From the Diabetes Branch, NIDDK, National Institutes
of Health, Bethesda, Maryland 20892 and the § Graduate
Genetics Program, George Washington University,
Washington, D. C. 20052
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ABSTRACT |
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Insulin receptor substrates (IRS) mediate
biological actions of insulin, growth factors, and cytokines. All four
mammalian IRS proteins contain pleckstrin homology (PH) and
phosphotyrosine binding (PTB) domains at their N termini. However, the
molecules diverge in their C-terminal sequences. IRS3 is considerably
shorter than IRS1, IRS2, and IRS4, and is predicted to interact with a distinct group of downstream signaling molecules. In the present study,
we investigated interactions of IRS3 with various signaling molecules.
The PTB domain of mIRS3 is necessary and sufficient for binding to the
juxtamembrane NPXpY motif of the insulin receptor in the
yeast two-hybrid system. This interaction is stronger if the PH domain
or the C-terminal phosphorylation domain is retained in the construct.
As determined in a modified yeast two-hybrid system, mIRS3 bound
strongly to the p85 subunit of phosphatidylinositol 3-kinase. Although
high affinity interaction required the presence of at least two of the
four YXXM motifs in mIRS3, there was not a requirement for
specific YXXM motifs. mIRS3 also bound to SHP2, Grb2, Nck,
and Shc, but less strongly than to p85. Studies in COS-7 cells
demonstrated that deletion of either the PH or the PTB domain abolished
insulin-stimulated phosphorylation of mIRS3. Insulin stimulation
promoted the association of mIRS3 with p85, SHP2, Nck, and Shc. Despite
weak association between mIRS3 and Grb2, this interaction was not
increased by insulin, and may not be mediated by the SH2 domain of
Grb2. Thus, in contrast to other IRS proteins, mIRS3 appears to have
greater specificity for activation of the phosphatidylinositol 3-kinase
pathway rather than the Grb2/Ras pathway.
Insulin binding to the extracellular domain of the insulin
receptor (IR)1 results in the
activation of the insulin receptor tyrosine kinase, which undergoes
autophosphorylation of tyrosine residues in its cytoplasmic domain and
subsequently phosphorylates several intracellular proteins including
insulin receptor substrates (IRS), Shc (1), Gab1 (2), pp120/HA4 (3, 4),
and p62doc (5). Upon phosphorylation, IRS
proteins serve as docking molecules for downstream signaling proteins
that contain SH2 domain(s) and therefore activate signal transduction pathways.
To date four different IRS molecules have been cloned (6-10). Recently
the roles of IRS1 and IRS2 in insulin signaling have been extensively
investigated (11). However less is known about the role of IRS3 in
insulin signal transduction (8, 9, 12-17). Sequence analysis reveals
that IRS3 has the same general architecture as IRS1, IRS2 and IRS4: a
pleckstrin homology (PH) domain at the N terminus, followed by a
phosphotyrosine binding (PTB) domain, and a C-terminal domain
containing multiple sites for tyrosine phosphorylation (designated as
the phosphorylation domain) (8, 9). Both PH and PTB domains of IRS3
exhibit a high degree of similarity to those in IRS1, IRS2, and IRS4.
Although the specific ligands for the PH domain of IRS proteins are
still unknown, the PH domains of IRS1 has been shown to be required for
its coupling to the activated insulin receptor (18-21). In studies
using the yeast two-hybrid systems, the PTB domains of IRS1 and IRS2
have been demonstrated to bind to the NPX(p)Y motif in the
juxtamembrane domain of the IR and IGF1R (22-26). The phosphorylation
domain of IRS3 is only about half the size of, and shares little if any homology with, IRS1, IRS2, and IRS4. Nevertheless, it has similar tyrosine motifs that can be phosphorylated by the insulin receptor tyrosine kinase in response to insulin stimulation, and subsequently bind to SH2 domains of signaling molecules such as p85, SHP2, and Grb2
(8, 9).
In the present study, we used the yeast two-hybrid system (27, 28) to
investigate the interaction of IRS3 with the insulin receptor, the
insulin receptor-related receptor, and the insulin-like growth factor-1
receptor and also to downstream SH2 domain-containing signaling
proteins. The minimal PTB domain of IRS3 binds relatively weakly to the
IR cytoplasmic domain, but the binding was markedly enhanced by the
presence of either the PH domain or the phosphorylation domain of IRS3.
