(Received for publication, May 8, 1995 )
From the
To characterize the structural basis for the interactions
between the insulin receptor (IR) and its major substrate, insulin
receptor substrate-1 (IRS-1), a segment of the NH The insulin receptor (IR) In non-treated cells,
IRS-1 is serine-phosphorylated, and, after insulin stimulation,
tyrosine and serine phosphorylation of IRS-1 is increased(13) .
IRS-1 has 21 potential tyrosine phosphorylation sites, including six in
YMXM motifs and three in YXXM motifs. These motifs
belong to a common consensus sequence (D/E)(D/E)Y(M/V)(P/D/E)M also
present in the platelet-derived growth factor, colony-stimulating
factor-1, and Kit receptors(16) . Phosphorylation of these Tyr
residues of IRS-1 by insulin receptor kinase is believed to create a
binding site for the SH2 domains of the p85 regulatory subunit of
phosphatidylinositol 3-kinase(17, 18) , Grb2, Nck, and
the protein tyrosine phosphatase SH-PTP2 (Syp)(19) . The
association between p85 and IRS-1 results in activation of
phosphatidylinositol 3-kinase (17, 18) in a mechanism
independent of the direct activation of phosphatidylinositol 3-kinase
by Ras(20) . Similarly, binding of the ras activator, son of
sevenless (mSos), to the Grb2 IRS-1 also contains a pleckstrin homology (PH) domain (23, 24) at its extreme NH To
study the role of the PH domain of IRS-1, a truncated form of IRS-1 was
generated where the first four ``blocks'' of the PH region
were deleted. Here we demonstrate that such a deletion selectively
impairs the ability of IRS-1 to undergo in vivo insulin-dependent phosphorylation and markedly inhibits the
association between the truncated form of IRS-1 and
phosphatidylinositol 3-kinase, but does not affect the capacity of the
mutated IRS-1 to serve as an in vitro substrate for the
insulin receptor kinase.
To generate a
construct expressing IRS-1 The initiation codon is indicated in bold. The correctness of
the construct (pcDNAI-IRS1 To generate pcDNAIII-IRS-1
Figure 1:
Insulin-dependent phosphorylation of
IRS-1 in COS-7 cells. COS-7 cells remained untransfected (lane
3), or were transfected with pcDNA-IR (IR) alone (lane 2) or in combination with pcDNA-IRS1 (IRS-1) (lane 1). After 36 h cells were incubated for 8 h in
serum-free Dulbecco's modified Eagle's medium and then
stimulated for 1 min with 10
Figure 2:
Reduced insulin-dependent phosphorylation
of IRS-1
Figure 3:
Phosphatidylinositol 3-kinase activity in
IRS-1 immunoprecipitates from insulin-treated COS-7 cells. COS-7 cells
were cotransfected with pcDNA-IR in combination with pcDNA-IRS1 or
pcDNA-IRS1
Figure 4:
In vitro phosphorylation of WT
IRS-1 and IRS-1
The present study provides evidence that deletion of four out
of six PH subdomains of IRS-1 largely impairs its capacity to serve as
an in vivo substrate for the insulin receptor kinase. The
reduced phosphorylation could not be accounted for by deletion of
tyrosine phosphorylation sites, because only 2 (Tyr The immediate outcome of the reduced
phosphorylation of IRS-1 Partial deletion of the PH region
could alter the overall structure of IRS-1 such that it fails to
interact with and to serve as a substrate for IR. Alternatively, the PH
domain itself could serve as the binding domain to IR. In such a case,
its deletion could prevent interactions between IRS-1 and IR, even
though the rest of the IRS-1 molecule still maintains its overall
native structure. To address these possibilities, we compared the
abilities of wild-type and mutated IRS-1 to serve as in vitro substrates for insulin receptor kinase. We could readily
demonstrate that both IRS-1 forms serve as comparable in vitro substrates for insulin receptor kinase, indicating that the
partial deletion of the PH region does not affect, in any major way,
the in vitro interactions between IRS-1 Although the solution structure
of two PH domains has been resolved (39, 40) , the
function of the PH domain is largely unknown. On the basis of
structural similarities between PH domains and lipid-binding proteins,
it has been proposed that the PH domain binds to lipophilic
molecules(39) . Consistent with this hypothesis, PH domains
were shown to bind to phosphatidylinositol
4,5-bisphosphate(41) . Alternatively, it has been suggested
that PH domains function in the recognition of phosphorylated Ser/Thr
residues in specific peptide sequences(42) . Indeed, a PH
domain localized at the COOH-terminal end of At present it is difficult to
reconcile these alternatives. However, our studies support the
hypothesis that the PH region facilitates in vivo binding of
IRS-1 to membrane phospholipids or other cellular constituents in close
proximity to the IR, whereas the actual interactions with the IR are
presumably mediated through other domains of the IRS-1 molecule. The
fact that partial deletion of the PH domain selectively impairs the in vivo interactions between the insulin receptor and IRS-1,
whereas their in vitro interactions are unaffected, strongly
supports this hypothesis.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-terminal
region of IRS-1 (Pro
-Pro
) was deleted.
