Cloning of the Mouse Insulin Receptor Substrate-3 (mIRS-3) Promoter, and Its Regulation by p53

Salvatore Sciacchitano, Andrea Orecchio, Luca Lavra, Silvia Misiti, Anna Giacchini, Massimo Zani, Daniele Danese, Aymone Gurtner, Silvia Soddu, Umberto Di Mario and Mario Andreoli

Chair of Endocrinology (S.Sc., A.O., L.L., S.M., A.G., M.A.), Second Faculty of Medicine, Università "La Sapienza" di Roma, Centro Ricerca Ospedale S. Pietro Fatebenefratelli, 00189 Roma, Italy; Department of Experimental Medicine and Pathology (M.Z.), Università "La Sapienza" di Roma, 00161 Roma, Italy; Italian Air Force, Medical Institute (D.D.), 00185 Roma, Italy; Molecular Oncogenesis Laboratory (A.G., S.So.), Regina Elena Cancer Institute, 00162 Roma, Italy; and Department of Clinical Sciences (U.D.M.), Università "La Sapienza" di Roma, 00161 Roma, Italy

Address all correspondence and requests for reprints to: Salvatore Sciacchitano, M.D., Ph.D., Chair of Endocrinology, Universita’ "La Sapienza" di Roma, Department of Experimental Medicine and Pathology, Viale Regina Elena, 324, 00161 Roma. E-mail: salvatore.sciacchitano{at}uniroma1.it.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The insulin receptor susbtrate-3 (IRS-3) is a member of a family of intermediate adapter proteins that function as major intracellular targets for phosphorylation by the activated insulin and IGF-I receptors. Among the four IRS proteins identified so far, IRS-3 exhibits a rather peculiar expression pattern during both the embryonic development and adult life, suggesting a different mechanism of regulation of its expression. In this study, we cloned the 5' flanking region of the mIRS-3 gene and analyzed its promoter activity. The mIRS-3 promoter is inhibited by wild-type p53, and this effect is completely abolished by cotransfection of a dominant negative p53. Tumor-derived p53 mutants show variable, but lower suppressing capability than wt p53. In addition, treatment with doxorubicin inhibits endogenous expression of mIRS-3 mRNA in C2C12 and 3T3-L1 cells. The DNA region spanning from nucleotides -287 and -178 in the mIRS-3 promoter is responsible for a 32.2% reduction of the mouse double minute 2 (MDM2) promoter activity, suggesting its involvement in the p53-mediated inhibitory effect. In conclusion, our study demonstrates that the mIRS-3 promoter is regulated by p53 at the transcriptional level. The inhibition of mIRS-3 promoter by wild-type p53, and its de-repression by tumor-derived p53 mutants, appears to be similar to that previously reported for the IGF-I receptor promoter, suggesting a common role of these two genes in p53-mediated cell growth and differentiation.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
INSULIN RECEPTOR SUBSTRATES (IRSs) play a central role in the transduction of metabolic and mitogenic signals stimulated by insulin and IGF-I. They serve as an important point in the intracellular pathway, activated by insulin-/IGF-I receptors, at which the two signals diverges to produce the multiplicity of their final biological effects. To date, four different IRS genes have been cloned, all of which share a common overall structure and the ability to activate the same intracellular signaling pathway [e.g. activation of phosphatidylinositol (PI) 3-kinase]. However, it has been reported that each substrate of this family may present a unique pattern of tissue distribution, and could mediate distinct functions. Experiments based on the targeted disruption of the IRS genes demonstrated that IRS-1 and IRS-2 play important roles in the regulation of growth and glucose homeostasis. Knockout mice lacking IRS-1 gene are growth retarded and moderately insulin resistant (1, 2). Mice deficient in IRS-2 gene develop diabetes early in life, due to the combination of severe insulin resistance and failure of pancreatic ß-cells to proliferate (3, 4). IRS-4 null mice exhibited mild defects in growth, reproduction, and glucose homeostasis (5). Surprisingly, IRS-3 knockout mice did not show any abnormalities in growth or in glucose homeostasis, suggesting that IRS-3 is not essential for these functions (6). Thus, even if several reports indicate that IRS-3 is able to mediate both mitogenic and metabolic functions induced by insulin and IGF-I, the exact role of IRS-3 is not clear yet. We recently purified, cloned, and characterized the structure of the IRS-3 gene in mouse (7). Expression studies, performed during mouse embryonic development, indicated that this gene is highly expressed in the first part of the embryonic life, and it progressively reduces its expression levels during the rest of the embryonic development (7). This pattern is similar to that observed for the product of the p53 tumor suppressor gene (8). The down-regulation of p53 at specific stages of embryonic development is considered an important step in embryonic growth and differentiation (9). p53 is a well-known regulator of cell-cycle checkpoints and apoptotic death, but considerable experimental evidence has accumulated, suggesting that a fine regulation of the p53 protein activity is required also for optimal development and differentiation (10, 11). In adult mice, mIRS-3 RNA is mainly expressed in adipose tissue and it is almost undetectable in skeletal muscle. Because the mechanisms of regulation of IRS-3 expression are currently unknown, we decided to clone its regulatory region and analyze its promoter activity. In this study, we demonstrate by transfection experiments using a p53 null cell line, the ability of wild-type (wt) p53 to inhibit mIRS-3 gene at the transcriptional level, whereas tumor-derived p53 mutants are responsible for a lower degree of this inhibitory effect. We cannot demonstrate a direct binding of p53 to specific DNA regulatory sequences, but we define a specific DNA region, responsible for the p53-mediated trans-repression. We observe the same inhibitory effect on endogenous mIRS-3 mRNA level after activation of p53 transcriptional properties by doxorubicin (Dox) treatment of two different cell lines. Our observation regarding p53 trans-repression of mIRS-3 promoter appears to be similar to that previously reported for the IGF-I receptor gene promoter (12). Based on these data, we postulate that IRS-3, one of the intracellular substrate for IGF-I receptor, could participate to p53-regulated cell growth and differentiation in a manner similar to that of IGF-I receptor.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cloning of the mIRS-3 5' Flanking Region and Primer Extension Analysis
A 1787-bp-long DNA sequence at the 5' flanking region of the mIRS-3 gene was subcloned into pBluescript SK- vector and sequenced (Fig. 1Go). This region lacks typical TATA boxes and shows many potential sites for ubiquitously transcription factors, such as SP1, AP1, AP2, and CAAT-box binding factor. These features suggest that, as already reported for the promoters of IRS-1 (13) and IRS-2 (14), IRS-3 might be a gene with housekeeping function. Sequence analysis of the mIRS-3 promoter region reveals the presence of many potential responsive elements (REs), including four decamers, highly related to the consensus DNA binding half-site for p53 (Pu-Pu-Pu-C-A/T-T/A-G-Py-Py-Py), which is known to mediate most of the stimulatory effects of p53 (15, 16). These four decamers are arranged head-to-head to form two potential p53 REs and are located in the region between nucleotides -1059 and -969 (Fig. 1Go). Each p53 RE consists of two half-sites, separated by 4 bp and 13 bp, respectively. Two major transcription starting sites (TSS), located at positions -179 and -357 respectively, have been identified by primer extension analysis (Fig. 2Go). The two major transcripts of the mIRS-3 gene, generated from these two TSS, differ by 178 bp and may account for the two bands previously observed by Northern blot analysis (7).



