomitantly
increased Gab1 association to SHP-2. In contrast, we did not observe
phosphorylation of Gab1
PH and association with SHP-2 either in
stimulated or in unstimulated cells. These data indicate that, in
intact cells, activated IRs induce Gab1 tyrosine phosphorylation and
its association with SHP-2. In addition, the Gab1 PH domain appears to
be crucial for the occurrence of these insulin actions. Taking our
results as a whole, we conclude that Gab1 is a direct substrate of the
IR, and that in intact cells Gab1 PH domain is necessary to allow Gab1
tyrosine phosphorylation and association with SHP-2 after insulin
stimulation. However, the PH domain does not appear to be required in a
cell-free system or in the yeast two-hybrid system.

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Figure 6. Gab1 Tyrosine Phosphorylation and Association of
Gab1 with SHP-2 in Intact Cells
293 EBNA cells were transfected with plasmids expressing various
HA-Gab1 forms. Then, cells were incubated with 10 mM sodium
orthovanadate for 20 min at 37 °C and thereafter for 5 min at 37 °C
in the absence or presence of 10-7 M insulin.
Cells were solubilized and extracted proteins were subjected to
immunoprecipitation with antibodies to hemagglutinin. The
immunoprecipitated proteins were separated by SDS-PAGE under reducing
conditions and transferred to an Immobilon P membrane. The membrane was
probed with antibodies to phosphotyrosine (upper part of the
membrane) and by antibodies to SHP-2 (lower part of the
membrane). A representative experiment is shown.
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DISCUSSION
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Upon hormone binding, the IR undergoes multisite
autophosphorylation on tyrosine residues, including the juxtamembrane
tyrosine 960, which becomes a key binding site for the receptor
substrates, IRS-1, IRS-2, IRS-3, and Shc (3, 19, 20, 21, 22). Recently, Gab1
has been added to the list of IR substrates (17). This newly identified
protein contains in its N terminus a PH domain that is highly
homologous to the IRS-1 PH domain. Its C-terminal part carries several
potential tyrosine phosphorylation sites included in motifs that are
expected to function as binding sites for SH2 domain-containing
proteins. In contrast with IRS-1, Gab1 does not possess a PTB
domain.
In the present work, using different approaches, we characterized
interaction between 1) Gab1 and IR, and 2) Gab1 and SH2
domain-containing proteins such as p85-regulatory subunit of PI3-K and
phosphotyrosine phosphatase, SHP-2.
With the classic two-hybrid system, we failed to detect interaction
between Gab1 and IR, or IGF-I R. Similarly, we did not find interaction
of Gab1 with p85 or with SHP-2.
Therefore, we engineered a modified two-hybrid vector in which
the IR ß cDNA was subcloned under control of the
methionine-repressible promoter MET25. We found that the presence of a
functional IR ß leads to interaction between Gab1 and p85, or between
Gab1 and SHP-2. Therefore, we conclude that phosphorylation of Gab1 by
IR is a prerequisite for the induction in the yeast two-hybrid system
of an efficient interaction with the SH2 domains of p85 and of
SHP-2.
Previous studies performed in our laboratory have shown that IRS-1
interacts with p85 in the classic yeast two-hybrid system,
i.e. without expression of IR. However, compared with the
interaction seen in the absence of IR, a 4-fold increase in interaction
of IRS-1 with p85 is seen when IR is expressed (data not shown). These
data suggest that IRS-1 tyrosine phosphorylation by tyrosine kinases
present in yeast is insufficient to generate the full-blown interaction
between IRS-1 and p85.
Holgado-Madruga et al. (17) have shown that Gab1 isolated
from insulin-induced A431 cells coimmunoprecipitated with the p85
regulatory subunit of PI3-K and with the phosphotyrosine phosphatase
SHP-2. Using the modified two-hybrid system and coimmunoprecipitation
in intact cells, we studied the phosphorylated Gab1 tyrosines possibly
involved in interaction between Gab1 and p85, or between Gab1 and
SHP-2. In relation to association with p85, tyrosines 447, 472, and 589
appear to be crucial for the interaction of Gab1 with PI3-K. Previous
studies have shown that tyrosyl-phosphorylated IRS-1 peptides
containing a single YXXM motif activate PI3-K in vitro.