This region contains the first four conserved boxes of a pleckstrin
homology (PH) domain, located at the NH
-terminal part of
IRS-1. COS-7 cells were then cotransfected with the genes coding for IR
and a wild-type (WT) or a mutated form of IRS-1. IRS-1
underwent significantly reduced insulin-dependent tyrosine
phosphorylation compared with WT IRS-1. The reduced in vivo tyrosine phosphorylation of IRS-1
was
accompanied by reduced association between IRS-1
and
its downstream effector p85 regulatory subunit of
phosphatidylinositol-3 kinase. In contrast, both WT IRS-1 and
IRS-1
underwent comparable insulin-dependent tyrosine
phosphorylation in vitro when incubated with partially
purified insulin receptor kinase. These findings suggest that the
overall structure of IRS-1 is not altered by deletion of its PH domain
and that the PH domain is not the main site for protein-protein
interactions between the insulin receptor and IRS-1, at least in
vitro. In conclusion, the PH region might facilitate in vivo binding of IRS-1 to membrane phospholipids or other cellular
constituents in close proximity to the IR, whereas the actual
interactions with the IR are presumably mediated through other domains
of the IRS-1 molecule. This could account for the fact that partial
deletion of the PH domain selectively impairs the in vivo interactions between the insulin receptor and IRS-1, whereas their in vitro interactions remain unaffected.
(
)is a
transmembrane glycoprotein composed of two extracellular
subunits
and two transmembrane
subunits linked by a disulfide bond to form
an
heterotetrameric structure. The
subunits contain the insulin-binding domain, whereas the
transmembrane
subunits function as tyrosine-specific protein
kinase (insulin receptor kinase), which undergoes autophosphorylation
following insulin binding (compare (1, 2, 3, 4) for reviews).
Autophosphorylation activates the insulin receptor kinase (5) and enables it to phosphorylate protein substrates,
including insulin receptor substrate-1 (IRS-1) (6) and Shc (7) . IRS-1 is a key element in insulin action, and mutations
of IR (e.g. mutation of Tyr
) that abolish IRS-1
phosphorylation inhibit insulin-induced metabolic and growth-promoting
effects(4, 8, 9) . IRS-1 and its homologue
called 4PS are also essential for insulin-, IGF-1-, and IL-4-stimulated
mitogenesis (10, 11, 12, 13) . In
contrast, the phenotype of mice lacking IRS-1 is surprisingly
mild(14, 15) . The mice suffer some retardation of
intrauterine growth and show impaired glucose tolerance. These data
suggest the existence of both IRS-1-dependent and IRS-1-independent
pathways for signal transduction of insulin.
IRS-1 complexes links the insulin
receptor to the ras signaling pathway(21) . Both Grb-2 and
phosphatidylinositol 3-kinase can simultaneously bind distinct
tyrosine-phosphorylated regions on the same IRS-1 molecule, suggesting
a mechanism whereby IRS-1 could serve as a docking site for multiple
SH2 proteins to further propagate the insulin signal(22) .
