A Novel Insulin Receptor Substrate Protein, xIRS-u, Potentiates Insulin Signaling: Functional Importance of Its Pleckstrin Homology Domain
Nicholas Ohan,
Mustafa Bayaa,
Parul Kumar,
Li Zhu and
X. Johné Liu
Ottawa Civic Hospital Loeb Research Institute Ottawa Civic
Hospital Department of Biochemistry (M.B., X.J.L.) Department
of Obstetrics & Gynaecology (X.J.L.) University of Ottawa
Ottawa, K1Y 4E9 Canada
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ABSTRACT
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A novel Xenopus insulin receptor
substrate cDNA was isolated by hybridization screening using the rat
insulin receptor substrate-1 (IRS-1) cDNA as a probe. The xIRS-u cDNA
encodes an open reading frame of 1003 amino acids including a putative
amino-terminal pleckstrin homology (PH) domain and
phosphotyrosine-binding (PTB) domain. The carboxy terminus of xIRS-u
contains several potential Src homology 2 (SH2)-binding sites, five of
which are in the context of YM/LXM (presumptive binding sites for
phosphatidylinositol 3-kinase). It also contains a putative binding
site for Grb2 (YINID). Pair-wise amino acid sequence comparisons with
the previously identified xIRS-1 and the four members of the mammalian
IRS family (1 through 4) indicated that xIRS-u has similar overall
sequence homology (3345% identity) to all mammalian IRS proteins. In
contrast, the previously isolated xIRS-1 is particularly similar (67%
identical) to IRS-1 and considerably less similar (3146%) to the
other IRS family members (2 through 4). xIRS-u is also distinct from
xIRS-1, having an overall sequence identity of 47%. These sequence
analyses suggest that xIRS-u is a novel member of the IRS family rather
than a Xenopus homolog of an existing member.
Microinjection of mRNA encoding a Myc-tagged xIRS-u into
Xenopus oocytes resulted in the expression of a 120-kDa
protein (including 5 copies of the 13-amino acid Myc tag). The
injection of xIRS-u mRNA accelerated insulin-induced MAP kinase
activation with a concomitant acceleration of insulin-induced oocyte
maturation. An amino-terminal deletion of the PH domain (xIRS-u
PH)
significantly reduced the ability of xIRS-u to potentiate insulin
signaling. In contrast to the full-length protein, injection of xIRS-u
(1299), which encoded the PH and PTB domain, or xIRS-u (1170),
which encoded only the PH domain, blocked insulin signaling in
Xenopus oocytes. Finally, xIRS-u (119299), which had a
truncated PH domain and an intact PTB domain, had no effect on insulin
signaling. This is the first report that the PH domain of an IRS
protein can function in a dominant negative manner to inhibit insulin
signaling.
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INTRODUCTION
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Insulin receptor substrate-1 (IRS-1) was first identified as a
major target of insulin receptor tyrosine kinase (1, 2). Recently,
several additional members of the IRS family have been identified and
cloned (3, 4, 5, 6, 7). All known members of the IRS family share a common
structural arrangement. They contain a pleckstrin homology (PH) domain
at the extreme amino terminus, followed by a phosphotyrosine-binding
(PTB) domain. In contrast, other substrates of the insulin receptor,
such as Shc (8), Gap-1 (9), and p62dok(10, 11), lack these
tandem PH-PTB domains. The carboxy-terminal halves of IRS proteins
contain multiple potential tyrosine phosphorylation sites implicated in
binding various Src homology 2 (SH2) domain-containing proteins. In
particular, each IRS family member contains several potential tyrosine
phosphorylation sites in the context of YM/LXM (X standing for any
amino acid). When phosphorylated on the tyrosine residue, these motifs
constitute binding sites for phosphatidylinositol 3-kinase (PI
3-kinase) (12).
The best studied member of this putative gene family is IRS-1. IRS-1 is
phosphorylated on multiple tyrosine residues after insulin stimulation.
Tyrosine-phosphorylated IRS-1 binds several signaling proteins through
their respective SH2 domains. These include PI 3-kinase (2), the
Grb2/mSos complex that activates Ras (13), and a phosphotyrosine
phosphatase SHP2 (14). Binding to PI 3-kinase or SHP2 results in
activation of the respective enzymes (15, 16). IRS-1 binding to
Grb2/mSos seems to be involved in the localization of the Ras activator
to the membrane (13, 17). The IRS-1 PH domain sensitizes IRS-1 to
tyrosine phosphorylation by the insulin receptor (18, 19, 20, 21). How
this is accomplished remains unclear. The IRS-1 PTB domain
seems to be responsible for mediating the interaction between IRS-1 and
the ligand-activated, autophosphorylated insulin receptor through an
autophosphorylation site (NPEpY-960) in the juxtamembrane region of the
receptor. This interaction may be important for activated insulin
receptor to recognize IRS-1 as a substrate, since mutation of this
autophosphorylation site (22) [and the equivalent tyrosine-950 of the
insulin-like growth factor-I receptor (23)] specifically abolishes
phosphorylation of IRS-1. The Shc and IRS-1 NPXpY binding domain (SAIN)
of IRS-1 (amino acids 313462), which resides carboxy-terminal to the
PTB domain, also contributes to recognizing the insulin receptor NPEpY
autophosphorylation site (24, 25). Although IRS-2 appears to have an
equivalent SAIN domain (26), the two new members of the IRS family,
IRS-3 and IRS-4, lack any sequence homology to the IRS-1 SAIN domain
(6, 7).
We have previously isolated a Xenopus IRS cDNA using rat
IRS-1 as a probe. This cDNA, originally designated as xIRS-L
(IRS-1-like), most likely represents the amphibian IRS-1 (see below)
and therefore is referred to hereafter as xIRS-1. In further cDNA
screenings, we isolated several related but distinct cDNAs from a
Xenopus oocyte cDNA library (27). In this study, one of
these cDNAs has been characterized and found to encode a protein that
is a novel member of the IRS family. We report here that injection of
mRNA encoding this IRS protein potentiates insulin signaling in
Xenopus oocytes and that its putative PH domain functions in
a dominant negative manner to inhibit insulin signaling in
Xenopus oocytes.