Furthermore, mutation of either SH2 domain significantly reduced
phosphopeptide binding to p85 and decreased PI3-K activation by IRS-1
by 50% (25). Taking these findings together, it is tempting to imagine
a two-step process in which first phosphotyrosine (e.g.
tyrosine 472) interacts with one of the two SH2 domains (amino- or
carboxy-terminal), and subsequently the other tyrosine (447 or 589)
binds to the second SH2 domain. This bivalent binding would stabilize
the interaction between Gab1 and p85 and would lead to full-blown PI3-K
activation by Gab1.
Tyrosines 183 and 627 are present in a potential binding site for the
SH2 domains of SHP-2 (YXXL or YXXI). We found that substitution of
tyrosine 627 on Gab1 abolishes interaction between Gab1 and SHP-2 in
yeast and in intact mammalian cells. In contrast, mutation of tyrosine
183 does not modify interaction of Gab1 with SHP-2. We have previously
demonstrated that IRS-1 phosphotyrosines 1172 and 1222, present in YXXL
and YXXI motifs, respectively, are the interaction sites of IRS-1 with
SHP-2 in intact cells (5). Contrary to IRS-1, only one site is
important for interaction of Gab1 with the SH2 domains of SHP-2. It
remains to be determined whether SHP-2 is capable of dephosphorylating
Gab1 and whether this dephosphorylation is dependent on its association
with Gab1.
Finally, we investigated the role of Gab1 PH domain in Gab1 interaction
with IR and its phosphorylation and association with SHP-2. It has been
shown previously in hematopoietic 32D cells, that the IRS-1 PH domain,
but not its PTB domain, is essential for insulin-stimulated IRS-1
tyrosine phosphorylation and subsequent stimulation of PI3-K (24).
Knowing that Gab1 contains only a PH domain and no PTB domain, we
examined the role of the Gab1 PH domain in the Gab1/IR interaction. We
found that in intact cells the Gab1 PH domain is essential for
insulin-induced Gab1 tyrosine phosphorylation and its association with
SHP-2. In contrast, when the IR is expressed at high levels or when
Gab1 and IR are colocalized, for example in in vitro
experiments and in yeast two-hybrid system, deletion of the PH domain
has no effect on Gab1 tyrosine phosphorylation by IRs. Our observations
are in agreement with recent studies in 32D hematopoietic cells showing
that, in the presence of low levels of receptor, the PH domain of IRS-1
is essential for insulin-stimulated IRS-1 tyrosine phosphorylation,
PI3-K activity, and p70s6k stimulation (24). In addition,
other studies have shown that deletion of the PH domain of IRS-1 has no
effect on in vitro phosphorylation by the purified IR (26).
Crystal structure analysis of PH domains found in several unrelated
proteins suggests that the end of the structure is open and may
represent an interaction site with another protein(s) (27). Further, it
has been demonstrated that PH domains may associate with membrane
phospholipids and thereby recruit signaling proteins to the membrane
(28, 29). Taking these findings together, we propose that the PH domain
of Gab1 may interact with membrane phospholipids to permit recruitment
of Gab1 to proximity of IR, resulting its subsequent phosphorylation
and transduction of insulin responses. Since Gab1 does not have a PTB
domain, it remains to be determined which region on Gab1 drives the
interaction with IR leading to its phosphorylation.
In summary, using a modified version of the yeast two-hybrid system, we
have demonstrated which phosphorylated tyrosine residues of Gab1 are
required for interaction of 1) Gab1 and p85 or 2) Gab1 and SHP-2.
Further, our data indicate that in intact mammalian cells, the Gab1 PH
domain plays an important role in mediation of interaction with IR.
A general picture emerges in which Gab1 is a direct substrate of the IR
and plays a role of adaptor for several SH2-containing proteins. In
addition, this newly identified IRS protein is also a substrate for
other tyrosine kinases such as EGF receptor and c-Met receptor. An
urgent challenge is to elucidate the precise role of Gab1 compared with
other IRS proteins in transmission of the insulin pleiotropic
effects.
 |
MATERIALS AND METHODS
|
---|
Materials
The yeast strain L40 and the plasmid encoding the Gal4
activation domain GAD-Raf were provided by A. Votjek (Seattle, WA)
(30); the yeast expression plasmid pACTII was from S. Elledge (Houston,
TX) (31). The pECE/HA-tagged expression vector and the antibodies to HA
were a gift from J. Pouysségur (Nice, France) (32). The pVJL9 3H
yeast expression plasmid was a gift from J. Camonis (Paris, France).