This interaction is dependent upon the intact NPX(p)Y motif
within the juxtamembrane domain of the IR. Deletion of either the PH
domain or the PTB domain abolished the in vivo tyrosine
phosphorylation of IRS3 by the IR in COS-7 cells. In addition, by
fusing the IR kinase domain to the phosphorylation domain of IRS3, we
used the yeast two-hybrid system to study the interaction of IRS3 with
downstream SH2 domain-containing signaling proteins such as p85 subunit
of PI 3-kinase, SHP2, Grb2, Nck, and Shc. In this modified yeast
two-hybrid system, IRS3 interacts strongly with the SH2 domains of p85,
moderately with Grb2, Nck, and the SH2 domains of Shc and SHP2, but
only weakly with the SH2 domains of PLC Materials--
The yeast MATCHMAKER LexA two-hybrid system (27,
28) reagents were purchased from CLONTECH. Yeast
Saccharomyces cerevisiae strain EGY48 (MAT cDNA Plasmid Constructs--
All cDNA constructs for
yeast transformation were generated by cloning PCR products into the
yeast two-hybrid vectors pLexA or pB42AD. PCR reactions were carried
out using high fidelity Pfu DNA polymerase (Stratagene, La
Jolla, CA) and primers with appropriate restriction sites incorporated
as needed. The following cDNA clones were used as templates for PCR
reactions: expressed sequence tag clones AA111517 (GenBank accession
number, mouse IRS3), AA100052 (IGF1R), R18818 (Grb2), AA517594
(Nck Yeast Transformation and Interaction Assays--
Yeast strain
EGY48 was cotransformed with plasmid constructs by the polyethylene
glycol/lithium acetate method according to CLONTECH's protocol. Transformants were grown on
appropriate SD glucose agar plates for 3 days at 30 °C. Five to 10 independent colonies were transferred to SD galactose/raffinose agar
plates and grown overnight at 30 °C to induce the expression of B42
fusion proteins. For colony lift filter assay for Expression in COS-7 Cells and Immunoprecipitation--
Wild type
IRS3, IRS3 Expression of IRS3 in NIH 3T3 Cells--
Wild type IRS3 was
cloned into pIRES1neo (CLONTECH) and the
insert containing the IRS3, the encephalomyocarditis virus IRES, and
the neomycin phosphotransferase gene was excised with EcoRI and XbaI, blunted, and cloned into the pBPV vector to yield
pBPV-IRS3-IRES-neo. NIH 3T3 cells were transfected with
pBPV-IRS3-IRES-neo, and Geneticin-resistant cell lines were screened
for IRS3 expression. Five stable cell lines were selected and combined.
After starvation, cells were stimulated with 100 nM insulin
for 5 min, and lysed with the lysis buffer as described above.
Immnuoprecipitation was performed as described above.
Interaction of IRS3 with IR, IRR, and IGF1R--
In this study, we
used the LexA-based yeast two-hybrid system (28) to characterize
interactions of IRS3 with IR, IRR, and IGF1R. One plasmid encoded
proteins with the DNA-binding domain of LexA fused to cytoplasmic
domains of each receptor (Table I, a).
The other construct contained the transcriptional activation domain of
B42 fused either to full-length IRS3 or various fragments of the
molecule (Table I, b). Interactions between the two proteins were
assayed semiquantitatively by observing blue color in a colony filter
assay and quantitatively with
When yeast cells were cotransformed with expression vectors for both
fusion proteins, (i.e., pLexA-IR and pB42AD-IRS3), a strong
blue color developed rapidly (within minutes) in the filter assay. As
shown in Fig. 1, in the quantitative
liquid
The PTB domains of both IRS1 and IRS2 have been shown to bind the
juxtamembrane domain of IR (22-26, 31-33). In addition, IRS2 (but not
IRS1) has an additional insulin receptor binding domain named receptor
binding domain 2 (25) or kinase regulatory loop binding domain (33)
that is distinct from the PTB domain. To map the domain(s) of IRS3
required for binding to IR, we characterized deletion mutants of IRS3
(Fig. 2). As shown in Fig. 2, the minimal PTB domain of IRS3 bound weakly to IR. Neither the isolated PH domain
nor the isolated phosphorylation domain of IRS3 interacted significantly with IR. Deletion of the PTB domain completely abolished binding to IR, while removal of either the PH domain or the
phosphorylation domain had little effect. Furthermore, the receptor
tyrosine kinase domain is required for the interaction inasmuch as the
kinase inactive mutant of IR fails to bind to IRS3. Moreover, similar to what has been observed for PTB domains of IRS1 and IRS2 (22-26, 31,
33), mutations in the juxtamembrane domain NPXY motif of IR
(N957A/Y960A) abolished the interaction of the insulin receptor with
either the isolated PTB domain (data not shown) or the full-length IRS3
(Fig. 2). Thus, the PTB domain of IRS3 binds to the NPXY motif in the juxtamembrane domain of the IR, and the binding can be
greatly enhanced by the presence of either the PH domain or the
phosphorylation domain.
Phosphorylation of IRS3 in COS-7 Cells--
Epitope-tagged IRS3
expressed in COS-7 cells contained an unexpectedly high phosphotyrosine
content, even when cells were incubated in the absence of insulin (Fig.
3). Nevertheless, when the transfected
cells were incubated in the presence of insulin (100 nM),
this led to approximately 50% increase in tyrosine phosphorylation of
IRS3. Since, the results from our yeast two-hybrid system showed that
the PTB domain of IRS3 is required for binding to IR, we inquired
whether deletion of the PTB domain would alter tyrosine phosphorylation
of IRS3 in COS-7 cells. As shown in Fig. 3, wild type IRS3 and the
mutants were expressed at a similar level in the COS-7 cells. As
expected, deletion of the PTB domain abolished phosphorylation of IRS3
in both the presence and absence of insulin. Interestingly, deletion of
the PH domain also abolished tyrosine phosphorylation of IRS3, both in
the presence or absence of insulin.
Interaction of IRS3 with the SH2 Domains of the p85 Subunit of PI
3-Kinase--
Both the PH domain and the PTB domain are highly
conserved among IRS proteins, but there is little conservation of the
phosphorylation domains. For instance, the IRS3 molecule is
substantially shorter than the other three members of the family.
