From the
The NH
During insulin stimulation, IRS-1
Deletion of the first 500 amino acids
of IRS-1 prevents phosphorylation of the COOH-terminal portion of the
molecule expressed in COS cells (6). A pleckstrin homology (PH) domain
exists between residues 13 and 115, which is absolutely identical among
the rat, mouse, and human isoforms
(7, 8, 9) ,
and is 62% identical in the recently cloned IRS-2.(
In this study we have investigated the role of the PH
domain in IRS-1 by deleting it and studying this mutant
(IRS-1
32D cells are an ideal system for
the examination of IRS-1 function, since they contain no endogenous IRS
proteins to interfere with the analysis of signaling by exogenous
expressed species
(2, 3, 5) . Activation of
endogenous insulin receptors stimulated tyrosyl phosphorylation of
IRS-1 in 32D/IRS-1 cells, whereas disruption of the PH domain reduced
tyrosine phosphorylation by more than 95% (Fig. 3A); no
tyrosyl phosphorylation of IRS-1 was observed in untransfected 32D
cells, since these cells do not contain
IRS-1
(2, 3, 5) . Insulin stimulated the
association of PI 3`-kinase with IRS-1 in 32D/IRS-1 cells. Moreover,
IRS-1 was essential for the activation of p70
Why does overexpression of the insulin receptor in
CHO
It remains unclear how the PH domain mediates its
function. It could involve direct protein-protein interactions between
the receptor and IRS-1; however, our attempts to measure specific
interactions between the purified insulin receptor and glutathione
S-transferase fusion proteins containing the PH domain have
been unsuccessful. Recent evidence suggests that PH domains from other
signaling molecules bind specific phospholipids
(13) . Further
work should shed light on whether the PH domain acts specifically to
target IRS-1 to the insulin receptor or more generally to the membrane
compartment where it encounters the tyrosine kinase.
IRS-1 contains
a region immediately downstream of the PH domain which has also been
implicated in the IR/IRS-1 interaction (IH2 region in
Fig. 1A)
(6) . The IH2 region is similar in IRS-1
and IRS-2, and like the PH domain, contains no tyrosine phosphorylation
sites (15).
The PH domain is 100% identical in mouse, human, and rat IRS-1, and
62% similar in IRS-1 and IRS-2. This is the highest level of PH domain
conservation between distinct proteins yet observed; this conservation
goes well beyond the requirement for similar folding recently revealed
by structural studies
(10, 11) and is consistent with a
specific function. The IH2 regions are also conserved between IRS-1 and
IRS-2. The COOH terminus, however, is poorly conserved between IRS-1
and IRS-2, however, with the critical exception of short elements
surrounding tyrosine phosphorylation sites.
We thank Jia-Huai Wang for helpful discussions and a
critical reading of the manuscript. We also thank Gus Gustafson, Mary
Elizabeth Patti, Ron Kahn, Jon Backer, and Lynne Yenush for helpful
discussions and sharing unpublished data. We thank Yitao Zhang and
Charles Knickley for excellent technical assistance.
terminus of insulin receptor substrate-1
(IRS-1) contains a pleckstrin homology (PH) domain. We deleted the PH
domain in IRS-1 (IRS-1
) and expressed the mutant in
Chinese hamster ovary and 32D cells. During insulin stimulation,
IRS-1
is poorly tyrosine-phosphorylated in CHO cells,
but undergoes serine/threonine phosphorylation. Similarly,
IRS-1
fails to undergo insulin-stimulated tyrosine
phosphorylation in 32D cells, which uncouples the activation of
phosphatidylinositol 3`-kinase and p70
from the
endogenous insulin receptors. Overexpression of the insulin receptor in
32D
cells, however, restores tyrosine phosphorylation of
IRS-1
and rescues insulin responses including
mitogenesis. Thus, while the PH domain is not required for the
engagement of downstream signals, it is one of the elements in the
NH
terminus of IRS-1 that is needed for a sensitive
coupling to insulin receptors, especially at ordinary receptor levels
found in most cells and tissues.