This plasmid has been described (33). Human SHP-2 cDNA was obtained
from E. Fischer (Seattle, WA). p85
cDNA was a gift from J. E.
Pessin (Iowa City, IA). Human IR cDNA was provided by A. Ullrich
(Munich, Germany). Antibodies to phosphotyrosine and to human Gab1 were
obtained from Upstate Biotechnology (Lake Placid, NY). Antibodies to
human SHP-2 were obtained from Santa Cruz Biotechnology (Santa Cruz,
CA). Synthetic complete (SC) minimal yeast media lacking the
appropriate amino acids were from BIO 101 (La Jolla, CA).
Oligonucleotides were purchased from Genset (Paris, France) and
chlorophenol red-ß-D-galactopyranoside (CPRG) was from
Boehringer Mannheim (Meylan, France). Insulin was kindly provided by
Novo-Nordisk (Copenhagen, Denmark). Triton X-100 and reagents for
SDS/PAGE were from Bio-Rad (Richmond, CA). All other chemical reagents
were obtained from Sigma Chemical Co. (St. Louis, MO).
Plasmid Construction
For most constructions, we introduced convenient restriction
endonuclease sites to each end of the desired cDNA fragment by PCR to
allow the in-frame insertion into the expression vector. The
full-length human Gab-1 cDNA was subcloned in frame with the Gal4
activation domain into the two-hybrid expression vector pACT II. The
coding sequence of the IR cytoplasmic domain (amino acids 944-1343)
(34) was amplified by PCR and then inserted in the plasmid pVJL9 3H
downstream of the repressible promoter MET25 in frame with the HA
epitope and a nuclear localization signal. The full-length human p85
and the n/c SH2 SHP-2 cDNA were subcloned in pVJL9 3H in frame with the
DNA-binding domain of lexA. The plasmid encoding GAD-IRS-1 construct
(IRS-1 amino acids 51235) was obtained as previously described (22).
All point mutations and deletions of different proteins were generated
by site-directed mutagenesis using the Stratagene QuikChange Kit (La
Jolla, CA). Point mutations were verified by DNA sequence analysis.
Yeast Strain, Culture Media, Transformation, and Reporter Gene
Expression
The genotype of the Saccharomyces cerevisiae reporter
strain L40 is MAT a, trp1, leu2, his3,
LYS2::lexA-HIS3,
URA3::lexA-lacZ (30). L40 were grown at
30 C in YPD media containing 1% (wt/vol) yeast extract, 2% (wt/vol)
Bacto-Peptone, and 2% (wt/vol) glucose, or in SC yeast media lacking
the appropriate auxotrophic amino acids.
Yeast L40 was transformed simultaneously with the two indicated
plasmids by the improved lithium acetate method of Gietz et
al. (35). The transformants were grown on SC plates lacking
tryptophan and leucine to select for the presence of pBTM116 and
pACTII, respectively. Where indicated, medium without methionine was
used to allow expression of IR ß-subunit.
After 48 h, the double transformants were patched on SC plates
lacking tryptophan, leucine, and methionine for ß-galactosidase
assays or on SC plates lacking tryptophan, leucine, methionine, and
histidine to study histidine prototrophy. After 2 days at 30 C, the
ß-galactosidase assay was performed by a color filter assay using
5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-gal)
as previously described (36). For quantitative studies of
ß-galactosidase activity, liquid culture assays using CPRG as a
substrate were carried out as described by Bartel et al.
(36). Yeast extracts were incubated with 8 mM CPRG, and the
increase in A574 was measured after 10 or 30 min. Results
were expressed as Millers units: one unit of ß-galactosidase was
defined as (A574 x 1000)/[A600 x volume (ml)
x time (min)] (37).