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Figure 1. Sequence Analysis of the 5' Flanking Region of the mIRS-3 Gene (GenBank Accession No. AF367626)

Nucleotide refers to the translation starting site, where +1 is numbering the adenosine of the ATG. Potential AP-1 binding sites are underlined. The transcription starting sites are indicated by arrows (->). The nucleotide sequence complementary to the two primers, used for primer extension analysis (Primer P-1 and Primer P-2) are underlined by arrows. The cutting positions of unique restriction enzymes are indicated with an inverted triangle and with the name of the enzyme. The nucleotide sequence of the four decamers, highly related to the consensus DNA binding half-site for p53, is shown in boxes.

 


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Figure 2. Primer Extension Analysis of the mIRS-3 Gene

Two oligonucleotides, Primer P-1 and Primer P-2 (for nucleotide sequences and position see Fig. 2Go) were end-labeled with [{gamma}32P] ATP and T4 polynucleotide kinase. About 15 µg of Total RNA, isolated from mouse liver, was mixed with the labeled primer and analyzed as described in Materials and Methods. The arrows to the right (<-) indicate the primer extension products. The sequence around the TSS is shown on the left.

 
Promoter Activity in Human Embryonic Kidney (HEK) 293 Cells
Transient transfection experiments and dual luciferase assays performed in HEK 293 cells, demonstrate that the 5' flanking region of the mIRS-3 gene possess clear promoter activity. A 14-fold induction is observed using the full-length promoter construct, compared with the empty vector. The promoter activity of the 5' progressively-deleted mIRS-3 promoter constructs is significantly reduced only when the deletion encompasses the sequence upstream to the two TSS (Fig. 3AGo). The initial analysis of the activity of mIRS-3 promoter constructs, designed to harbor a 300-bp deletion at their 5' ends has been also extended by including two additional constructs that contained both putative p53 REs [p1093-IRS3-luciferase reporter gene (LUC)], or only one of them (p1038-IRS3-LUC). The p1093-IRS3-LUC, shows the highest reporter gene activity among all pIRS3-LUC promoter constructs examined (Fig. 3AGo). The removal of one potential p53 RE (p1038-IRS3-LUC) results in the same promoter activity observed transfecting the full-length promoter construct (p1787-IRS3-LUC), whereas the removal of both p53 REs (p922-IRS3-LUC) causes a 50% suppression of the promoter activity. These data suggest the presence of a positive regulatory element in the DNA region, spanning nucleotide -1093 bp and -922 bp, from the ATG, where the two potential p53 REs are located. The activity of the reporter gene, driven by the mIRS-1 promoter (pIRS1-LUC), has been evaluated in the same cells and gives comparable, but lower results than that observed with mIRS-3 promoter (Fig. 3BGo).