Therefore, it is important to study structure-function relationships
that determine the signaling specificity of IRS3. We used the yeast
two-hybrid system to investigate interactions of phosphorylated IRS3
with downstream signaling molecules. As expected, when yeast cells were
cotransformed with pLexA-IRS3 plus pB42AD-p85ncSH2, we did
not detect an interaction between IRS3 and p85 presumably due to the
lack of IRS3 phosphorylation in this system. However, constitutive
expression of c-Src in the yeast three hybrid system (34) did not
permit detection of an interaction between IRS3 and p85 (data not
shown). Presumably, these YXXM motifs in IRS3 molecules were
poor substrates for phosphorylation by c-Src. Thus, we modified the
yeast two-hybrid system so that the insulin receptor tyrosine kinase
could phosphorylate tyrosine residues in IRS3 (Fig.
4). To accomplish this, we fused the IR kinase domain with the phosphorylation domain of IRS3, and the chimera
was then fused to the LexA domain to yield
pLexA-IRk-IRS3 construct (Table I). When yeast
cells were cotransformed with pLexA-IRk-IRS3 and p85 SH2
domains (pB42AD-p85ncSH2), we could detect strong
interaction. As judged by the
Four Tyr residues of mIRS3 are located in YXXM motifs:
Tyr341, Tyr350, Tyr361, and
Tyr390 (9). We used our modified yeast two-hybrid system to
investigate which of the phosphotyrosine residues bind to the SH2
domains of p85. When phenylalanine (Phe) was substituted for all four of these Tyr residues, this essentially abolished the interaction between p85ncSH2 and IRk-IRS3 (Fig. 4). We also
investigated the effects of single, double, and triple substitutions.
In general, there was good correlation between the number of Tyr
residues that were retained and the apparent strength of the
interactions (Fig. 4). However, there were some subtle differences that
suggest there might be specificity with regard to which Tyr residues
interact with p85ncSH2. For example, single substitutions of
Phe for either Tyr341 or Tyr350 had little if
any effect upon the apparent strength of the interaction. In contrast,
substitution of Phe for either Tyr361 or Tyr390
led to a larger reduction in the apparent strength of the interaction. These subtle differences raise the possibility that Tyr361
and Tyr390 might bind p85ncSH2 more effectively
than Tyr341 and Tyr350. Indeed, when we
eliminated three of the four Tyr residues, p85ncSH2 appeared
to interact more strongly with two constructs containing either
Tyr361 or Tyr390 than the two constructs
containing only Tyr341 or Tyr350
(p < 0.05, paired Student's test). However, we did
not carry out detailed studies to rule out alternative explanations;
for example, there might be differences in the levels of expression of
the various fusion proteins, or there might be differences in the
stoichiometry of phosphorylation of the individual tyrosine residues in
the fusion proteins.
In the previous experiments, our p85 construct contained both the N-
and C-terminal SH2 domains arranged in tandem. To determine which of
the SH2 domains mediated the interaction with phosphorylated IRS3, we
tested constructs containing only one of the two SH2 domains. No
interaction between IRk-IRS3 and the individual SH2 domains
(p85nSH2 or p85cSH2) was detected using the
liquid Interaction of IRS3 with Other Proteins Containing SH2
Domains--
We used a similar approach to study the interactions of
phosphorylated IRS3 with SH2 domains derived from SHP2, Grb2, Nck, Shc,
or PLC Specificity of the Modified Yeast Two-hybrid System--
Because
IRS3 contains multiple potential sites of tyrosine phosphorylation,
this raises the question of which phosphotyrosine residues interact
with which SH2 domains. In the case of p85, our mutational studies
support the conclusion that the tandem SH2 domains interact with
phosphotyrosine residues in the context of YXXM motifs (Fig.
4). Like p85, SHP2 also contains two SH2 domains. The C terminus of
IRS3 (amino acid residues 405-495) contains at least two motifs
(Y467VDL and Y490ASI) that conform to consensus
binding motifs for the SH2 domains of SHP2 but not of p85 (35-39).
Yeast cells were co-transformed with
pLexA-IRk-IRS3ct together with vectors encoding
fusion proteins of B42 with SHP21-218,
p85ncSH2, Grb2, or Shc201-458. IRk-IRS3ct interacted strongly with
SHP21-218, as indicated by the observation that the blue
color developed within 12 h. This is consistent with the
hypothesis that the two SH2 domains of SHP2 interact with two tyrosine
motifs in the C terminus of IRS3 (i.e. Y467VDL
and Y490ASI). In contrast, the interaction of
IRk-IRS3ct was weaker with p85ncSH2
(requiring 48 h for color development) and even weaker for Grb2
and Shc201-458, both of which required 72 h for color
development (Table IV).