Immunoblot Analysis of Expression and Tyrosine Phosphorylation of
Hybrid IR and GAD-Gab1 in Yeast
A single colony of each strain expressing the different IR and
Gab1 hybrids was cultured in 100 ml selective medium at 30 C until the
cells reached a density of 1.2 x 107 cells/ml. The
cells were pelleted and washed with water (10 ml). The dry pellet was
frozen on dry ice and then at -20 C for 10 min, resuspended in 500
µl of lysis buffer (50 mM HEPES, 150 mM NaCl,
10 mM EDTA, 10 mM
Na4P2O7, 2 mM vanadate,
100 mM NaF, 1% (vol/vol) Triton X-100, 0.5 mM
phenylmethylsulfonylfluoride, 100 U/ml aprotinin, and 20
µM leupeptin) and vortexed with glass beads (425600
µm). Fusion proteins (HA-IR and GAD-Gab1) were immunoprecipitated
using antibodies to HA. After separation of the samples by SDS-PAGE
under reducing conditions, the proteins were transferred to a
polyvinylidene difluoride membrane (Immobilon, Millipore Corp.,
Bedford, MA). The membrane was probed with antibodies to
phosphotyrosine (1 µg/ml) and, as expression and immunoprecipitation
control, with antibodies to HA, as previously described (5). Finally,
antibody binding was visualized using [125I] protein A
and quantified using the PhosphoImager system (Bio-Rad).
Cell Culture and Transfection of 293 EBNA Cells
293 EBNA cells are human embryo kidney cells that constitutively
express the EBNA-1 protein from the Epstein Barr Virus (Invitrogen, San
Diego, CA). These cells were grown in DMEM supplemented with 5%
(vol/vol) FCS in the presence of 500 µg/ml geneticin (G418, GIBCO,
Grand Island, NY). Cells were transfected as described by Chen and
Okayama (38). Briefly, exponentially growing cells were trypsinized,
seeded at 3 x 106 cells per 10-cm plate, and
incubated overnight in 10 ml of growth medium. Then 10 µg of
supercoiled DNA were mixed with 0.5 ml of 0.25 M
CaCl2 and 0.5 ml of 2 x BBS (buffered saline
containing 50 mM BES, 280 mM NaCl, 1.5
mM Na2HPO4, pH 6.95). The mixture
was incubated for 30 min at room temperature before being added
dropwise to the cells. After incubation for 1518 h at 35 C under 3%
CO2, the medium was removed, and cells were incubated with
growth medium for 8 h and then starved for 14 h in DMEM
containing 0.5% (vol/vol) FCS.
Tyrosine Phosphorylation of Gab1 in Intact Cells
Transfected 293 EBNA cells in 10-cm plates were stimulated with
insulin (10-7 M) for 5 min at 37 C and
solubilized on ice in lysis buffer B [50 mM HEPES, 150
mM NaCl, 10 mM EDTA, 10 mM
Na4P2O7, 2 mM vanadate,
100 mM NaF, 1% (vol/vol) Triton X-100, 0.5 mM
phenylmethylsulfonyl fluoride, 100 IU/ml Aprotinin, and 20
µM leupeptin]. Gab-1 was immunoprecipitated during 90
min at 4 C with antibodies to HA (ascites fluid 1:100) or with
antibodies to human Gab1 (2 µg/plate) preadsorbed on protein
G-Sepharose beads. Samples were analyzed by SDS-PAGE followed by
Western blotting with antibodies to phosphotyrosine (1 µg/ml) as
previously described (5). Proteins were revealed using
[125I]protein A followed by autoradiography.
Association of Gab-1 with SHP-2 in Intact Cells
Transfected 293 EBNA cells in 10-cm plates were incubated with
10 mM vanadate for 10 min before stimulation with insulin
(10-7 M) for 5 min at 37 C. After
solubilization of the cells in ice-cold lysis buffer B and
immunoprecipitation with antibodies to HA or with antibodies to Gab1,
the proteins were separated by SDS-PAGE and immunoblotted with
antibodies to HA, to Gab1, or to SHP-2 depending on the experiment.
PI3-Kinase Assay
Transfected 293 EBNA cells in 10-cm plates were stimulated with
insulin (10-7 M) for 5 min at 37 C. The PI3-K
activity was measured after immunoprecipitation of Gab1 with antibodies
to Gab1 as previously described (39). The phospholipids were analyzed
by TLC and autoradiography [32P]phosphate. Incorporation
into phosphatidylinositol 3-phosphate was quantified using the
PhosphoImager system (Bio-Rad).
In Vitro Phosphorylation of Gab1 by Wheat Germ
Agglutinin (WGA)-Purified IRs
Antibodies to HA were incubated with protein G-Sepharose for 45
min at 4 C. The pellets were washed twice with 50 mM HEPES,
150 mM NaCl, pH 7.6. Lysates from Gab1-transfected cells
were incubated with the HA antibody-containing pellets for 90 min at 4
C. The Gab1-containing pellets were washed twice with 50 mM
HEPES, 150 mM NaCl, containing 1% (vol/vol) Triton X-100.