-terminal
region(25) . This domain of about 100 amino acids is found in a
number of signaling proteins including mSos and Gap(26) . It
consists of six weakly conserved blocks separated by insertions of
different lengths. This domain has been termed ``pleckstrin
homologue'' because it was originally detected as an internal
repeat in pleckstrin, a 47-kDa protein that is the major substrate of
protein kinase C in platelets(27) . It is unlikely that the PH
domain could function catalytically because of its small size and lack
of conserved residues. Instead, it may be involved in molecular
recognition similarly to SH2 and SH3 domains. As with these domains,
the PH domain does not occupy a specific position in the host molecule,
suggesting that it is functionally independent(28) .
Materials
Restriction enzymes were purchased
from Boeheringer Mannheim and Promega. Radiolabeled nucleotides and
[S]methionine were from Amersham Corp. All other
reagents were from Sigma unless stated otherwise. Wheat germ agglutinin
coupled to agarose was from BioMakor (Kiryat Weizmann, Rehovot,
Israel).
Antibodies
Antibodies to IRS-1 (anti-IRS-1) were
raised in rabbits according to standard procedures (29) by
injection of a keyhole limpet hemocyanin-coupled peptide
CYASINFQKQPEDRQ that corresponds to the carboxyl-terminal 14 amino
acids of rat liver IRS-1 (6) (and an additional Cys residue at
the NH-terminal site). Antibodies were affinity-purified
from the serum by adsorption onto a column of peptide coupled to
Affi-Gel 10, elution with 100 mM HCl glycine, pH 2.7, and
immediate neutralization. Monoclonal mouse anti-Tyr(P) antibodies
(PY-20) were purchased from Affinity Research Products (Nottingham, UK)
and were used at 1:1000 dilution.
Cloning of the Mouse IRS-1 Gene
The IRS-1 gene was
cloned from a 129SV mouse genomic library made in the Fix vector
(Stratagene). The library was screened with a 0.7-kilobase pair cDNA
probe corresponding to base pairs 670-1314 of the rat IRS-1 sequence (6) . Positive clones were identified, isolated by three rounds
of screening, digested with different restriction enzymes, and
subcloned into a pGEM-11Z vector (Promega). The insert (denoted
pGEM-11Z-gIRS1) represented the sequence from -2.7 to 4.7
kilobase pairs of the IRS-1 gene. Because the coding segment of IRS-1
lacks introns, the plasmid contained the entire coding region of the
129SV mouse IRS-1 gene (base pairs 478-4700, numbered according to the
rat IRS-1 sequence(6) ). It should be noted that, similar to
the human IRS-1 gene(30) , the mouse genomic sequence also
contains an intron located 20 nucleotides 3` to the TAG termination
codon.
Generation of IRS-1 Expression Vectors
To generate
expression vectors for IRS-1, sequences upstream of the ATG initiation
codon were deleted because they contain several in-frame initiation and
termination codons that might interfere with efficient translation (10) . In order to delete these regions, the sequence from 512
to 812, including the ATG start codon at 589 and the XhoI site
at 785, was amplified by polymerase chain reaction (PCR). The 5` primer
(5`-GGAAGTCGACAAGCTTAGCATGGCGAG-3`) was located at nucleotide 586 and
included HindIII and SalI sites. The 3` primer
(5`-CCGCTTGTTGAGTTGAAACAGCT-3`) was located at nucleotide 812 of the
IRS-1 gene. Both pGEM-11Z-gIRS1 and the PCR product were digested with HindIII and XhoI, and the 5` end of the genomic IRS-1
was replaced with the PCR fragment (nucleotides 586-785). Proper,
in-frame ligation of the PCR product into this new plasmid
(pGEM-11Z-cIRS1) was confirmed by restriction map analysis and
sequencing. To allow expression of the IRS-1 molecule in COS-7 cells,
the IRS-1 gene (0.58-4.7 kilobase pairs) was excised from
pGEM-11Z-cIRS1 with HindIII and SalI, gel-purified,
and ligated into pcDNAI/Amp or pcDNAIII (Invitrogen) to generate
pcDNAI-IRS-1 or pcDNAIII-IRS-1, respectively., where 60 amino acids
from the NH
-terminal region of IRS-1
(Pro
-Pro
) are deleted, pcDNAI-IRS-1 was
digested with HindIII and XhoI, and a 200-base-pair
piece was deleted and replaced by a double-stranded synthetic
oligonucleotide containing matching overhangs:
) was verified by
restriction mapping.