Association of IRS3 with SH2 Domain-containing Proteins in
Mammalian Cells--
Next, we carried out studies in COS-7 cells to
test some of the predictions derived from the yeast two-hybrid system
experiments. COS-7 cells transiently expressing myc-tagged mIRS3 were
incubated for 10 min in the presence or absence of insulin. The cell
lysates were subjected to immunoprecipitation with antibodies directed against p85, SHP2, Grb2, Nck, or Shc. The immunoprecipitates were analyzed by SDS-polyacrylamide gel electrophoresis followed by immunoblotting with anti-myc antibody. In lysates derived from insulin-stimulated cells, we detected co-immunoprecipitation of IRS3
with p85, Shc, SHP2, Nck, and Grb2; in contrast, little if any IRS3 was
coimmunoprecipitated with PLC The yeast two-hybrid system provides a powerful technique to study
protein-protein interactions. This system has been applied previously
to characterize structure-function relationships of both IRS1 and IRS2,
specifically, their interactions with IR and IGF1R (22-26, 33). We
have used a similar approach to define the domains of IRS3 that
interact with the insulin receptor, the IGF-1 receptor, and the insulin
receptor-related receptor. In addition, we have modified the yeast
two-hybrid approach to allow us to study interactions with downstream
signaling molecules that interact via their SH2 domains with
phosphotyrosine residues in IRS3. The majority of the predictions based
upon observations obtained with the yeast two-hybrid system were
confirmed by carrying out appropriate studies in mammalian cells.
Interactions of IRS3 with Receptor Tyrosine Kinases--
Using the
yeast two-hybrid system, we demonstrated that the PTB domain of IRS3
(like the PTB domains of IRS1 and IRS2) is essential for binding to the
NPX(p)Y motif within the juxtamembrane domain of the insulin
receptor in a phosphorylation-dependent manner. However,
the isolated minimal PTB domain binds weakly to the IR, and this
binding is markedly enhanced by the presence of either the PH domain or
the phosphorylation domain. On the other hand, neither the isolated PH
domain nor the isolated phosphorylation domain could interact directly
with the IR cytoplasmic domain. Since the sequence downstream of the
minimal PTB domain in IRS3 has no detectable homology with IRS1, IRS2,
or IRS4, we suggest that the PTB domain of IRS3 is the only domain that
binds directly to IR. Rather, the effects of both the PH and
phosphorylation domains to promote the interaction of PTB domain with
the IR may be explained by favorable effects upon either the folding of
the fusion protein or the stability of the molecule.
As predicted by the yeast two-hybrid system, deletion of the PTB domain
prevented insulin-stimulated phosphorylation of IRS3 in COS-7 cells
(Fig. 3). Furthermore, deletion of the PH domain also inhibited
phosphorylation of IRS3. Similar results were reported previously in
studies in which the PH domain of IRS1 was deleted or replaced with PH
domains derived from proteins other than IRS family members (18-21).
Thus, both the PH domain and the PTB domain or IRS3 are required for
normal phosphorylation of IRS3 by the insulin receptor in mammalian cells.
Multiple factors may contribute to determining which IRS molecules are
phosphorylated by specific receptor tyrosine kinases. Thus, we compared
the strength of the interactions between IRS3 and various members of
the insulin receptor family of tyrosine kinases. The interaction of
IRS3 was approximately twice as strong with the IR or IGF1R as with
IRR. In addition, we compared the strength of the interaction of the IR
with IRS1, IRS2, and IRS3. In the yeast two-hybrid system, the
interaction of IR with IRS2 or IRS3 was approximately twice as strong
as with IRS1. These data are consistent with observations with
endogenous IRS proteins in rat adipocytes. Rat adipocyte IRS3 binds
more strongly than IRS1 to immobilized peptides containing
phosphorylated NPX(p)Y motifs (15). Furthermore, IRS3 was
phosphorylated more rapidly than IRS1 in rat adipocytes (15). In
contrast, although the yeast two-hybrid system suggests that IR binds
to IRS2 with greater affinity than to IRS1, IRS1 appears to be
phosphorylated more heavily than IRS2 in rat adipocytes (15, 16, 40).
It is likely that this discrepancy is accounted for by
differences in the level of expression of the two
substrates. However, it is possible that other factors
(e.g. subcellular localization) might also contribute.
Interaction of IRS3 with Downstream Effector Molecules--
It is
necessary to modify the yeast two-hybrid system to analyze an
interaction between proteins if the interaction requires one of the
proteins to undergo post-translational modification (in this case,
phosphorylation). For example, in the yeast three-hybrid system (34,
41-44), expression of a tyrosine kinase has enabled screening of
libraries for tyrosine phosphorylation-dependent interactions between proteins (41, 44), and studying tyrosine phosphorylation-dependent interactions between two proteins
(34). To our knowledge, the yeast three-hybrid system has not been
applied to study the interaction of IRS proteins with downstream
signaling proteins containing SH2 domains. In the present study, we
utilized a modified yeast two-hybrid system. Rather than express a
separate tyrosine kinase to phosphorylate the substrate, we fused the
phosphorylation domain of IRS3 directly to the IR kinase domain. This
chimera was fused to the C terminus of the transcription activation
domain LexA to yield a chimeric LexA-IRk-IRS3ct
fusion. Both the LexAop-LacZ gene in the p8op-LacZ plasmid
vector and the LexAop-LEU2 gene in the yeast EGY48
chromosome allow multiple LexA fusion proteins to bind to the LexA
operators simultaneously. This binding results in activation of the IR
kinase, possibly by phosphorylation in an intramolecular manner (22).