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RESULTS
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Molecular Cloning and Sequence Analysis of xIRS-u
Using the entire coding region of rat IRS-1 as a probe, we
isolated several positive clones from a Xenopus oocyte cDNA
library. Southern blotting revealed that many of these clones were
distinct from the previously isolated xIRS-1 (28) (not shown). One of
these potentially novel cDNAs, clone 15-5, was subcloned into
pBluescript for DNA sequence determination. Clone 15-5 contained 2.3-
and 0.5-kb EcoRI fragments. The 2.5-kb fragment corresponded
to the 3'-end of the cDNA insert, including an open-reading frame of
704 amino acids and about 400 bp of the 3'- untranslated region. The
0.5-kb EcoRI fragment corresponded to the 5'- end of the
cDNA and was entirely within the coding region. To clone the remaining
5'-coding region of xIRS-u, we rescreened the oocyte cDNA library using
the 0.5-kb EcoRI fragment as a probe. Several clones were
isolated and, one (15-5b7) was found to contain the remaining 5'-
coding sequence of xIRS-u. Based on the overlapping sequence between
clone 15-5 and clone 15-5b7, the entire coding sequence of xIRS-u was
assembled (Fig. 1A
).



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Figure 1. xIRS-u Is a Novel IRS Protein
A, The nucleotide sequence of xIRS-u with the corresponding amino
acid sequence in one-letter code. The shaded blocks
(from amino terminus to carboxy terminus) indicate the PH domain, the
PTB domain, and the SAIN domain, respectively. Blocks in
black are potential tyrosine phosphorylation sites that conform
to the binding consensus sequence for known SH2 domain-containing
proteins (see text for details). Also indicated are the two possible
translation initiation sites and several key restriction sites in the
cDNA mentioned in the text. B, Pair-wise amino acid sequence
comparisons (BESTFIT, Genetic Computer Group, Madison, WI) of xIRS-u
and xIRS-1 to the mammalian IRS family members. Shown are percent
identity between each pair. C, Sequence alignment using PILEUP (Genetic
Computer Group, Madison, WI). For PH domains and PTB domains,
blocked amino acids indicate identity in at least four
of the six sequences. For SAIN domains, blocked amino
acids indicate identity in at least three of the four
sequences. The number at the beginning of each sequence
indicates the position of the first residue in the full-length
protein.
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Two possible translation initiation sites were found in the xIRS-u cDNA
(Fig. 1A
). The first ATG codon is preceded by TCC (-3 to -1), which
is not a good consensus sequence for eukaryotic translation initiation
(29). The second ATG, which is 19 codons downstream, is preceded by a
good Kozak consensus (AGA, -3 to -1) (Fig. 1A
) (29). Although the
actual translation initiation codon used in vivo is not
clear, we chose the first ATG as the putative translation initiation
site to avoid possible truncation of xIRS-u at the amino terminus. In
addition, the cDNA contains a unique NcoI site at the first
ATG; and therefore a Myc-tagged full-length xIRS-u was conveniently
generated (see below). As a result of this somewhat arbitrary choice,
the xIRS-u cDNA contains an open reading frame of 1003 amino acids with
an estimated molecular mass of 110 kDa.
Pair-wise sequence comparisons (Fig. 1B
) indicate that xIRS-u has
similar overall sequence homology to all members of the mammalian IRS
family (3345% identical in predicted amino acid sequence). In
contrast, the previously isolated xIRS-1 (28) is particularly similar
to IRS-1 (67%) and much less similar to the other mammalian IRS
members (3146%). This pattern of sequence homology is also reflected
when individual domains are compared (Fig. 1C
). xIRS-u encodes a
putative PH domain (amino acids 63166) that is almost equally similar
to the PH domains of IRS-1 (67%) (2), IRS-2 (69%) (5), and IRS-4
(63%) (6). In addition, xIRS-u encodes a putative PTB domain (amino
acids 193293) that is almost equally similar to those of IRS-1
(72%), IRS-2 (68%), and IRS-4 (67%). In contrast, The PH and PTB
domains of xIRS-1 are particularly similar (87 and 90% identical,
respectively) to their counterparts in IRS-1 (Fig. 1C
), and much less
similar to their counterparts in other IRS members (IRS-2, IRS-3, or
IRS-4) (Fig. 1B
). Unlike the PH and the PTB domains, which are readily
identifiable in all IRS members, the SAIN domains have only been
identified in IRS-1 (24) and IRS-2 (26). The two new members, IRS-3 and
IRS-4, lack detectable SAIN domains (6, 7) based upon sequence
comparison. xIRS-u contains an identifiable SAIN domain between amino
acids 347 and 499 that is 58% identical to that of IRS-1, compared
with a 34% identity between the SAIN domains of IRS-1 and IRS-2 (Fig. 1C
). This contrasts with the xIRS-1 SAIN domain, which is very similar
to the rIRS-1 SAIN domain, exhibiting 88% sequence identity. Taken
together, these differences suggest that xIRS-u is a new member of the
IRS family, rather than an amphibian homolog of an existing IRS member.
We chose xIRS-u (unique or undetermined designation) to reflect the
fact that a putative mammalian homolog has not yet been identified.
Like other IRS proteins, xIRS-u also contains multiple (5) tyrosine
residues in the context of the YM/LXM motif (Fig. 1A
). Phosphorylation
of such motifs creates binding sites for the SH2 domains of PI 3-kinase
(12). In addition, xIRS-u contains a putative phosphorylation site
(Y789INID) that may represent a binding site for the GRB2
SH2 domain (30).