,
the IRS1
gene was excised from pcDNAI-IRS-1
with HindIII and SalI,
gel-purified, and ligated into pcDNAIII (Invitrogen). To generate a
plasmid for transient expression of IR in COS-7 cells, the IR coding
sequence was excised from pGEM4Z-IR with XhoI. pcDNAI plasmid
was linearized with XhoI, gel purified, dephosphorylated with
calf intestinal alkaline phosphatase, and ligated to the IR coding
sequence. The correctness of the construct (pcDNA-IR) was verified by
restriction mapping.
Transient Expression of the IRS-1 Gene in COS-7
Cells
COS-7 cells were cotransfected using DEAE-dextran (31) with pcDNA-IRS1, pcDNA-IRS1, or pcDNA-IR
(alone or in the combinations indicated in the figure legends). After
36 h the cells were starved for 8 h in serum-free Dulbecco's
modified Eagle's medium and stimulated for 1 min with
10
M insulin. Cells were then washed three
times with phosphate-buffered saline and were frozen in liquid
nitrogen. Cell extracts were made in 250 µl of lysis buffer I
containing 0.15 M sucrose, 50 mM Hepes, pH 7.4, 80
mM
-glycerophosphate, 10 mM NaF, 1% Triton
X-100, 0.1% SDS, 2 mM each of EDTA, EGTA, and sodium
orthovanadate, 10 µg/ml aprotinin, 5 µg/ml leupeptin, 10
µg/ml trypsin inhibitor, and 1 mM phenylmethylsulfonyl
fluoride. The cell lysates were centrifuged for 15 min at 4 °C in
an Eppendorf centrifuge at 12,000
g, and protein
concentrations were determined with the Bradford dye binding
assay(32) . 50 µg of protein from the supernatants were
loaded on a 7.5% SDS-PAGE and immunoblotted with anti-phosphotyrosine
antibodies or with anti-IRS-1 antibodies.
Stable Expression of the IRS-1 Gene in CHO
Cells
CHO cells were transfected by electroporation (33) with pcDNAIII-IRS1, pcDNAIII-IRS1, or
pcDNA-IR. 4
10
cells were electroporated at 960
microfarads and 250V using 10 µg of DNA. Following transfection,
cells were incubated in nonselective medium. 48 h later cells were
divided into 8 plates, and G418 (Life Technologies, Inc., 500 µg/ml
active units) was added for selection of stable colonies.
G418-resistant clones that overexpress IRS-1 or IRS1
were isolated and further propagated. For in vitro phosphorylation by insulin receptor kinase, cell extracts were
prepared from cells grown to confluence in 15-cm plates. The cells were
freeze-thawed three times in 800 µl of lysis buffer containing 0.15 M sucrose, 50 mM Hepes, pH 7.4, 80 mM
-glycerophosphate, 10 mM NaF, 2 mM each of EDTA,
EGTA, and sodium orthovanadate, 10 µg/ml aprotinin, 5 µg/ml
leupeptin, 10 µg/ml trypsin inhibitor, and 1 mM phenylmethylsulfonyl fluoride. The cell lysates were centrifuged
for 15 min at 4 °C in an Eppendorf centrifuge at 12,000
g, and protein concentrations were determined with the
Bradford dye binding assay (32) .