WGA-purified IRs (300 fmol) (40) were incubated for 45 min with insulin
(10-7 M) before being added to the
Gab1-containing pellets. The phosphorylation reaction was initiated by
addition of 30 µM ATP, 8 mM
MgCl2, 4 mM MnCl2. After 30 min,
the pellets were washed three times with 50 mM HEPES, 150
mM NaCl, 10 mM EDTA, 10 mM
Na4P2O7, 2 mM vanadate,
100 mM NaF, 10% (vol/vol) glycerol, and 1% (vol/vol)
Triton X-100. Samples were resuspended into Laemmli sample buffer and
separated by SDS-PAGE followed by Western blotting with antibodies to
phosphotyrosine (1 µg/ml) as previously described (5). Proteins were
visualized using [125I]protein A followed by
autoradiography.
 |
ACKNOWLEDGMENTS
|
---|
We thank A. Vojtek and S. Elledge for L40 strain and yeast
plasmids, E. Fischer for SHP-2 cDNA, M. F. White and C. R.
Kahn for rat IRS-1 cDNA, A. Ullrich for human IR cDNA, J. E.
Pessin for p85
cDNA, J. Pouysségur for PECE-HA vector, and J.
Camonis for pVJL-modified vector. We also thank V. Baron and C. Sable
for critical reading of the manuscript.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Emmanuel Van Obberghen, INSERM U145, Faculté de Médecine, avenue de Valombrose, 06107 Nice Cédex 2, France. E-mail: vanobbeg{at}unice.fr
This work was supported by funds from INSERM, Université de
Nice-Sophia-Antipolis, Association pour la Recherche contre le Cancer
(ARC Grant 6432), Ligue Nationale contre le Cancer, and Groupe LIPHA
(Contract 9323). Stephane Rocchi has a student-fellowship from Ligue
Nationale contre le Cancer.
Received for publication October 3, 1997.
Revision received March 5, 1998.
Accepted for publication April 8, 1998.
 |
REFERENCES
|
---|
-
Jongsoon L, Pilch PF 1994 The insulin receptor:
structure, function, and signaling. Am J Physiol 266:C319C334
-
Van Obberghen E 1994 Signalling through the insulin
receptor and the insulin-like growth factor-1 receptor. Diabetologia 37:125134
-
Lavan BE, Lane WS, Lienhard GE 1997 The 60-kDa
phosphotyrosine protein in insulin-treated adipocytes is a new menber
of the insulin receptor substrate family. J Biol Chem 272:1143911443[Abstract/Free Full Text]
-
Myers M, White M 1996 Insulin signal
transduction and the IRS proteins. Annu Rev Pharmacol Toxicol 36:615658[CrossRef][Medline]
-
Rocchi S, Tartare-Deckert S, Mothe I, Van Obberghen
E 1995 Identification by mutation of the tyrosine residues in the
insulin receptor substrate-1 affecting association with the tyrosine
phosphatase 2C and phosphatidylinositol 3-kinase. Endocrinology 136:52915297[Abstract]
-
Mothe I, Delahaye L, Filloux C, Pons S, White MF,
Van Obberghen E 1997 Interaction of the regulatory subunit of PI
3-kinase,p55pik, with insulin-like growth factor-I
signaling proteins. Mol Endocrinol, in press
-
Kotani K, Yonezawa K, Hara K, Ueda H, Kitamura Y,
Sakaue H, Ando A, Chavanieu A, Calas B, Grigorescu F, Nishiyama M,
Waterfield MD, Kasuga M 1994 Involvement of phosphoinositide 3-kinase
in insulin- or IGF-1-induced membrane ruffling. EMBO J 13:23132321[Abstract]
-
Okada T, Kawano Y, Sakakibara T, Hazeki O, Ui M 1994 Essential role of phosphatidylinositol-3-kinase in insulin-induced
glucose transport and antilipolysis in rat adipocytes. Studies with a
selective inhibitor wortmannin. J Biol Chem 269:35683573[Abstract/Free Full Text]
-
Cheatham B, Vlahos CJ, Cheatham L, Wang L, Blenis
J, Kahn R 1994 Phosphatidylinositol 3-kinase activation is required for