The activated IR kinase subsequently phosphorylates these tyrosine
motifs in IRS3 allowing their binding to the SH2 domain of B42 fusion
proteins. Eventually, the transcription of both the
LexAop-LEU2 and LexAop-LacZ genes is activated,
allowing the yeast cells to grow in leucine-deficient plates and
express
Using this modified yeast two-hybrid system, we demonstrated that
tyrosine phosphorylated IRS3 binds to the SH2 domains of the p85
subunit of PI 3-kinase, SHP2, Nck, and Grb2, but not PLC-
It has been demonstrated that Grb2 can associate with IRS3 in rat
adipose cells (14, 17) and rat hepatoma cells overexpressing insulin
receptor (HTC-IR) (14).2 It
has been suggested that Grb2 may bind to a putative binding motif
(i.e. Y321VNP) in rat IRS3 (8). Unlike rat IRS3,
mouse IRS3 does not possess an obvious motif for binding to Grb2 (9).
The corresponding sequence in mouse IRS3 is Y319FKP, which
does not conform to the consensus sequence for Grb2 binding. Consistent
with the absence of an obvious binding site, insulin did not enhance
the interaction of recombinant mIRS3 with Grb2 in COS-7 cells. However,
we did detect a weak interaction between Grb2 and IRS3 in cells
incubated in the absence of insulin. Because of the presence of
proline-rich sequences in IRS3, it seems likely that the
insulin-independent interaction may involve the SH3 domain of Grb2.
However, it is also possible that insulin-independent tyrosine
kinases may have phosphorylated IRS3 at low stoichiometry, and this
could explain the low level interaction of IRS3 and Grb2 observed in
COS-7 cells in the absence of insulin.
SHP2 is a phosphotyrosine phosphatase containing two tandem SH2
domains. Its SH2 domains bind preferentially to phosphotyrosines in
(p)Y-hydrophobic-X-hydrophobic motifs, such as
Y-(I/V)-X-(V/I/L/P) (35). In IRS1, the SHP2 binding motif
was previously demonstrated to be Y1172IDL (36, 47). More
recently it has been demonstrated that both Y1172IDL and
Y1222ASI motifs in IRS1 are necessary for bivalent high
affinity binding to the tandem SH2 domains of SHP2 (37-39). Originally
the Y467VDL motif in mouse IRS3 and the Y466VDL
motif in rat IRS3 were suggested to bind SHP2 (8, 9). Recently we
noticed that both mouse and rat IRS3 have an additional potential SHP2
binding motif (i.e. YASI) located in the
C-terminal tail. Our results from the yeast two-hybrid system and
immunoprecipitation clearly indicate that SHP2 binds to IRS3 with
considerable affinity. Other groups have also demonstrated that SHP2
could associate with IRS3 in rat adipose cells (14, 17) and rat
hepatoma cells overexpressing insulin receptor (HTC-IR) (14). We
propose that the two tandem SH2 domains in SHP2 can simultaneous bind
to the YVDL and YASI motifs in IRS3 when both tyrosine residues are
phosphorylated. However we cannot exclude the possibility that other
tyrosine motifs in IRS3 also participate in SHP2 binding.
Role of IRS3 in Intracellular Signaling--
The existence of
multiple IRS molecules raises the interesting question of whether each
IRS molecule serves a distinct function. All four molecules (IRS1,
IRS2, IRS3, and IRS4) possess homologous PH and PTB domains. These
domains appear to be required for bind to the receptors, thereby
permitting efficient phosphorylation of the substrate molecule.
However, IRS proteins differ in several respects. First, each IRS
protein has a characteristic pattern of expression in specific tissues
and at specific times of development. Second, it has been suggested
that there may be differences in the subcellular localization of
individual IRS molecules (48, 49). Finally, IRS proteins differ in
their C-terminal sequences. Several tyrosine phosphorylation sites seem
to be conserved in all four IRS molecules (e.g.
YXXM motifs that provide binding sites for PI 3-kinase).
This shared structural motif correlates with the observation that all
four IRS proteins are capable of mediating the metabolic actions of
insulin in rat adipose cells (45). However, there appears to be some
specificity with respect to interactions with other downstream
proteins. For example, unlike other IRS proteins, murine IRS3 does not
appear to possess a binding motif for the SH2 domain of Grb2.
Accordingly, IRS3 might lack the ability to mediate the activation of
Ras, and therefore might be predicted to be specialized in triggering
the metabolic actions of insulin rather than mediating mitogenic activity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1. These interactions
required intact kinase activity of the insulin receptor. Moreover, the
high affinity binding of IRS3 with p85 required at least two intact
YXXM motifs in IRS3 and two tandem SH2 domains in p85.
Furthermore, using an immunoprecipitation approach, we showed that
insulin stimulated the association of IRS3 with p85, Nck, SHP2, Shc but
not with PLC
1 or Grb2 in COS-7 cells.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
trp1, his3, ura3, 6LexAop-LEU2, LYS2)
pretransformed with p8op-lacZ was used as the host for the in
vivo interaction studies.
), W53926 (SHP2) and AA081539 (p66Shc), rat PLC
1 cDNA (a
gift from Dr. S. G. Rhee), and bovine PI 3-kinase p85
(from Dr.