Northern blot analysis revealed the presence of a single maternal
message (oocytes, eggs, and cleavage embryos) of about 7 kb. This
message was slightly smaller than that of xIRS-1 (28) (Fig. 2
and data not shown). Interestingly, the
level of this message was much lower in midblastula [during which
transcription of the embryonic genome starts (31)] and gastrula
embryos and the level increased again during the early stages of
neurulation. This pattern of expression suggested that xIRS-u
transcription was activated during embryogenesis, and therefore it may
play a role in early embryogenesis. Densitometric analysis of the
stained gel confirmed that the amounts of 18S or 28S ribosomal RNA were
within 80% of each other (not shown).

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Figure 2. Expression of xIRS-u mRNA during Embryogenesis
Total RNA isolated from unfertilized eggs or embryos at the various
stages were analyzed by Northern blotting using a 2.5-kb
EcoRI insert of clone 15-5 (Fig. 1A , nucleotide 1030 to
the 3'-end) as a probe. A duplicate gel was stained with ethidium
bromide to reveal the amount of the two ribosomal RNAs.
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Expressing Full-Length and Deletion Mutants of xIRS-u in
Xenopus Oocytes
For the functional characterization of xIRS-u, we chose pCS2+MT
(32) as an expression vector. Insertion of xIRS-u (or its mutants) at
the NcoI site would result in a plasmid encoding five copies
of the Myc tag (MT) followed by the xIRS-u sequence. pCS2+MT also
contains an SV40 polyA sequence after the cDNA insert to increase mRNA
stability in oocytes. Five xIRS-u constructs were used in this study
(Fig. 3A
). xIRS-u represents the
full-length protein initiating at amino acid 1. xIRS-u PH-PTB contains
amino acids 1299. xIRS-u
PH was derived from xIRS-u with the first
118 amino acids deleted. A similar mutation in IRS-1 is known to
compromise the function of its PH domain and hence the function of
IRS-1 (20).
PH-PTB was derived from PH-PTB with the same
amino-terminal (1118) deletion. xIRS-u PH domain (amino acids 1170)
was also derived from PH-PTB construct, by an internal deletion that
removed amino acids 171299. Oocytes injected with the various xIRS-u
mRNAs were incubated overnight. Western blotting analysis was used to
assess the accumulation of Myc-tagged proteins (Fig. 3B
). Injection of
xIRS-u mRNA resulted in the accumulation of a prominent protein of
about 120 kDa, recognizable by anti-Myc antibodies. The size of this
protein was within the range of the predicted molecular mass of the
Myc-tagged xIRS-u (110 kDa plus about 7 kDa from the 5 copies of the
13-amino acid Myc tag). Several smaller protein bands were detected
(Fig. 3B
, lane 2), the identity of which are not known. xIRS-u
PH
produced a protein of about 86 kDa, consistent with the amino-terminal
deletion of 118 amino acids. Several smaller protein bands were also
detected. xIRS-u PH-PTB produced a protein of about 57 kDa, slightly
larger than the predicted molecular mass (estimated at
40 kDa
including the Myc tag). The size of
PH-PTB was in line with a
deletion of the amino-terminal 118 amino acids. In addition, xIRS-u PH
domain appeared to be correctly expressed (Fig. 7B
).

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Figure 3. Expression of xIRS-u and Several xIRS-u Truncation
Mutants in Xenopus Oocytes
A, The various constructs used in this study are schematically
represented. These constructs were made in the pCS2+MT vector and thus
are fused to five copies of the Myc tag. The approximate positions of
several putative SH2 binding sites are also indicated. B, Groups of at
least 20 oocytes were injected with water (control) or mRNA (10 ng/per
oocyte except for lane 5 in which 3 ng were injected). After an
overnight incubation, oocytes were lysed. Expression of the various
proteins was determined by Western blotting using an anti-Myc
antibody.
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Figure 7. xIRS-u PH Domain Inhibits Insulin-induced xMAP
Kinase Activation
Groups of 30 or more oocytes were injected with water (control) or 3.3
ng (1/3) or 10 ng (PH) xIRS-u PH mRNA. The injected oocytes were
incubated overnight in OR2. After the addition of insulin, oocytes were
further incubated for 15 h before being scored for GVBD (Fig. 7C )
or lysed for xMAP kinase Western blot analysis (A). Protein expression
was analyzed by anti-Myc Western blot (B).
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xIRS-u Potentiates Insulin Signaling in Xenopus
Oocytes
To examine a possible role of xIRS-u in insulin-induced oocyte
maturation, we attempted to repeat an experiment by Chuang et
al. (33, 34) in which the authors demonstrated that oocytes
retrieved from non-PMSG-primed frogs did not respond to insulin unless
they were injected with recombinant IRS-1. We did not observe a
consistent difference in insulin response before or after the donor
frogs were primed with PMSG, with the exception that oocytes from
recently primed frogs (within 35 days) responded more rapidly (not
shown). Furthermore, oocytes from recently primed frogs also responded
more rapidly to progesterone than those from the same frogs before PMSG
priming (not shown). In addition, we previously determined that PMSG
priming did not change the level of xIRS-1 mRNA or proteins in oocytes
(C. Cummings and X.J. Liu, unpublished). We believe that the effect of
priming is likely on the general metabolic activity of oocytes rather
than on any specific component(s) in the insulin pathway. Therefore,
for subsequent studies, we used oocytes from PMSG-primed frogs, as was
the case in almost all published literature.
The quality of oocytes varies among donor frogs and determines the
maximum insulin response. We discarded oocytes that did not respond to
insulin or those that responded very poorly (<50%). The exact time
for insulin-induced germinal vesicle breakdown (GVBD) also varies among
individual frogs, as well as the time that has lapsed since PMSG
priming. Typically, we observed that uninjected oocytes or oocytes
injected with water reached 50% maximum GVBD 812 h after the
addition of insulin. In several independent experiments using different
donor frogs, we observed a marked acceleration of insulin-induced GVBD
in oocytes injected with xIRS-u mRNA, which reached 50% maximum
response 510 h after the addition of insulin (not shown). An increase
in maximum insulin response was also observed after xIRS-u mRNA
injection, in oocytes that did not otherwise reach 100% response (not
shown).