Immunoprecipitation
Anti-IRS-1 antibodies (at a
1:10 dilution) were added to 60 µl of 50% protein A-Sepharose in
0.1 M Tris buffer, pH 8.5, and were incubated for 1 h at 4
°C. Cell extracts, prepared in buffer I, were incubated for 2 h
with the antibody-protein A-Sepharose complex. Immunocomplexes were
washed, suspended in sample buffer(34) , resolved on 7.5%
SDS-PAGE (35) , and transferred to nitrocellulose for Western
blotting.Protein Electrophoresis and
Blotting
Immunoblotting was carried out essentially as
described(18) . The blotted proteins were incubated with
monoclonal anti-Tyr(P) or polyclonal anti IRS-1 antibodies at 4 °C
for 16 h, followed by intensive washing. To detect antibody binding, a
horseradish peroxidase-conjugated goat anti-mouse antibodies ECL kit
(Amersham) was used according to the manufacturer's instructions. Partial Purification of Insulin Receptor
Insulin
receptors were partially purified from rat liver plasma membranes. The
preparation of membranes, solubilization in Triton X-100, and affinity
chromatography of insulin receptors on wheat germ agglutinin coupled to
agarose were carried out as described previously(36) .In Vitro Phosphorylation of IRS-1
The reaction was
carried out essentially as described previously(37) . Briefly,
60-µl aliquots of partially purified insulin receptors
(50-200 µg/ml) were incubated in the presence or absence of
10M insulin (in 50 mM Hepes, 0.1%
bovine serum albumin, 0.1% Triton X-100, pH 7.6) for 30 min, 22
°C). Cytosolic extracts derived from CHO cells that overexpress
either WT-IRS-1 or IRS-1
were added, and
phosphorylation in a final volume of 450 µl was initiated with 80
µl of a ``reaction mix'' to yield the following final
concentrations: 50 mM Hepes, 1 mM ATP, 10 mM magnesium acetate, 4 mM manganese acetate, and 0.05%
Triton X-100. Reactions were allowed to proceed for 10 min at 22 °C
and were terminated by adding EDTA to a final concentration of 20
mM. IRS-1 was precipitated with anti-IRS-1 antibodies, and the
samples were further processed as described above.
Phosphatidylinositol 3-Kinase Assay
Assay of
phosphatidylinositol 3-kinase activity was performed as described
previously.(18) . Briefly, whole cell lysates were prepared as
above, and the amount of IRS-1 in each extract was estimated based upon
immunoblots with IRS-1 antibodies. Equal amounts of IRS-1 were immune
precipitated with anti-IRS-1 antibodies, and phosphatidylinositol
3-kinase activity was assayed in the immunoprecipitates using
phosphatidyl inositol and [-
P]ATP as
substrates. The reaction was carried out for 10 min at 24 °C.
Reaction products were extracted and separated by thin layer
chromatography. Autoradiograms were scanned by densitometry.
Insulin-dependent Tyrosine Phosphorylation of IRS-1 in
COS-7 Cells
Stimulation of untransfected COS-7 cells with
insulin resulted in a very low insulin-dependent tyrosine
phosphorylation of both IR and IRS-1 (Fig. 1, lane 3).
Transfection of these cells with an IR expression plasmid yielded an
insulin-dependent prominent band whose molecular mass (95 kDa) is
consistent with that of native subunits of the insulin receptor
and a fainter band whose molecular mass (180 kDa) is consistent with
that of IRS-1 (Fig. 1, lane 2). When the cells were
cotransfected with expression plasmids for both IR and WT-IRS-1, the
level of insulin-induced IR phosphorylation remained largely unaltered,
whereas IRS-1 phosphorylation was markedly (16-fold) enhanced (Fig. 1, lane 1). These findings show that transient
expression of either IR or IRS-1 generates functional full-length
proteins. Moreover, the expressed IRS-1 serves as a substrate for the
insulin receptor kinase. Comigration of the native (Fig. 1, lane 2) and transfected forms of IRS-1 (Fig. 1, lane 1) suggests that transfected IRS-1 undergoes the same
post-translational modification as the native endogenous one.
M insulin.
Cell extracts were prepared; 50 µg of protein were resolved on 10%
SDS-PAGE under reducing conditions and transferred to nitrocellulose.