K. Hara). Human pB42AD-IRS1 and mouse pB42AD-IRS2 constructs were generous gifts from Dr. Thomas A. Gustafson. To generate IRK-IRS3 chimeric constructs, IRS3 YXXM mutants were digested with
BglII and SalI, and the inserts were cloned into
the pLexA-IRk construct such that the C-terminal sequence of
IRS3 (amino acids 338-495) was fused in-frame to the IR kinase domain
(amino acids 941-1271). The PCR products were cloned into the yeast
two-hybrid vectors pLexA or pB42AD as listed in Table I. The IRS3
YXXM motif Tyr
Phe point mutation mutants,
IRS3
PH, IRS3
PTB, IR juxtamembrane NPXY motif mutant (N957A/Y960A; Ullrich numbering; Ref. 29) and IR kinase-dead mutant (K1018A) were generated by site-directed mutagenesis. The detailed cloning and mutagenesis strategies for the
above constructs are available upon request. All constructs were
verified by sequencing using an Applied Biosystems Inc. Prism 377 DNA
automatic sequencer.
-galactosidase
activity, the colonies were transferred to Whatman 3MM filter paper and frozen in liquid nitrogen for 10-15 s, placed on top of another filter
that was presoaked with 5 ml of Z-buffer/X-gal/2-mercaptoethanol (60 mM Na2HPO4·7H2O, 40 mM NaH2PO4·H2O, 100 mM KCl, 1 mM
MgSO4·7H2O, pH 7.0, 0.27% (v/v)
2-mercapthoethanol and 0.334% (w/v X-gal), and incubated at 30 °C.
For the liquid
-galactosidase activity assay, at least
six independent yeast colonies were picked and cultured in appropriate
SD medium with galactose/raffinose (in which the glucose was replaced
by galactose/raffinose as carbon source) overnight with shaking at
30 °C. The culture was diluted in the same medium and regrown for
additional 3 h at 30 °C. Yeast cells were harvested by low
speed centrifugation, suspended in Z-buffer, and lysed by three cycles
of freezing in liquid nitrogen, followed by thawing at 37 °C. The
-galactosidase activity was measured using
o-nitrophenyl
-D-galactoside as substrate,
and the results were expressed as Miller units (30) according to the
CLONTECH protocol. The liquid
-galactosidase
activity was defined arbitrarily as 100% for the interaction between
IR and IRS3. The LacZ activity assay on Gal/Raf/-UWHL/X-gal plates,
which is based on the activation of both LEU2 and LacZ reporter genes, was used to assess the in vivo interaction of IRS3 with SH2
domain-containing proteins. In this assay at least six independent
yeast colonies on Glu/-UWH plates were streaked onto
Gal/Raf/-UWHL/X-gal plates (which were the defined minimal dropout
plates with 2% galactose and 1% raffinose as a carbon source, with
X-gal but without uracil, histidine, tryptophan, and leucine). The
plates lacked uracil, tryptophan, and histidine to maintain the
p8op-LacZ, pB42AD fusion, and pLexA fusion plasmids and lacked leucine
to permit only leucine prototropic colonies to grow. The plates were
incubated at 30 °C and examined for growth (leucine prototropy) and
blue color development in yeast colonies (LacZ gene activation) over
the next few days.
PH, and IRS3
PTB were subcloned
into pCDNA3-myc-his A (Invitrogen), which provides the constructs with an in-frame myc tag at the C terminus, to yield pCDNA3.1-IRS3, pCDNA3.1-IRS3
PH, and
pCDNA3.1-IRS3
PTB, respectively. COS-7 cells were
transfected with DNA using LipofectAMINE Plus (Life Technologies,
Inc.). 16 h after transfection, cells were starved for 4 h,
then stimulated with 100 nM insulin, and lysed with lysis
buffer (20 mM Tris-HCl, pH7.5, 150 mM NaCl,
10% glycerol, 1% Triton X-100, 1 mM EDTA, 10 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, and 1 µg/ml each of
aprotinin, leupeptin and pepstatin). For immunoprecipitation, lysates
were incubated overnight with appropriate antibodies, and the immune
complexes were captured with protein A (or plus protein G)-agarose
beads (Amersham Pharmacia Biotech). After washing once with lysis
buffer and three times with phosphate-buffered saline buffer, the beads were boiled in SDS-sample buffer. Proteins were separated by
SDS-polyacrylamide gel electrophoresis, and transferred to
polyvinylidene difluoride membrane. Western blotting was performed
using either anti-myc (9E10, Santa Cruz Biotechnology) or
anti-phosphotyrosine (4G10, Upstate Biotechnology, Inc.) antibodies.
Bands were visualized using SuperSignal chemiluminescent substrates (Pierce).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase assays (see "Experimental Procedures").
cDNA constructs
-galactosidase assay, the interaction of IR with IRS3 was
comparable to the interaction with IRS2, but approximately twice as
strong as the interaction with IRS1. Relative to the interaction of
IRS3 with IR,
-galactosidase activity was 110% and 47% in yeast
transformed with pB42AD-IRS3 and either pLexA-IGF1R or pLexA-IRR,
respectively. As negative control, when yeast cells were transformed
with a single construct of either IR or IRS3, the
-galactosidase
activity was negligible (about 2% of maximal activity in yeast
co-transformed with pB42AD-IRS3 and pLexA-IR; Fig. 1). Similarly when
yeast cells were co-transformed with a LexA or a B42 construct (with
insert) paired with the appropriate empty expression vector, the
-galactosidase activity was barely detectable (data not shown).
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Fig. 1.
IRS3 interacts with IR, IGF1R, and IRR.