To better quantify insulin signaling in Xenopus oocytes, we
employed anti-xMAP kinase immunoblotting to determine the ratio of
activated mitogen-activated protein (MAP) kinase vs. total
MAP kinase. xMAP kinase activation, together with the activation of
maturation-promoting factor, are both hallmark biochemical events
upstream of GVBD during hormone-induced oocyte maturation (35, 36). The
ratio of active xMAP kinase over total xMAP kinase, as determined by
Western blotting, is a particularly accurate indication of GVBD in a
population of oocytes (37, 38). Injection of xIRS-u mRNA did not induce
xMAP kinase activation in the absence of insulin stimulation (Fig. 4
, 0 insulin). Acceleration of
insulin-induced xMAP kinase activation was detectable by 7 h
postinsulin addition in oocytes injected with xIRS-u mRNA compared with
control oocytes in which no xMAP kinase activation was seen. At
10.5 h after insulin stimulation, xMAP kinase activation reached
almost maximum levels in oocytes injected with xIRS-u mRNA, whereas
only minimum activation had occurred in control oocytes. After a longer
(15 h) incubation, however, xMAP kinase activation in both groups of
oocytes reached maximum levels. These results suggest that
overexpression of xIRS-u (Fig. 3B
, lane 2) accelerates insulin
signaling in Xenopus oocytes.

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Figure 4. xIRS-u Accelerates Insulin-Induced MAP Kinase
Activation
A, Oocytes injected with water or xIRS-u mRNA (at least 150 oocytes in
each group) were incubated overnight in OR2. Thirty oocytes from each
group were left in OR2 for the duration of the experiments. The
remaining oocytes were incubated with insulin. At the indicated time
after the addition of insulin, 30 oocytes were withdrawn from each
group and lysed for a MAP kinase Western blotting assay. B,
Densitometric analysis of data presented in panel A. Data are presented
as active MAP kinase (upper band)/total MAP kinase (sum
of upper and lower bands).
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xIRS-u PH Domain Inhibits Insulin Signaling in Xenopus
Oocytes
Previous studies have demonstrated that IRS-1 lacking the PH
domain is a poorer substrate than the full-length IRS-1 for
ligand-activated insulin receptor and therefore is a poorer mediator of
insulin signaling (18, 19, 20). To assess the role of the putative PH
domain of xIRS-u, we compared the ability of xIRS-u and xIRS-u
PH to
potentiate insulin-induced MAP kinase activation (Fig. 5
). Enhancement of insulin-induced MAP
kinase activation was observed in oocytes injected with both xIRS-u and
xIRS-u
PH mRNA such that at 10 h after insulin stimulation xMAP
kinase was fully activated in oocytes injected with either xIRS-u (lane
5) or xIRS-u
PH (lane 6) while only partial activation was observed
in control oocytes (lane 4). It is interesting to note that xIRS-u
PH was less effective than xIRS-u in enhancing insulin-induced MAP
kinase activation (Fig. 5
, comparing lanes 2 and 3). These results
suggest that deletion of the PH domain compromises the ability of
xIRS-u to potentiate insulin signaling.

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Figure 5. xIRS-u PH Is Less Efficient Than xIRS-u in
Enhancing Insulin Signaling
Groups of about 80 oocytes were injected with water (control), xIRS-u
mRNA, or xIRS-u PH mRNA. Forty oocytes were withdrawn and lysed at
5 h after the addition of insulin. The remaining 40 were lysed
10 h after the addition of insulin. Lysates were subjected to a
MAP kinase Western blotting assay.
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Tanaka et al. (39) have demonstrated that overexpression of
the amino-terminal region of IRS-1 containing the PH and PTB domains
inhibits insulin signaling, suggesting that it functions as a dominant
negative inhibitor of endogenous IRS-1. It is unclear from their
studies if the PH domain or the PTB domain or both are required for the
inhibition. We first tested whether xIRS-u (1299), which contained
both the putative PH and PTB domains, was able to inhibit insulin
signaling in Xenopus oocytes (Fig. 6A
). While the control sample xMAP kinase
was almost completely activated after 15 h of insulin stimulation,
as expected, injection of the xIRS-u PH-PTB mRNA abolished
insulin-induced xMAP kinase activation (Fig. 6
, compare lanes 3 and 1)
with a concomitant inhibition of insulin-induced GVBD (not shown, but
see Fig. 7C
). The inhibition by the
PH-PTB construct was significant even when the concentration of the
mRNA was reduced to one third (Fig. 6A
, compare lanes 4 and 1) [with a
corresponding reduction in the expression of the protein (Fig. 3B
, lanes 4 and 5)]. In contrast, xIRS-u (119299), which contained a
truncated PH domain but still retained an intact PTB domain, neither
inhibited insulin-induced MAP kinase activation (lane 5) nor
accelerated it (not shown). To ensure that the inhibitory effect of
xIRS-u PH-PTB was specific to the insulin pathway, we assessed its
effect on progesterone signaling in Xenopus oocytes (Fig. 6B
). Injection of xIRS-u (1299) mRNA blocked insulin-induced MAP
kinase activation (Fig. 6B
, compare lanes 3 and 4) but did not affect
progesterone-induced MAP kinase activation (compare lanes 5 and 6) or
progesterone-induced GVBD (not shown, but see Fig. 7C
).

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Figure 6. xIRS-u PH-PTB Inhibits Insulin-Induced MAP Kinase
Activation
A, Groups of at least 30 oocytes were injected with water (control) or
mRNA encoding the indicated regions of xIRS-u (10 ng mRNA/oocyte except
for lane 4 in which 3 ng were injected per oocyte). Oocytes were
incubated with insulin overnight (15 h) before being lysed for MAP
kinase Western blot analysis. B, Groups of about 100 oocytes were
injected with water (control) or mRNA encoding xIRS-u PH-PTB. After an
overnight incubation in OR2, each group of injected oocytes was further
split into three groups of at least 30 oocytes each. Groups 1 and 2
were left in OR2, 3 and 4 were incubated with insulin, and 5 and 6 were
incubated with progesterone. After another overnight incubation,
oocytes were lysed and subjected to MAP kinase Western blot analysis.