(
)
becomes tyrosine-phosphorylated and binds to the Src
homology-2 domains in several signaling proteins (SH2
proteins)
(1) . As a consequence of docking SH2 proteins, IRS-1
mediates multiple downstream signals, including the direct activation
of PI 3`-kinase and SH-PTP2, and the stimulation of mitogen-activated
protein kinase and p70
; IRS-1 at least partially
regulates mitogenesis, chemotactic signaling, and glucose
transport
(1, 2, 3, 4, 5) . Since
IRS-signaling proteins are not engaged by most growth factor receptors,
specific interactions between IRS-signaling proteins and receptors must
mediate productive coupling.
)
PH domains were originally recognized as a repeat in pleckstrin
and later found in various signal transduction proteins
(7) .
Although the amino acid sequence of various PH domains is poorly
conserved, the PH domain in spectrin and pleckstrin has a common
structure composed of three
-sheets and an
-helix arranged
around a hydrophobic core
(10, 11) . Although the exact
function of PH domains is obscure, recent studies suggest that they
mediate protein-protein interactions or bind phospholipids
(12-14).
) in CHO
cells, which contain
endogenous IRS-1, and in 32D cells, which lack endogenous IRS-signaling
proteins. Our results suggest that the PH domain is not required for
the engagement of downstream signals; rather, it is one of the elements
that mediates the coupling between the insulin receptor and
IRS-signaling proteins.
Construction of IRS-1
The cDNA
for rat IRS-1 in pBluescript
(15) was digested with
BspEI and BspMI, and the resulting fragment
containing the majority of IRS-1 was religated in the presence of
linkers formed by annealing the oligonucleotides
5`-CCG/GAG/GAT/CCC/CTT/AAG-3` and 5`-CCT/CCT/TAA/GGG/GAT/CCT-3`. The
resulting mutant cDNA was subcloned into pCMVhis for expression using
SacI and HindIII
(3) .
Cell Lines
Chinese hamster ovary (CHO) cells
expressing the human insulin receptor (CHO) or the human
insulin receptor and IRS-1 (CHO
/IRS-1) have been
described
(4, 15) . CHO cell lines were maintained in
Ham's F-12 medium containing 10% fetal bovine serum. CHO
cells were transfected with IRS-1
cDNA by the
calcium phosphate method and selected in 10 mM
histidinol
(4, 15) . Surviving cells were cloned and
maintained in the presence of 10 mM histidinol. 32D cells and
cell lines expressing IRS-1 (32D/IRS-1), IR (32D
), or IR
and IRS-1 (32D
/IRS-1) have been described
previously
(2) . 32D cell lines were maintained in RPMI 1640
media supplemented with 10% fetal bovine serum and 5%
WEHI-3-conditioned media (a source of IL-3). 32D and 32D
cells were electroporated with the cDNA for IRS-1
and selected with 5 mM histidinol to obtain cell lines
expressing the PH deletion mutant
(2, 3) . Cell lines
were selected for expression by immunoblotting lysates of
histidinol-resistant cell lines with
IRS-1 antibodies. Expression
of the human insulin receptor was confirmed by
IR immunoblotting
cell lines co-expressing the insulin receptor. For experiments,
CHO
cells were made quiescent by incubation in Ham'
F-12 medium supplemented with 0.5% bovine serum albumin for 18-24
h; 32D cells were made quiescent by incubation in unsupplemented
Dulbecco's modified Eagle's medium for 4 h.
Antibodies and Growth Factors
IRS-1
antibodies were rabbit polyclonal antisera against a COOH-terminal
peptide of IRS-1; they were used at a 1:300 dilution to immunoblot and
1:100 to immunoprecipitate
(16) . Being directed against the COOH
terminus of IRS-1, this antibody recognizes IRS-1 and IRS-1
equivalently.
IR antibodies were rabbit antisera raised
against a glutathione S-transferase fusion protein containing
the intracellular
-subunit of the human insulin receptor; they
were used 1:300 to immunoblot.
PY antibodies were mouse monoclonal
4G10 purified from tissue culture supernatant by chromatography on
Protein A-Sepharose or affinity-purified rabbit polyclonal
antibodies
(17) ; both antibodies immunoblotted at 1:300
dilution. Rabbit antisera against p70
and their use have
been described
(18) . Insulin was from Calbiochem (San Diego,
CA).