insulin stimulation of pp70, DNA synthesis, and glucose transporter
translocation. Mol Cell Biol 14:49024911[Abstract]
-
Hara K, Yonezawa K, Sakaue H, Ando A, Kotani K,
Kitamura T, Kitamura Y, Ueda H, Stephens L, Jackson TR, Hawkins PT,
Dhand R, Clark AE, Holman GD, Waterfield MD, Kasuga M 1994 1-phosphatidylinositol 3-kinase activity is required for
insulin-stimulated glucose transport but not for RAS activation in CHO
cells. Proc Natl Acad Sci USA 91:74157419[Abstract]
-
Kanai F, Ito K, Todaka M, Hayashi H, Kamohara S,
Ishii K, Okada T, Hazeki O, Ui M, Ebina Y 1993 Insulin-stimulated Glut4
translocation is relevant to the phosphorylation of IRS-1 and the
activity of PI-3-kinase. Biochem Biophys Res Commun 195:762768[CrossRef][Medline]
-
Milarsky KL, Saltiel AR 1994 Expression of
catalytically inactive Syp phosphatase in 3T3 cells blocks stimulation
of mitogen-activated protein kinase by insulin. J Biol Chem 269:2123921243[Abstract/Free Full Text]
-
Noguchi T, Matosaki T, Horita K, Fujioka Y, Kasuga M 1994 Role of SH-PTP2, a protein-tyrosine phosphatase with Src Homology
2 domains, in insulin-stimulated ras activation. Mol Cell Biol 14:66746682[Abstract]
-
Xiao S, Rose DW, Sasaoka T, Maegawa H, Burke TRJ,
Roller PP, Shoelson SE, Olefsky JM 1994 Syp (SH-PTP2) is a positive
mediator of growth factor-stimulated mitogenic signal transduction.
J Biol Chem 269:2124421248[Abstract/Free Full Text]
-
Yamauchi K, Milarski KL, Saltiel AR, Pessin JE 1995 Protein-tyrosine-phosphatase SHPTP2 is a required positive effector for
insulin downstream signaling. Proc Natl Acad Sci USA 92:664668[Abstract]
-
Kuhné MR, Zhao Z, Rowles J, Lavan BE, Shen
S-H, Fischer EH, Lienhard GE 1994 Dephosphorylation of insulin receptor
substrate 1 by tyrosine phosphatase PTP2C. J Biol Chem 269:1583315837[Abstract/Free Full Text]
-
Holgado-Madruga M, Emlet DR, Moscatello DK,
Godwin AK, Wong AJ 1996 A Grb2-associated docking protein in EGF- and
insulin -receptor signalling. Nature 379:560564[CrossRef][Medline]
-
Weidner K, Di Cesare S, Sachs M, Brinkmann V 1996 Interaction between Gab1 and the c-Met receptor tyrosine kinase is
responsible for epithelial morphogenesis. Nature 384:173176[CrossRef][Medline]
-
Gustafson TA, He W, Craparo A, Schaub CD, ONeill
TJ 1995 Phosphotyrosine-dependent interaction of Shc and insulin
receptor substrate-1 with the NPEY motif of the insulin receptor via a
novel Non-SH2 domain. Mol Cell Biol 15:25002508[Abstract]
-
ONeill TJ, Craparo A, Gustafson TA 1994 Characterization of an interaction between insulin receptor substrate 1
and the insulin receptor by using the two-hybrid system. Mol Cell Biol 14:64336442[Abstract]
-
Sawka-Verhelle D, Tartare-Deckert S, White MF, Van
Obberghen E 1996 Insulin receptor substrate-2 binds to the insulin
receptor through its phosphotyrosine-binding domain and through a newly
identified domain comprising amino acids 591786. J Biol Chem 271:59805983[Abstract/Free Full Text]
-
Tartare-Deckert S, Sawka-Verhelle D, Murdaca J, Van
Obberghen E 1995 Evidence for a differential interaction of SHC and the
insulin receptor substrate-1 (IRS-1) with the insulin-like growth
factor-1 (IGF-I) receptor in the yeast two-hybrid system. J Biol
Chem 270:2345623460[Abstract/Free Full Text]
-
Myers MGJ, Grammer TC, Brooks J, Glasheen EM, Wang
LM, Sun XJ, Blenis J, Pierce JH, White MF 1995 The pleckstrin homology
domain in insulin receptor substrate-1 sensitizes insulin signaling.