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To further determine whether the xIRS-u PH domain could function
independently to inhibit insulin signaling in Xenopus
oocytes, we injected oocytes with 10 ng or 3.3 ng (one third) of xIRS-u
PH mRNA. Injection of the PH domain, but not water (control),
significantly inhibited insulin-induced MAP kinase activation (Fig. 7A
), with a concomitant inhibition of insulin-induced GVBD (Fig. 7C
).
In contrast, injection of the PH domain did not affect MAP kinase
activation (Fig. 7A
) or GVBD (Fig. 7C
) induced by progesterone.
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DISCUSSION
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We have identified a Xenopus cDNA encoding a novel
member of the IRS family, which we have termed xIRS-u. This conclusion
is based mainly upon pair-wise comparisons of the deduced amino acid
sequence of xIRS-u with those of other known members (Fig. 1
, B and C).
Whereas the previously cloned xIRS-1 is particularly similar to
mammalian IRS-1 (67% identical) and much less similar to other
mammalian IRS family members (3146%), xIRS-u has similar sequence
homology to all mammalian IRS family members (3345%). These
comparisons clearly suggest that xIRS-u represents a previously
unidentified member of the IRS family rather than a Xenopus
homolog of an existing mammalian IRS gene. Cloning the putative
mammalian homolog of xIRS-u, which is underway in our laboratory, is of
significant interest.
Xenopus laevis has a pseudo tetraploid genetic makeup (40),
and many genes are represented in nonallelic duplicates, including, for
example, the proinsulin gene (41) and the insulin receptor gene (42).
However, the duplicate loci of the same gene are usually more than 90%
identical in the coding regions (40, 41, 42). The relatively low degree of
sequence identity (47%) between xIRS-u and xIRS-1 clearly suggests
that they are distinct genes rather than nonallelic copies of the same
gene. Although it is unclear how many IRS genes exist in the
Xenopus laevis genome (for that matter in the human genome),
we have isolated partial cDNAs that likely represent other IRS family
members from the same Xenopus oocyte cDNA library (Liu
et al., unpublished). Therefore, Xenopus laevis,
like mammals, likely contains multiple IRS genes.
Previous studies have demonstrated that deletion of the PH domain from
IRS-1 or IRS-2 (18, 19, 20) diminishes tyrosine phosphorylation of the
respective IRS proteins by ligand-activated insulin receptors and
reduces the ability of the IRS proteins to mediate insulin-induced
mitogenesis (19). Interestingly, deletion of the PTB domain has much
less effect on either IRS tyrosine phosphorylation or insulin-induced
mitogenesis (19). These findings suggest that the PH domain plays an
essential role in IRS-1 (and IRS-2) phosphorylation by the insulin
receptor, whereas the PTB domain is not absolutely required. Perhaps in
support of such a notion is the fact that a recently identified insulin
receptor substrate, Gab1, contains a PH domain but not a PTB domain
(9). Our data (Fig. 5
) also support such a notion in demonstrating that
xIRS-u lacking a functional PH domain was much less effective in
accelerating insulin signaling in Xenopus oocytes.
Furthermore, we demonstrated that the PH domain of xIRS-u, but not the
PTB domain, can function in a dominant-negative fashion to inhibit
insulin-induced MAP kinase activation and insulin-induced oocyte
maturation (Figs. 6
and 7
). The inability of the xIRS-u PTB domain to
inhibit insulin-induced MAP kinase activation in Xenopus
oocytes is in contrast to a recent report (25) that suggests that
either the PTB domain or the SAIN domain of IRS-1 can function in a
dominant negative manner to inhibit insulin-induced MAP kinase
activation in 3T3 L1 adipocytes. We cannot explain this apparent
discrepancy at the present time. However, our studies represent the
first demonstration that the PH domain of an IRS protein alone can
function in a dominant negative manner to inhibit insulin signaling.
These results suggest that the PH domain of IRS proteins must interact
with other components (proteins or lipid) in the insulin-signaling
pathway.
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MATERIALS AND METHODS
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Molecular Cloning and cDNA Characterization
Standard molecular cloning procedures (43) were followed as
outlined below. The full-length rat IRS-1 cDNA was excised from
pBluescript (2) by EcoRI digestion and agarose purified. The
purified cDNA was then used as a template to synthesize random-primed
32P-dCTP-labeled cDNA probes. The hybridization buffer
consisted of 50% formamide, 5x SSPE [1x SSPE is 0.8 M
NaCl, 10 mM NaH2PO4, and 1
mM EDTA (pH 7.7)], 1x Denhardts solution, 90 mg/ml
dextran sulfate, and 0.1% SDS. Hybridization was carried out at 42 C
overnight. The filters were washed several times with large volumes of
increasingly dilute SSC containing 0.1% SDS at increasing
temperatures, until the background became stable. Positive clones were
confirmed and purified by secondary and tertiary screenings using the
same IRS-1 probe. Bacterial phage DNA was isolated from the purified
clones and digested with EcoRI to excise the cDNA inserts.
The purified inserts were subcloned into pBluescript previously
linearized with EcoRI, and their sequences were determined
by dideoxy DNA sequencing using a semiautomatic DNA sequencer (ABI
Advanced Biotechnologies, Columbia, MD). Any sequence ambiguity was
clarified by sequencing in the opposite direction.
