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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 (33–45% 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 (31–46%) 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 {Delta}PH) significantly reduced the ability of xIRS-u to potentiate insulin signaling. In contrast to the full-length protein, injection of xIRS-u (1–299), which encoded the PH and PTB domain, or xIRS-u (1–170), which encoded only the PH domain, blocked insulin signaling in Xenopus oocytes. Finally, xIRS-u (119–299), 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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 313–462), 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1AGo).





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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.

 
Two possible translation initiation sites were found in the xIRS-u cDNA (Fig. 1AGo). 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. 1AGo) (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. 1BGo) indicate that xIRS-u has similar overall sequence homology to all members of the mammalian IRS family (33–45% 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 (31–46%). This pattern of sequence homology is also reflected when individual domains are compared (Fig. 1CGo). xIRS-u encodes a putative PH domain (amino acids 63–166) 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 193–293) 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. 1CGo), and much less similar to their counterparts in other IRS members (IRS-2, IRS-3, or IRS-4) (Fig. 1BGo). 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. 1CGo). 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. 1AGo). 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. 2Go 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. 1AGo, 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.

 
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. 3AGo). xIRS-u represents the full-length protein initiating at amino acid 1. xIRS-u PH-PTB contains amino acids 1–299. xIRS-u {Delta}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). {Delta}PH-PTB was derived from PH-PTB with the same amino-terminal (1–118) deletion. xIRS-u PH domain (amino acids 1–170) was also derived from PH-PTB construct, by an internal deletion that removed amino acids 171–299. 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. 3BGo). 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. 3BGo, lane 2), the identity of which are not known. xIRS-u {Delta}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 {Delta}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. 7BGo).



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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. 7CGo) or lysed for xMAP kinase Western blot analysis (A). Protein expression was analyzed by anti-Myc Western blot (B).

 
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 3–5 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 8–12 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 5–10 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. 4Go, 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. 3BGo, 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).

 
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 {Delta}PH to potentiate insulin-induced MAP kinase activation (Fig. 5Go). Enhancement of insulin-induced MAP kinase activation was observed in oocytes injected with both xIRS-u and xIRS-u {Delta}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 {Delta}PH (lane 6) while only partial activation was observed in control oocytes (lane 4). It is interesting to note that xIRS-u {Delta}PH was less effective than xIRS-u in enhancing insulin-induced MAP kinase activation (Fig. 5Go, 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 {Delta}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 {Delta}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.

 
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 (1–299), which contained both the putative PH and PTB domains, was able to inhibit insulin signaling in Xenopus oocytes (Fig. 6AGo). 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. 6Go, compare lanes 3 and 1) with a concomitant inhibition of insulin-induced GVBD (not shown, but see Fig. 7CGo). The inhibition by the PH-PTB construct was significant even when the concentration of the mRNA was reduced to one third (Fig. 6AGo, compare lanes 4 and 1) [with a corresponding reduction in the expression of the protein (Fig. 3BGo, lanes 4 and 5)]. In contrast, xIRS-u (119–299), 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. 6BGo). Injection of xIRS-u (1–299) mRNA blocked insulin-induced MAP kinase activation (Fig. 6BGo, 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. 7CGo).



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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.

 
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. 7AGo), with a concomitant inhibition of insulin-induced GVBD (Fig. 7CGo). In contrast, injection of the PH domain did not affect MAP kinase activation (Fig. 7AGo) or GVBD (Fig. 7CGo) induced by progesterone.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1Go, 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 (31–46%), xIRS-u has similar sequence homology to all mammalian IRS family members (33–45%). 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. 5Go) 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. 6Go and 7Go). 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 {alpha}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 Denhardt’s 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. 1AGo, 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 1–299 (PH-PTB) of xIRS-u (Fig. 1AGo). To construct the full-length Myc-tagged xIRS-u (amino acids 1–1003), the EcoRI-EcoRI fragment (Fig. 1AGo, 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 {Delta}PH-PTB) and the full-length xIRS-u (to generate xIRS-u {Delta}PH), the NcoI-EcoRI fragment (encoding amino acids 1–299) was replaced with a PCR-amplified fragment encoding amino acids 119–299. 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. 1AGo) and an XbaI of the vector, resulting in expression of the first 170 amino acids corresponding almost precisely to the PH domain (Fig. 1AGo). 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) 3–10 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 2–5 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 manufacturer’s 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.


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