The entire cytoplasmic domain of the IR, IGF1R, or IRR was fused to the
LexA DNA-binding domain in the pLexA vector. Various insulin receptor
substrates were fused to the B42 activation domain in the pB42AD
vector. The interactions were assessed using a liquid -galactosidase
assay. The data (Miller units) were normalized to the interaction of IR
with IRS3 (as 100%).
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Fig. 2.
The PTB domain of IRS3 interacts with the
NPX(p)Y motif in the IR. These experiments were
carried out as in Fig. 2 with various deletion mutants of IRS3. In
addition, the kinase-dead mutant of IR (IRKD)
and an IR mutant in which the juxtamembrane tyrosine phosphorylation
site was mutated (IRJMm) were included in this
experiment.
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Fig. 3.
Phosphorylation of IRS3 in COS-7 cells
requires both intact PH and PTB domains. COS-7 cells were
transfected with vector pCDNA3.1,
pCDNA3.1-IRS3 PH,
pCDNA3.1-IRS3
PTB, or pCDNA3.1-IRS3. After
starvation, cells were treated with or without insulin for 10 min. Cell
lysates were analyzed by SDS-polyacrylamide gel electrophoresis,
followed by immunoblotting with antibodies
-myc (9E10) and
-pY
(4G10) antibodies.
-galactosidase assay, the interaction
was 60% as strong as the interaction between the IR cytoplasmic domain
and full-length IRS3 (Fig. 4). In contrast, p85ncSH2 did not
interact directly with the isolated IR kinase domain when it was not
fused to the IRS3 phosphorylation domain. Taken together, these
observations demonstrate that the tandem SH2 domains of p85 bind to
phosphotyrosine residues in the phosphorylation domain of IRS3.
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Fig. 4.
At least two YXXM motifs in
IRS3 are required for the strong interaction of IRS3 with p85. The
IR kinase domain was fused to the phosphorylation domain of various
IRS3 YXXM mutants as described in Table I, b. The
interaction was analyzed using a liquid -galactosidase assay with
o-nitrophenyl
-D-galactoside as the
substrate. The data (Miller units) were normalized to the interaction
of IR with IRS3 (as 100%).
-galactosidase assay. Therefore, both SH2 domains of p85 must
be present to permit high affinity interaction with IRS3. However, by
increasing the assay sensitivity, we succeeded in demonstrating a low
affinity interaction of IRS3 with the individual SH2 domains. In this
method, we combined a genetic selection for the ability to grow in the absence of leucine with an assay for
-galactosidase activity. If
yeast cells were co-transformed with plasmids encoding interacting proteins, this favored growth on leucine-deficient plates, and also led
to the expression of
-galactosidase activity. This combination (i.e. a higher yield of yeast cells plus a higher specific
activity of
-galactosidase) amplified the signal, thereby increasing
the sensitivity of the assay for low affinity interactions. Using this
modified assay system, we transformed yeast with two plasmids: pLexA-IRk-IRS3cp (a plasmid encoding a fusion
protein containing the IR kinase domain plus amino acid residues
338-495 of IRS3) and pB42AD-p85ncSH2 (a plasmid encoding a
fusion protein containing both the N-terminal and C-terminal SH2
domains of p85) (Table II). In yeast
expressing both proteins (i.e. on galactose plates), a blue
color was detectable within 12 h of incubation. In contrast, when
only one protein was expressed (i.e. on glucose plates), the
yeast colonies remained white for at least 72 h of incubation. When plasmids encoding fusion proteins with only one SH2 domain (pB42AD-p85nSH2 or pB42AD-p85cSH2) were
substituted in the experiment, then 48 h were required for the
development of visible blue color. Control experiments provided
evidence for the specificity of the assay. We did not detect the
development of a blue color, even after 48 h in yeast transformed
singly with any of the following constructs in the absence of an
interacting construct: pLexA-IRk; pB42AD-p85nSH2,
or pB42AD-p85cSH2. Furthermore, in the absence of the
additional phosphorylation sites derived from IRS3, the IR kinase
domain did not interact directly with either the single SH2 domains of
p85 (p85nSH2 or p85cSH2) or both SH2 domains
arranged in tandem (p85ncSH2). Finally, receptor tyrosine
kinase activity is required for the interaction of IRk-IRS3
with the tandem SH2 domains (p85ncSH2) (Table II).
IRS3 interacts more strongly with tandem SH2 domains than with
individual SH2 domains
1. In yeast co-expressing the IRk-IRS3cp
fusion protein together with fusion proteins containing SH2 domains for
SHP2, Grb2, Nck, and Shc, we did detect evidence for interaction (Table III). Because yeast expressing the
IRk domain did not interact with any of the constructs, we
conclude that the SH2 domains do not bind directly to phosphotyrosine
residues in the insulin receptor kinase domain. Assuming that the
length of time required to develop blue color is inversely related to
the affinity of the interaction, we conclude that IRS3 binds strongly
to the tandem SH2 domains of p85, and less strongly to
SHP21-218, Grb2, Nck, and Shc201-458, but we
did not obtain convincing evidence for an interaction with PLC
1547-853.
The modified yeast two-hybrid system can be used to study the
interaction of IRS3 with other SH2 domain-containing proteins
-1. The interaction assays were carried out
as described in Table IV.
Specificity of the modified yeast two-hybrid system
1 (Fig.