Construction of Expression Plasmids
An NcoI-EcoRI fragment of the xIRS-u cDNA
insert (Fig. 1A
, nucleotides 197-1035) was ligated into pCS2+MT (32)
after digestion of the vector plasmid with the same two enzymes. The
resulting plasmid encoded five copies of the 13-amino acid Myc tag
followed by amino acids 1299 (PH-PTB) of xIRS-u (Fig. 1A
). To
construct the full-length Myc-tagged xIRS-u (amino acids 11003), the
EcoRI-EcoRI fragment (Fig. 1A
, nucleotide 1029 to
the 3'-end of the cDNA) was ligated to pCS2+MT/PH-PTB after
EcoRI digestion of the latter. To delete the amino-terminal
118 amino acids from both the PH-PTB (to generate
PH-PTB) and the
full-length xIRS-u (to generate xIRS-u
PH), the
NcoI-EcoRI fragment (encoding amino acids 1299)
was replaced with a PCR-amplified fragment encoding amino acids
119299. xIRS-u PH was derived from pCS2+MT/PH-PTB by deleting a
fragment of xIRS-u cDNA between a unique SacI restriction
site (Fig. 1A
) and an XbaI of the vector, resulting in
expression of the first 170 amino acids corresponding almost precisely
to the PH domain (Fig. 1A
). All plasmids were linearized with a
NotI restriction enzyme after the SV40 poly-A sequence in
the vector and then transcribed using SP6 RNA polymerase (Ambion,
Austin, TX). The synthesized mRNA was dissolved in water to a
concentration of 1 mg/ml, as estimated by comparison to RNA standards
of known concentration (GIBCO-BRL, Gaithersburg, MD).
Animal and Oocyte Manipulation
All procedures involving live oocytes were carried out in a room
maintained at 18 C. Sexually mature and oocyte-positive X.
laevis females were purchased from NASCO (Ft. Atkinson, WI) and
maintained according to local animal care guidelines. The frogs were
injected with PMSG (Sigma Chemical Co., St Louis, MO; 50 IU/frog) 310
days before oocyte retrieval. A fragment of ovary was surgically
removed under hypothermia. Stage VI oocytes (44) were manually
defolliculated according to Smith (45). Unless otherwise stated, 10 ng
(in 10 nl) of mRNA were microinjected per oocyte. Oocytes to be
microinjected were incubated in oocyte incubation medium OR2 (82.5
mM NaCl, 2.5 mM KCl, 1 mM
CaCl2, 1 mM MgCl2, 1 mM
Na2HPO4, 5 mM HEPES, pH 7.8) that
lacked CaCl2. Injected oocytes were incubated in OR2
(containing CaCl2) for 6 h to overnight before the
addition of hormones or a second microinjection.
Progesterone (final concentration, 20 µM) stimulation of
oocytes was carried out in OR2. Insulin stimulation (5
µM) was carried out in OR2 lacking K+ ions to
ensure that oocytes responded maximally to insulin, (46, 47).
Elimination of K+ ions from OR2 had no effect on oocyte
viability, and it did not induce or inhibit oocyte maturation by itself
(37, 46, 47). To assay for meiotic maturation, oocytes were incubated
overnight with insulin or progesterone. GVBD was determined by the
appearance of a white spot at the center of the animal hemisphere and
confirmed, when in doubt, by bisecting the oocytes after fixation in
5% trichloroacetic acid and observing the presence (GVBD negative) or
absence (GVBD positive) of a germinal vesicle.
Embryo Methods
Embryonic culture and related methods were according to Sive
et al. (48) as described below. Frogs were injected with
human CG (hCG, 1000 U per frog) and kept at 18 C overnight to induce
ovulation. Eggs were collected in MBS (5 mM HEPES, pH 7.8,
88 mM NaCl, 1 mM KCl, 0.7 mM
CaCl2, 1 mM MgSO4, 2.5
mM NaHCO3) with an additional 20 mM
NaCl (high salt-MBS). To fertilize eggs, we removed excess high
salt-MBS and rubbed a pinched testis over the eggs. An excess volume of
0.1x MBS was then added. After 30 min, fertilized eggs were treated
with 2% cysteine (pH 8) for 25 min until the jelly coat was removed.
Dejellied embryos were rinsed with an excess volume of 0.1x MBS and
incubated in 0.1x MBS to various developmental stages (49) before
being lysed for RNA isolation using RNAzol (Tel-Test Inc., Friendswood,
TX) according to the manufacturers instructions.
Other Methods
For protein isolation, oocytes were lysed by forcing them
through pipette tips in PBS lysis buffer (10 mM sodium
phosphate, pH 7.5, 150 mM NaCl, 1% Triton X-100, 10
µg/ml each of leupeptin and aprotinin, 1 mM
phenylmethylsulfonate, and 1 mM sodium orthovanadate; 10
µl lysis buffer per oocyte). The homogenate was centrifuged in an
Eppendorf centrifuge for 15 min at 4 C. The clarified lysates were
collected, mixed with SDS sample buffer, and analyzed by SDS-PAGE
followed by Western blotting with anti-xMAP kinase antiserum (used at
1:2000 dilution) and anti-Myc antibody (9E10). The immunoblots were
developed using an ECL kit (Amersham, Arlington Heights, IL).
 |
ACKNOWLEDGMENTS
|
---|
We thank Cathy Cummings for excellent technical assistance; Dr.
Jonathan Cooper and Dr. Dave Turner for anti-xMAP kinase serum and the
pCS2+MT vector, respectively; and Dr. Morris White for the rat IRS-1
cDNA.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Johné Liu, Ottawa Civic Hospital Loeb Research Institute, Ottawa Civic Hospital, 1053 Carling Avenue, Ottawa, K1Y 4E9, Canada. E-mail:
johne{at}civich.ottawa.on.ca
Abbreviations: OR2: oocyte incubation medium, PI: phosphatidylinositol,
SAIN: Shc and IRS-1 NPXpY binding domain, SH2: Src homology 2.
This study was supported by operating grants from the National Cancer
Institute of Canada and Cancer Research Society of Canada (to X.J.L.).
X.J.L. is a scholar of the Medical Research Council of Canada.
Received for publication January 26, 1998.
Revision received March 26, 1998.