Immunoblotting with anti-Tyr(P) was performed as described under
``Experimental Procedures.''
Partial Deletion of the PH Region Impairs Insulin-induced
Tyrosine Phosphorylation of IRS-1
To study the functional role
of the PH region of IRS-1, a truncated form of the IRS-1 gene was
generated where 61 amino acids encoding for the NH-terminal
region of the PH domain (Pro
-Pro
) were deleted
(IRS-1
). WT or mutant mouse IRS-1 genes were then
transiently expressed in COS-7 cells together with the IR gene.
Although both WT and mutated IRS-1 molecules were expressed at
comparable levels (detected by immunoblotting with anti IRS-1
antibodies) (Fig. 2, bottom), IRS-1
underwent significantly reduced insulin-dependent tyrosine
phosphorylation (detected by immunoblotting with anti-Tyr(P)
antibodies) (Fig. 2, top). Analysis by densitometry
revealed that the insulin-induced increase in tyrosine phosphorylation
of WT-IRS-1 is 13-fold greater than the increase in
IRS-1
. These results therefore suggest that the PH
domain of IRS-1 is required for normal in vivo tyrosine
phosphorylation of IRS-1 by IR.
. COS-7 cells were cotransfected with
pcDNA-IR in combination with pcDNA-IRS1 (WT) or
pcDNA-IRS1
(
PH). After 36 h cells were
incubated for 8 h in serum-free Dulbecco's modified Eagle's
medium and then stimulated for 1 min with 10
M insulin. Cell extracts were prepared; 50 µg of protein were
resolved on 10% SDS-PAGE under reducing conditions and transferred to
nitrocellulose. Immunoblotting with anti-Tyr(P) (Blot
-P-Ty, top) and anti-IRS-1 antibodies (Blot
-IRS-1, bottom) was performed as described under
``Experimental Procedures.'' The intensity of the bands
corresponding to tyrosine-phosphorylated IRS-1 and IRS1
was quantitated by densitometry. These values were: 9 for
IRS-1
without insulin, 109 for IRS-1
with insulin, 11 for IRS-1 without insulin, and 1433 for IRS-1
with insulin.
IRS-1
In view of
the fact that phosphatidylinositol 3-kinase associates with and is
activated upon interaction with tyrosine-phosphorylated
IRS-1(18) , the functional consequences of the reduced
phosphorylation of IRS-1Exhibits Reduced
Association with Phosphatidylinositol 3-Kinase
were assessed by assaying
for the phosphatidylinositol 3-kinase activity associated with the
truncated form of IRS-1. As shown in Fig. 3, the reduced
tyrosine phosphorylation of IRS-1
was accompanied by
reduced phosphatidylinositol 3-kinase activity detected in
IRS-1
immunoprecipitates (Fig. 3). Although
comparable amounts of both WT IRS-1 and IRS-1
were
precipitated by anti IRS-1 antibodies (not shown), the increase in
phosphatidylinositol 3-kinase activity, associated with
insulin-stimulated IRS-1, was 2-fold higher than the activity
associated with the insulin-stimulated IRS-1
. These
results indicate that deletion of the PH region of IRS-1 impairs its
interactions with downstream effectors like phosphatidylinositol
3-kinase.
. Following stimulation for 1 min with
10
M insulin, cell extracts were prepared
and immunoprecipitated with anti-IRS-1 antibodies. Phosphatidylinositol
3-kinase activity was assayed in the immunoprecipitates by in vitro phosphorylation of phosphatidylinositol as described under
``Experimental Procedures.'' The intensity of the spots
corresponding to phosphatidylinositol 3-phosphate was quantitated by
densitometry. These values were: 10 for IRS-1
without
insulin, 116 for IRS-1
with insulin, 14 for IRS-1
without insulin, and 234 for IRS-1 with
insulin.
IRS-1
To rule out
the possibility that IRS-1Is Phosphorylated in
Vitro to Levels Comparable to Those of WT IRS-1
fails to undergo in
vivo phosphorylation due to gross alterations in its overall
conformation, we compared the abilities of wild-type and mutated IRS-1
to serve as in vitro substrates for insulin receptor kinase.