5). In some cases (Nck, SHP2, and Shc),
there was a large effect of insulin. The large magnitude of the effect
is probably accounted for by the fact that only low levels of IRS3 were
co-immunoprecipitated from lysates of cells incubated in the absence of
insulin. In the case of p85, considerable quantities of p85 were
associated with IRS3 even in the absence of insulin stimulation so that
insulin exerted a relatively small effect to further increase the
association of p85 with IRS3. In contrast, insulin did not increase the
relatively low level association of Grb2 with IRS3. As noted
previously, mIRS3 does not possess an obvious binding site for the SH2
domain of Grb2 (9). Thus, it seems likely that the insulin-independent association between Grb2 and mIRS3 is not mediated by the SH2 domain of
Grb2, but more likely by another mechanism (e.g. possibly via the SH3 domains of Grb2). In addition, myc-tagged IRS3 obtained from lysates of insulin stimulated COS-7 cells could be "pulled down" by GST fusions of p85ncSH2, p85nSH2,
p85cSH2, SHP21-218, Grb2, Nck, and
Shc201-458, but not by GST-PLC
1547-853 (data not shown). Finally, when immunoprecipitation was carried out
using stable NIH3T3 cell lines that overexpress IRS3, we have observed
similar patterns of association between IRS3 and above-mentioned SH2
domain-containing proteins (data not shown).
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Fig. 5.
Coimmunoprecipitation of IRS3 with p85, SHP2,
Grb2, Shc, and Nck in COS-7 cells. COS-7 cells were transfected
with myc-tagged IRS3, and the lysates were immunoprecipitated with
antibodies against p85, SHP2, Grb2, PLC 1, Shc, and Nck. Anti-myc
(9E10) antibody was used for immunoblotting. The figure shows the
region of the immunoblot corresponding to myc-tagged IRS3.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase activity, respectively.
1. This is
consistent with other data obtained in mammalian cells obtained in this
and other studies (12-17, 45). Wild type IRS3 interacts strongly with
the p85 SH2 domains via four tandem YXXM motifs in the
phosphorylation domain of IRS3. By substituting Phe for Tyr residues
within these YXXM motifs, we demonstrated that at least two
intact tandem YXXM motifs in IRS3 were required for high
affinity binding to the tandem SH2 domains of p85, and that the
individual SH2 domains of p85 alone were not sufficient to bind
strongly to wild type IRS3. This is in agreement with the conclusion
from other groups that at least two tyrosine phosphorylation sites are
necessary to confer high affinity binding of tandem SH2 domains from
several signaling molecules such as p85, SHP2, ZAP-70, Syk, and PLC
1
(38, 39, 46). Our data indicate that Tyr361 and
Tyr390 in IRS3 confer higher binding affinity for SH2
domains of p85 than Tyr341 and Tyr350, as
mutation of either Tyr361 or Tyr390 has a
greater adverse impact than mutation of Tyr341 and
Tyr350 upon the interaction of IRS3 with the SH2 domains of
p85. It is noteworthy that Tyr341 is contained in the
sequence YITM (lacking a Met residue at the +1 position). In contrast,
Tyr350, Tyr361, and Tyr390 are
contained in YMXM motifs that conform to the optimal motif (Y(M/V)XM) to bind the SH2 domain of p85. In addition,
Ottinger et al. demonstrated that the spacing between two
YXXM motifs is not critical for high affinity binding of
tandem SH2 domains in p85. For example, in comparing bisphosphoryl
peptides containing two pYXXM motifs, the length of the
sequence separating the two pTyr residues did not affect binding to the
tandem SH2 domains in p85. Accordingly, we speculate that in the
mammalian cells p85 could bind to any pair of YXXM motifs in
IRS3 with high affinity, although the relative contribution of each
YXXM motifs remains unknown. Given that a large portion of
phosphorylated IRS3 binds to p85 in mammalian cells (12-17, 45), we
postulate that at least two if not all four YXXM motifs are
phosphorylated rapidly by the insulin receptor upon insulin
stimulation. Subsequently, these phosphorylated YXXM motifs
serve as high affinity sites for the simultaneous binding of tandem p85
SH2 domains.
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ACKNOWLEDGEMENTS |
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We thank Dr. Thomas Gustafson (Metabolex Inc,
CA) for the generous gifts of pB42AD-IRS1 and pB42AD-IRS2 constructs,
and Dr. K. Hara for the PI 3-kinase pSR-p85 cDNA construct. We
thank George Poy for oligoprimer synthesis and automated DNA sequencing.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: NIDDK, National Institutes of Health, Bldg. 10, Rm. 9S-213, Bethesda, MD 20892. Tel.: 301-496-4658; Fax: 301-402-0573; E-mail: simeon_taylor{at}nih.gov.
1
1 The abbreviations used are: IR, insulin
receptor; IRR, insulin receptor-related receptor; IGF1R, insulin-like
growth factor-1 receptor; IRS, insulin receptor substrate; PH,
pleckstrin homology; PTB, phosphotyrosine binding; SH2, Src-homology 2;
SD, synthetic dropout; X-gal,
5-bromo-4-chloro-3-indolyl--D-galactoside; Raf, raffinose; PI, phosphatidylinositol; PLC, phospholipase; PCR, polymerase chain reaction; IRES, internal ribosome entry site.
2 The 60-kDa protein in rat hepatoma HTC-IR cells was recently identified as IRS3 by immunological approaches by Dr. G. E. Lienhard's group (personal communication).
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REFERENCES |
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