Accepted for publication April 21, 1998.
 |
REFERENCES
|
---|
-
White MF, Maron R, Kahn CR 1985 Insulin rapidly
stimulates tyrosine phosphorylation of a Mr-185,000 protein in intact
cells. Nature 318:183186[Medline]
-
Sun XJ, Rothenberg P, Kahn CR, Backer JM, Araki E, Wilden PA,
Cahill DA, Goldstein BJ, White MF 1991 Structure of the insulin
receptor substrate IRS-1 defines a unique signal transduction protein.
Nature 352:7377[CrossRef][Medline]
-
Tobe K, Tamemoto H, Yamauchi T, Aizawa S, Yazaki Y, Kadowaki
T 1995 Identification of a 190-kDa protein as a novel substrate for the
insulin receptor kinase functionally similar to insulin receptor
substrate-1. J Biol Chem 270:56985701[Abstract/Free Full Text]
-
Araki E, Lipes MA, Patti M-E, Bruning JC, Haag III B, Johnson
RS, Kahn CR 1994 Alternative pathway of insulin signalling in mice with
targeted disruption of the IRS-1 gene. Nature 372:186190[CrossRef][Medline]
-
Sun XJ, Wang L-M, Zhang Y, Yenush L, Myers Jr MG, Glasheen E,
Lane WS, Pierce JH, White MF 1995 Role of IRS-2 in insulin and cytokine
signalling. Nature 377:173177[CrossRef][Medline]
-
Lavan BE, Fantin VR, Chang ET, Lane WS, Keller SR, Lienhard
GE 1997 A novel 160-kDa phosphotyrosine protein in insulin-treated
embryonic kidney cells is a new member of the insulin receptor
substrate family. J Biol Chem 272:2140321407[Abstract/Free Full Text]
-
Lavan BE, Lane WS, Lienhard GE 1997 The 60-kDa
phosphotyrosine protein in insulin-treated adipocytes is a new member
of the insulin receptor substrate family. J Biol Chem 272:1143911443[Abstract/Free Full Text]
-
Pelicci G, Lanfrancone L, Grignani F, McGlade J, Cavallo F,
Forni G, Nicolette I, Grignani F, Pawson T, Pelicci PG 1992 A novel
transforming protein (SHC) with an SH2 domain is implicated in
mitogenic signal transduction. Cell 70:93104[Medline]
-
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]
-
Carpino N, Wisniewski D, Strife A, Marshak D, Kobayachi R,
Stillman B, Clarkson B 1997 p62dok: a constitutively
tyrosine-phosphorylated, GAP-associated protein in chromic myelogenous
leukemia progenitor cells. Cell 88:197204[Medline]
-
Yamanashi Y, Baltimore D 1997 Identification of the Abl and
RasGAP-associated 62 kDa protein as a docking protein, Dok. Cell 88:205211[Medline]
-
Songyang Z, Shoelson SE, Chaudhuri M, Gish G, Pawson T, King
F, Rorberts T, Ratnofsky S, Schaffhausen B, Cantley LC 1993 SH2 domains
recognize specific phosphopeptide sequences. Cell 72:767778[Medline]
-
Skolnik EY, Batzer A, Li N, Lee C-H, Lowenstein E,
Mohammadi M, Margolis B, Schlessinger J 1993 The function of GRB2
in linking the insulin receptor to Ras signaling pathways. Science 260:19531955[Medline]
-
Kuhné MR, Pawson T, Lienhard GE, Feng G-S 1993 The
insulin receptor substrate 1 associates with the SH2-containing
phosphotyrosine phosphatase Syp. J Biol Chem 268:1147911481[Abstract/Free Full Text]
-
Backer JM, Myers Jr MG, Shoelson SE, Chin DJ, Sun X-J,
Miralpeix M, Hu P, Mardolis B, Skolnik EY, Schlessinger J, White MF 1992 Phosphatidylinositol 3'-kinase is activated by association with
IRS-1 during insulin stimulation. EMBO J 11:34693479[Abstract]
-
Lechleider RJ, Sugimoto S, Bennett AM, Kashishian AS, Cooper
JA, Shoelson SE, Walsh CA, Neel BG 1993 Activation of the
SH2-containing phosphotyrosine phosphatase SH-PTP2 by its binding site,
phosphotyrosine 1009, on the human platelet-derived growth factor
receptor ß. J Biol Chem 268:2147821481[Abstract/Free Full Text]
-
Skolnik EY, Lee CH, Batzer A, Vicentini LM, Zhou M, Daly RJ,
Myers Jr MG, Backer JM, Ullrich A, White MF, Schlenssinger J 1993 The
SH2/SH3 domain-containing protein GRB2 interacts with
tyrosine-phosphorylated IRS-1 and Shc: implications for insulin control
of ras signalling. EMBO J 12:19291936[Abstract]
-
Myers Jr MG, Grammer TC, Brooks J, Glasheen EM, Wang LM, Sun
XJ, Blenis J, Pierce JH, White MF 1995 The pleckstrin homology domain
in IRS-1 sensitizes insulin signaling. J Biol Chem 270:1171511718[Abstract/Free Full Text]
-
Yenush L, Makati KJ, Smith-Hall J, Ishibashi O, Myers Jr MG,
White MF 1996 The pleckstrin homology domain is the principle link
between the insulin receptor and IRS-1. J Biol Chem 271:2430024306[Abstract/Free Full Text]
-
Voliovitch H, Schindler DG, Hadari YR, Taylor SI, 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]
-
Burks DJ, Pons S, Towery H, Smith-Hall J, Myers Jr MG, Yenush
L, White MF 1997 Heterologous pleckstrin homology domains do not couple
IRS-1 to the insulin receptor. J Biol Chem 272:2771627721[Abstract/Free Full Text]
-
White MF, Livingston JN, Backer JM, Lauris V, Dull TJ, Ullrich
A, Kahn CR 1988 Mutation of the insulin receptor at tyrosine 960
inhibits signal transmission but does not affect its tyrosine kinase
activity. Cell 54:641649[Medline]
-
Yamasaki H, Prager D, Gebremedhin S, Melmed S 1992 Human
insulin-like growth factor I receptor 950 tyrosine is required for