Immunoblotting
Proteins were denatured by boiling
in Laemmli sample buffer containing 100 mM dithiothreitol and
resolved by SDS-PAGE. Gels were transferred to nitrocellulose membranes
(Schleicher & Schuell) in Towbin buffer containing 0.02% SDS and
20% methanol
(19) . Membranes were blocked, probed, and developed
as described previously, and visualized using I-Protein A
(Amersham Corp.) or horseradish peroxidase-conjugated goat anti-mouse
secondary antibodies (Cappel) and the Renaissance system (DuPont
NEN)
(3, 18) . Blots were exposed to Kodak X-AR film or
imaged on a Molecular Dynamics PhosphorImager.
Metabolic Labeling of CHO
Quiescent CHOCells
cell lines were washed
twice with phosphate-free RPMI 1640 medium and incubated for 3 h in
phosphate-free RPMI 1640 medium supplemented with 0.2 mCi/ml
[
P]orthophosphate (DuPont NEN). Cells were
stimulated with 100 nM insulin for 10 min and lysed in
ice-cold 100 mM Tris-HCl, pH 7.4, containing 1% Triton X-100,
100 mM NaF, 10 mM sodium pyrophosphate, 2 mM
sodium orthovanadate, 5 mM EDTA, 1 mM
phenylmethylsulfonyl fluoride, and 10 mg/ml each of leupeptin and
aprotinin. Insoluble material was removed by centrifugation at 10,000
g for 10 min, and
IRS-1 antibodies were added for
1 h at 4 °C. Immune complexes were collected on Protein A-Sepharose
(Pharmacia Biotech Inc.) and washed three times in ice-cold 50
mM HEPES, pH 7.4, containing 1% Triton X-100, 150 mM
NaCl, 100 mM NaF, and 2 mM sodium orthovanadate.
Immune complexes were resolved by 7.5% SDS-PAGE and visualized on a
Molecular Dynamics PhosphorImager.
PI 3`-Kinase, p70
In vitro phosphorylation of
phosphatidylinositol was carried out in immune complexes as described
previously and quantitated on a Molecular Dynamics
PhosphorImager
(3, 20) . In vitro kinase assays
for p70, and Mitogenesis
Assays
were carried out as described previously and
quantitated on a PhosphorImager (5, 21). Mitogenesis was measured by
thymidine incorporation into DNA as described
previously
(2, 3, 5) .
RESULTS AND DISCUSSION
Alignment of the NH terminus of IRS-1 with
various signaling proteins reveals a PH domain between residues 13 and
115
(7, 12) . The function of this PH domain was
investigated by deleting it (in-frame) from the IRS-1 cDNA
(IRS-1
) (Fig. 1A). IRS-1 and
IRS-1
were expressed to equivalent levels in 32D
cells and in CHO
and 32D
cells overexpressing
the insulin receptor. The 32D cells were used because they do not
contain endogenous IRS-signaling proteins. IRS-1
migrated at the expected molecular mass in all of the cell lines
during SDS-PAGE, suggesting that it was stably expressed and Ser/Thr
phosphorylated in a manner similar to wild-type IRS-1
(Fig. 1B).
Figure 1:
PH
domain deletion IRS-1. A, a linear
model of IRS-1 with the location of interesting domains and potential
tyrosine phosphorylation sites is shown. The PH domain of IRS-1 extends
from amino acids 13-115 (1, 7). Amino acids 6-155 are
removed from IRS-1
. The PH domain (IRS homology
region (IH)-1) and another region (IH2) share high identity between
IRS-1 and IRS-2 (see footnote 2). The location of potential tyrosine
phosphorylation sites is shown; asterisks indicate known sites
of tyrosine phosphorylation (25). B, parental
CHO
, 32D, or 32D
cells and CHO
,
32D, and 32D
cell lines expressing IRS-1 or
IRS-1
were lysed, resolved by SDS-PAGE, and
immunoblotted with
IRS-1 antibodies. Note, although endogenous
IRS-1 was detectable in CHO
cells on long exposure, it is
not seen in the figure as a consequence of the short exposure needed to
prevent overexposing the exogenous IRS-1 isoforms. No endogenous IRS-1
was detectable in 32D cell lines, as reported previously (2, 3).