Increasing amounts of cytosolic extracts derived from CHO cells that
overexpress either IRS-1
or WT IRS-1 were subjected
to phosphorylation by partially purified insulin receptor kinase. As
shown in Fig. 4(top) both forms of IRS-1 underwent
insulin-dependent tyrosine phosphorylation in a dose-dependent manner.
However, there was no significant difference between the extent of
their phosphorylation (when normalized according to the amount of
IRS-1) (Fig. 4, bottom). Hence, IRS-1
appears to maintain the overall structural organization of WT
IRS-1 to the extent that it serves as a comparable in vitro substrate for insulin receptor kinase.
by insulin receptor kinase. Top, aliquots (100-300 µl) of cytosolic extracts
derived from CHO cells overexpressing WT IRS-1 (lanes
A-D) or IRS-1
(lanes E-H)
were subjected to in vitro phosphorylation by partially
purified insulin receptor kinase in the presence (+) or the
absence (-) of insulin as described under ``Experimental
Procedures.'' IRS-1 immunoprecipitates were resolved by means of
SDS-PAGE and blotted with either Tyr(P) or IRS-1 antibodies. Bottom, the intensity of the bands was quantified by
densitometry, and the results are presented in arbitrary units. P-Tyr, Tyr(P);
, IRS-1;
,
IRS-1
.
and
Tyr
) out of 21 such potential sites were deleted in
IRS-1
.
is reduced
phosphatidylinositol 3-kinase activity that remains associated with the
mutated form of IRS-1. This result is somewhat expected in view of the
fact that the SH2 domains of p85, the regulatory subunit of
phosphatidylinositol 3-kinase, presumably interact with Tyr residues
460, 608, 939, and 987 of IRS-1, placed in the context of YMXM
or YXXM motifs (38) . This association results in
activation of pp110, the catalytic unit of phosphatidylinositol
3-kinase, by an as yet unknown mechanism. Our results therefore suggest
that the reduced phosphorylation of IRS-1
also
involves impaired phosphorylation of at least some of these residues.
Still, we cannot rule out the possibility that deformation of the
overall native structure of IRS-1 induced by the truncation impairs the
capability of IRS-1 to interact with phosphatidylinositol 3-kinase. The
reduction in phosphatidylinositol 3-kinase activity is less dramatic
than the reduction in Tyr(P) content of IRS-1
. This
apparent discrepancy could be attributed to the fact that the residual
amount of tyrosine-phosphorylated IRS-1
is still high
enough to complex with the limited amount of available endogenous
phosphatidylinositol 3-kinase.
and
insulin receptor kinase. These findings serve to make two points: (i)
the overall structure of IRS-1
is not grossly
modified by the mutation and (ii) the PH domain is not the main site
for protein-protein interactions between the insulin receptor and
IRS-1, at least in vitro.
-adrenergic receptor
kinase is essential for targeting
-adrenergic receptor kinase to
the plasma membrane(25) , presumably through interaction with
the
-subunits of heterotrimeric G-proteins. These findings
have set the stage for the hypothesis that, similar to SH2 and SH3
domains, PH regions might be involved in protein-protein interactions.
Support for this notion is provided by the fact that, when tested in vitro, the PH domain of IRS-1, expressed as a GST-fusion
protein, interacts, though to a rather low extent, with
G
(43) . In contrast, a 356-amino-acid region
encompassed by amino acids 160-516 of IRS-1 outside the PH region
is sufficient for interactions with the receptor in the yeast
two-hybrid assay(44) .
,
IRS-1 with a segment of the NH
-terminal region
(Pro
-Pro
) deleted; PH, pleckstrin
homologue; WT, wild-type; PCR, polymerase chain reaction; PAGE,
polyacrylamide gel electrophoresis; CHO, Chinese hamster ovary.
We thank Dr. Ronit Sagi-Eisenberg for helpful
discussions and a critical review of this manuscript. We thank Yael
Biener and Keren Paz for helpful advice and invaluable assistance.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.