somatotroph growth factor signal transduction. J Biol Chem 267:2095320958[Abstract/Free Full Text]
-
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]
-
Sharma PM, Egawa K, Gustafson TA, Martin JL, Olefsky JM 1997 Adenovirus-mediated over-expression of IRS-1 interacting domains
abolishes insulin-stimulated mitogenesis without affecting glucose
transport in 3T3L1 adipocytes. Mol Biol Cell 17:73867397
-
He W, Craparo A, Zhu Y, ONeill TJ, Wang LM, Pierce JM,
Gustafson TA 1996 Interaction of insulin receptor substrate-2 with the
insulin and IGF-1 receptors: evidence for two distinct
phosphotyrosine-dependent interaction domains within IRS-2. J Biol
Chem 271:1164111645[Abstract/Free Full Text]
-
Rebagliati MR, Weeks DL, Harvey RP, Melton DA 1985 Identification and cloning of localized maternal RNAs from
Xenopus eggs. Cell 42:769777[Medline]
-
Liu XJ, Sorisky A, Zhu L, Pawson T 1995 Molecular cloning of
an amphibian insulin receptor substrate-1-like cDNA and involvement of
phosphatidylinositol 3-kinase in insulin-induced Xenopus
oocyte maturation. Mol Cell Biol 15:35633570[Abstract]
-
Kozak M 1987 An analysis of 5'-noncoding sequences from 699
vertebrate messenger RNAs. Nucleic Acids Res 15:81258132[Abstract]
-
Myers Jr MG, Sun XJ, White MF 1994 The IRS-1 signalling
system. Trends Biochem Sci 19:289294[CrossRef][Medline]
-
Almouzni G, Wolffe AP 1995 Constraints on transcriptional
activator function contribute to transcriptional quiescence during
early Xenopus embryogenesis. EMBO J 14:17521765[Abstract]
-
Turner DL, Weintraub H 1994 Expression of achaete-scute
homology 3 in Xenopus embryos converts ectodermal cells
to a neural fate. Genes Dev 8:14341447[Abstract]
-
Chuang L-M, Myers Jr MG, Seidner GA, Birnbaum MJ, White MF,
Kahn CR 1993 Insulin receptor substrate 1 mediates insulin and
insulin-like growth factor I-stimulated maturation of
Xenopus oocytes. Proc Natl Acad Sci USA 90:51725175[Abstract]
-
Chuang L-M, Myers Jr MG, Backer JM, Shoelson SE, White MF,
Kahn CR 1993 Insulin-stimulated oocyte maturation requires insulin
receptor substrate 1 and interaction with the SH2 domains of
phosphatidylinositol 3-kinase. Mol Cell Biol 13:66536660[Abstract]
-
Smith LD 1989 The induction of oocyte maturation:
transmembrane signaling events and regulation of cell cycle.
Development 107:685699[Medline]
-
Matten W, Daar I, Vande Woude GF 1994 Protein kinase A acts at
multiple points to inhibit Xenopus oocyte maturation. Mol
Cell Biol 14:44194426[Abstract]
-
Cummings C, Zhu L, Sorisky A, Liu XJ 1996 A peroxovanadium
compound induces Xenopus oocyte maturation: inhibition by a
neutralizing anti-insulin receptor antibody. Dev Biol 175:338346[CrossRef][Medline]
-
Posada J, Yew N, Ahn NG, Vande Woude GF, Cooper JA 1993 Mos
stimulates MAP kinase in Xenopus oocytes and activates a MAP
kinase kinase in vitro. Mol Cell Biol 13:25462553[Abstract]
-
Tanaka S, Wands JR 1996 A carboxy-terminal truncated insulin
receptor substrate-1 dominant negative protein reverses the human
hepatocellular carcinoma malignant phenotype. J Clin Invest 98:21002108[Abstract/Free Full Text]
-
Graf J-D, Kobel HR 1991 Genetics of Xenopus laevis.
In: Kay BK, Peng HB (eds) Methods in Cell Biology. Xenopus
laevis: Practical Uses in Cell and Molecular Biology. Academic
Press, New York, vol 36:1931
-
Shuldiner AR, Phillips S, Roberts Jr CT, LeRoith D, Roth J 1989 Xenopus laevis contains two nonallelic preproinsulin
genes. J Biol Chem 264:94289434[Abstract/Free Full Text]
-
Scavo L, Shuldiner AR, Serrano J, Dashner R, Roth J, De Pablo
F 1991 Genes encoding receptors for insulin and insulin-like growth
factor I are expressed in Xenopus oocytes and embryos. Proc
Natl Acad Sci USA 88:62146218[Abstract]
-
Sambrook J, Fritsch EF, Maniatis T 1989 Molecular Cloning: A
Laboratory Manual, ed 2. Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, NY
-
Dumont JN 1971 Oogenesis in Xenopus laevis
(Daudin). J Morphol 136:153180
-
Smith LD, Xu W, Varnold RL 1991 Oogenesis and oocyte
isolation. In: Kay BK, Peng HB (eds) Methods in Cell Biology.
Xenopus laevis: Practical Uses in Cell and Molecular
Biology. Academic Press, New York, vol 36:4560
-
Cicirelli MF, Tonks NK, Diltz CD, Weiel JE, Fischer EH, Krebs
EG 1990 Microinjection of a protein-tyrosine-phosphatase inhibits
insulin action in Xenopus oocytes. Proc Natl Acad Sci USA 87:55145518[Abstract]
-
Tonks NK, Cicirelli MF, Diltz CD, Krebs EG, Fischer EH 1990 Effect of microinjection of a low-Mr human placenta protein tyrosine
phosphatase on induction of meiotic cell division in Xenopus
oocytes. Mol Cell Biol 10:458463[Medline]
-
Sive HL, Grainger RM, Harland RM 1996 Early Development of
Xenopus laevis. Course Manual, ed 4. Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, NY
-
Nieuwkoop PD, Faber J 1994 Normal Table of Xenopus
laevis. Garland Publishing, New York