Migration of molecular size standards, IRS-1, and IRS-1
is indicated.
Insulin stimulated the expected tyrosyl
phosphorylation of the insulin receptor -subunit in
CHO
, CHO
/IRS-1, and
CHO
/IRS-1
cells
(Fig. 2A). During insulin stimulation, tyrosine
phosphorylation of endogenous IRS-1 was clearly observed in CHO
cells, and overexpression of IRS-1 in these cells resulted in a
striking increase, as described previously
(4) . However,
insulin-stimulated tyrosine phosphorylation of IRS-1
was greatly reduced (>95%) in
CHO
/IRS-1
cells. Normal tyrosine
phosphorylation of endogenous IRS-1 was detected in the
CHO
/IRS-1
cells during insulin
stimulation (Fig. 2A). Thus, deletion of the PH domain
from IRS-1 abrogates its tyrosine phosphorylation in CHO
cells but does not interfere with the phosphorylation of the
endogenous wild-type IRS-1.
Figure 2:
Phosphorylation of IRS-1
in CHO
cells. A, quiescent CHO
cell
lines were incubated in the absence or presence of 100 nM
insulin for 5 min and lysed. Lysates were resolved by SDS-PAGE, and
tyrosyl phosphoproteins were analyzed by immunoblotting with
PY.
B, quiescent CHO
cell lines were labeled for 3 h
with [
P]orthophosphate and incubated in the
absence or presence of 100 nM insulin for 10 min. Cells were
lysed and immunoprecipitated with
IRS-1. Immunoprecipitates were
resolved by SDS-PAGE and imaged on a Molecular Dynamics PhosphorImager.
Phosphoamino acid analysis confirmed the presence of Ser(P) and Thr(P)
on all IRS-1 isotypes, but significant amounts of Tyr(P) were found
only in wild-type IRS-1 (not shown). The migration of IRS-1,
IRS-1
, IRS-1
, and molecular size
standards is indicated. These experiments are representative of
multiple independent assays performed with multiple independently
derived clones.
We investigated the Ser/Thr
phosphorylation of IRS-1 and IRS-1 in CHO
cells by monitoring levels of [
P]phosphate
incorporated during insulin stimulation (Fig. 2B).
Solely as a control for normal levels of Ser/Thr phosphorylation, we
included another IRS-1 mutant, IRS-1
, which contains an
intact PH domain but is not tyrosyl-phosphorylated due to the
substitution of phenylalanine for tyrosine in all of its putative
tyrosyl phosphorylation sites.
(
)
Insulin
stimulated the phosphorylation of IRS-1, IRS-1
, and
IRS-1
as monitored by decreased mobility and increased
[
P]phosphate content (Fig. 2B).
IRS-1 is highly serine-phosphorylated in the basal state and undergoes
tyrosine and Ser/Thr phosphorylation during insulin
stimulation
(4, 15) . Since IRS-1
and
IRS-1
are not tyrosine-phosphorylated in either the basal
state or during insulin stimulation in CHO
cells, the
phosphorylation of these species reflects only Ser/Thr phosphorylation.
Thus, IRS-1
is recognized normally by Ser/Thr
kinases, suggesting that its structure is not globally disrupted.
Therefore, removal of the PH domain interfered specifically with the
tyrosine phosphorylation of IRS-1.
by insulin
(Fig. 3C); however, disruption of the PH domain of IRS-1
completely inhibited these events in 32D cells
(Fig. 2C).
Figure 3:
Signaling by IRS-1 and
IRS-1 in 32D cells. A, quiescent 32D cell
lines were incubated in the absence or presence of insulin for 5 min
and lysed. Lysates were resolved by SDS-PAGE and analyzed by
immunoblotting with
PY antibodies. Migration of molecular size
standards and IRS-1 is indicated, along with the expected position of
IRS-1
. B, quiescent 32D cell lines were
stimulated with insulin for 5 min, lysed, and immunoprecipitated with
IRS-1 antibodies. Immunoprecipitates were washed and assayed for
associated PI 3`-kinase activity. C, quiescent 32D cell lines
were stimulated with insulin for 30 min and lysed. p70
was immunoprecipitated and its activity assayed in an in
vitro immune complex assay. Activity in B and C was quantified on a PhosphorImager and is shown as arbitrary
units. Each assay in this figure is representative of at least three
independent experiments with multiple independently derived cell
lines.