J Biol Chem 270:1171511718[Abstract/Free Full Text]
-
Yenush L, Makati KJ, Smith-Hall J, Ishibashi O,
Myers MGJ, White MF 1996 The pleckstrin homology domain is the
principal link between the insulin receptor and IRS-1. J Biol Chem 271:2430024306[Abstract/Free Full Text]
-
Rordorf-Nikolic T, Van Horn DJ, Chen D, White MF,
Backer JM 1995 Regulation of phosphatidylinositol 3'-kinase by tyrosyl
phosphoproteins. J Biol Chem 270:36623666[Abstract/Free Full Text]
-
Voliovitch H, Schindler D, Hadari Y, Taylor S,
Accili D, Zick Y 1995 Tyrosine phosphorylation of insulin receptor
substrate-1 in vivo depends upon the presence of its
pleckstrin homology region. J Biol Chem 270:1808318087[Abstract/Free Full Text]
-
Ferguson KM, Lemmon MA, Schessinger J, Sigler
PB 1994 Cristal structure at 2.2 A resolution of the pleckstrin
homology domain from human dynamin. Cell 79:199209[Medline]
-
Harlan JE, Hajduk P, Yoon HS, Fesik SW 1994 Pleckstrin homology domains bind to
phosphatidylinositol-4,5-bisphosphate. Nature 371:168170[CrossRef][Medline]
-
Pitcher JA, Touhara K, Payne ES, Lefkowitz RJ 1995 Pleckstrin homology domain-mediated membrane association and
activation of the beta-adrenergic receptor kinase requires coordinate
interaction with G beta gamma subunits and lipid. J Biol Chem 270:1170711710[Abstract/Free Full Text]
-
Vojtek AB, Hollenberg SM, Cooper JA 1993 Mammalian
ras interacts directly with the serine/threonine kinase raf. Cell 74:205214[Medline]
-
Harper JW, Adami GR, Wei N, Keyomarsi K, Elledge SJ 1993 The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1
cyclin-dependent kinases. Cell 75:805816[Medline]
-
Meloche S, Pagés G, Pouysségur J 1992 Functional expression and growth factor activation of an epitope-tagged
p44 mitogen-activated protein kinase, p44mapk. Mol Biol
Cell 3:6371[Abstract]
-
Tirode F, Malaguti C, Romero F, Attar R, Camonis J,
Egly JM 1997 A conditionally expressed third partner stabilizes or
prevents the formation of a transcriptional activator in a three-hybrid
system. J Biol Chem 272:2299522999[Abstract/Free Full Text]
-
Ullrich A, Bell JR, Chen E, Herrera R,
Petruzzelli LM, Dull TJ, Gray A, Coussens L, Liao YC, Tsubokawa M,
Mason A, Seeburg PH, Grunfeld C, Rosen OM, Ramachandran J 1985 Human
insulin receptor and its relationship to the tyrosine kinase family of
oncogenes. Nature 313:756761[Medline]
-
Gietz D, St Jean A, Woods RA, Schiestl RH 1992 Improved method for high efficiency transformation of intact yeast
cells. Nucleic Acids Res 20:14251426[Medline]
-
Bartel PL, Chien C, Sternglanz R, Fields S 1993 Using the two hybrid system to detect protein-protein interactions. In:
Hartley DA (ed) Cellular Interactions in Development: A Practical
Approach Oxford University Press, Oxford, U.K., pp 153179
-
Miller JH 1972 Experiments in Molecular Genetics.
Cold Spring Harbor Laboratory Press, Plainview, NY
-
Chen C, Okayama H 1987 High-efficiency
transformation of mammalian cells by plasmid DNA. Mol Cell Biol 7:27452752[Medline]
-
Giorgetti S, Ballotti R, Kowalski-Chauvel A, Cormont
M, Van Obberghen E 1992 Insulin stimulates
phosphatidylinositol-3-kinase in rat adipocytes. Eur J Biochem 207:599606[Abstract]
-
Gual P, Baron V, Alengrin F, Mothe I, Van Obberghen
E 1996 Insulin receptor-induced phosphorylation of cellular and
synthetic substrates is regulated by the receptor beta-subunit
C-terminus. Endocrinology 137:34163422[Abstract]