Unlike CHO cells,
overexpression of the insulin receptor in 32D
cells
restored normal insulin signaling by IRS-1
; both
IRS-1 and IRS-1
were tyrosine-phosphorylated to equal
levels in 32D
cells during insulin stimulation
(Fig. 4A). During insulin stimulation, IRS-1 and
IRS-1
associated normally with PI 3`-kinase
(Fig. 4B) and both mediated the activation of
p70
(Fig. 4C). Futhermore, insulin
stimulated mitogenesis equivalently in 32D
cells
expressing either wild-type IRS-1 or IRS-1
(Fig. 4D). Thus, overexpression of the insulin
receptor in 32D
cells restored IRS-1
phosphorylation and, with it, signaling.
Figure 4:
Signaling by IRS-1 and
IRS-1 in 32D
cells. A,
quiescent 32D
cell lines were incubated in the absence or
presence of insulin for 5 min and lysed. Lysates were resolved by
SDS-PAGE and analyzed by immunoblotting with
PY antibodies.
Migration of molecular size standards, IRS-1, IRS-1
,
and a degradation product of IRS-1 (IRS-1
) is indicated.
B, quiescent 32D
cell lines were stimulated with
insulin for 5 min, lysed, and immunoprecipitated with
IRS-1
antibodies. Immunoprecipitates were washed and assayed for associated
PI 3`-kinase activity. C, quiescent 32D
cell
lines were stimulated with insulin for 30 min and lysed. p70
was immunoprecipitated and its activity assayed in an in
vitro immune complex assay. Activity in B and C was quantified on a PhosphorImager and is shown as arbitrary
units. D, cell lines were incubated in various concentrations
of insulin for 48 h, followed by the addition of
[
H]thymidine for another 3 h. Cells were
harvested onto glass filters, and incorporated nucleotide was
quantified by scintillation counting. Each assay in this figure is
representative of at least three independent experiments with multiple
independently derived cell lines.
Our results suggest
that the PH domain is essential for sensitive coupling between IRS-1
and the insulin receptor. Despite the inability of IRS-1 molecules with
PH domain deletions to become tyrosine-phosphorylated and mediate
signaling in the low insulin receptor environment of 32D cells, these
mutations are rescued by overexpression of the insulin receptor in
32D cells. Therefore, disruption of the PH domain does not
damage the ability of IRS-1 to mediate downstream signals, as
IRS-1
is phosphorylated normally and signals
effectively in the presence of high levels of insulin receptor in
32D
cells. Since the PH domain comprises an independent
protein module
(10, 11) , it is not surprising that the
remaining portions of IRS-1
are properly folded and
retain biological function. The observation that a mutant IRS-1
molecule containing a small deletion (30 amino acids) at the beginning
of the PH domain also abrogates tyrosyl phosphorylation in 32D cells
provides further evidence that these results do not stem from the
disruption of sequences downstream of the PH domain.(
)
cells not rescue insulin-stimulated tyrosyl
phosphorylation of IRS-1
? Unlike 32D cells,
CHO cells contain endogenous IRS-1, which possesses an intact PH
domain. The PH domain must provide a significant advantage during
competition for a limited number of insulin receptors, possibly by
displacing the low affinity IRS-1
away from the
receptor.
The IH
region appears to be a
phosphotyrosine binding domain like the one in
Shc
(26, 27)
; this region binds the NPEY
motif in the insulin receptor
(6, 22) ,
and
its deletion partially inhibits IR-dependent phosphorylation (data not
shown). This supports previous results showing that the
NPXY
motif is essential for insulin signaling
and IRS-1 phosphorylation
(15, 23, 24) . The role
of the PH domain and the IH2 regions in IRS engagement by other
systems, such as IL-4 and growth hormone, remains to be determined.
Thus, the PH
domain and the IH2 regions are likely to cooperate in mediating IR/IRS
recognition. Impairment of PH domain or IH2 region interactions is
expected to alter the sensitivity of the insulin response, and could
contribute to insulin resistance.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.