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Figure 3. Functional Analysis of the mIRS-3 Promoter Constructs in HEK 293 Cells

The upper panel (A) shows the promoter activity of each mIRS-3 promoter construct. The two TSS are indicated by arrows (->), and the two putative p53 responsive elements (RE) are shown as filled triangles ({blacktriangleup}). The lower panel (B) shows the result of the same experiment, performed using the pIRS1-LUC promoter construct. The results of each experiment are normalized against the activity of the pRL vector, and are expressed as fold of induction above the basal level of the corresponding promoterless reporter gene (pGL3-LUC for IRS-3 and pGL2-LUC for IRS-1). Results represent the mean ± SD of three independent experiments, each performed in triplicate.

 
Analysis of p53 Binding to mIRS-3 Promoter
No direct binding of p53 to the putative p53 REs present in the mIRS-3 promoter sequence has been demonstrated in our in vitro EMSA, and the binding of p53, with its supershift using anti-p53 antibodies, has been observed only using our positive control (data not shown). The negative result of our EMSA has been confirmed in three different experiments, using as a probe either the 100-bp-long DNA fragment, spanning all the two different potential p53 REs or only one of them. The comparison between the nucleotide sequence of our putative p53 REs and the consensus sequence reported in the literature (16), shows that a perfect match is present only in the first decamer, while the other three show one mismatch each (Fig. 1Go). The lack of interaction between p53 and the putative REs in the mIRS-3 promoter by EMSA may be due to these differences in the nucleotide sequence. However, as already reported, p53 can modulate the activity of various TATA-less promoters (12, 17, 18, 19, 20, 21) without involving any direct interaction between p53 and DNA. To verify this hypothesis, and to demonstrate the presence of p53 in the DNA complex that binds in vivo to the proximal region of the IRS-3 promoter, we decided to perform a chromatin-cross-linked immunoprecipitation assay (ChIP) using proliferating C2C12 cells (22). PCR amplification of a 145-bp-long DNA fragment in the proximal region of the mIRS-3 promoter, demonstrate the presence of the mIRS-3 promoter DNA sequence in the p53-bound cross-linked chromatin of proliferating C2C12 cells. This result indicates that p53 participates in vivo to the complexes binding the mIRS-3 promoter in muscle cells, thus contributing to its transcriptional regulation.

Inhibitory Effect of p53 on mIRS-3 Promoter Activity
To analyze the effects of p53 on the mIRS-3 promoter activity, coexpression studies were performed in a p53 null cell system, using different IRS-3 promoter/ reporter constructs, together with wt p53 expression plasmid. Surprisingly, our results indicate that the cotransfection of wt p53 into the human non-small cell lung cancer H1299 cells markedly inhibits mIRS-3 promoter activity (Fig. 4AGo). This effect is not related to the presence or the absence of anyone of the four potential p53 REs present in this region because it can be detected using all the mIRS-3 promoter constructs, whose activities are suppressed by approximately 50% (Fig. 4AGo). Experiments performed using the p1487-IRS3-LUC promoter construct, result in a total suppression of promoter activity. Because the same promoter construct proved to be active in our experiments performed in HEK-293 cells, we postulate the occurrence of a cell-type specific mechanism of regulation, acting through the interaction of trans-factor with cis-elements present in the 300-bp region upstream to nucleotide -1487, yet to be identified. Murine IRS-1 promoter, analyzed in the same cells, shows very low level of its activity, compared with that observed with mIRS-3, and no effect can be induced by cotransfection of wt p53 (Fig. 4AGo). The inhibitory effect of wt p53 on mIRS-3 promoter activity is dose dependent, being maximum using 0.75 µg of transfected DNA, and no additional inhibitory effect can be obtained by increasing the amount of transfected p53 DNA (Fig. 4BGo). Cotransfection of both wt p53 and dominant negative p53 (dnp53) completely abolishes this inhibitory effect, strongly suggesting that p53 is responsible for the repression of mIRS-3 gene expression in our cell system (Fig. 4CGo). To further characterize the inhibitory action of p53 on the mIRS-3 promoter activity, we analyzed three different p53 mutants, deriving from human cancers, and known to alter the transcription properties of the p53 protein. The Arg175His p53 mutant proves to be a strong inhibitor of mIRS-3 promoter, with a 74% reduction, comparable to that observed using the wt p53 (Fig. 4CGo). The Arg273His p53 mutant is less effective in this inhibitory activity, causing a 43% reduction of the basal promoter activity. No inhibition has been observed using the third p53 mutant (Glu220Ser), which demonstrates to have completely lost the ability to suppress our promoter. No effect of wt p53 has been observed on the levels of luciferase and renilla/luciferase activities. The results of our transfection experiments, using the H1299 cells, suggest that the DNA sequence of mIRS-3 promoter, spanning from -287 to -178 bp, could be the most likely candidate region for the presence of a putative p53-responsive element, responsible for its negative regulation. This promoter fragment, in fact, is common to all promoter constructs inhibited by p53, and is absent in the only promoter construct p178-IRS3-LUC, whose reporter activity is too low to be further inhibited by p53 (Fig. 4AGo). To test the ability of this region to confer to an heterologous promoter a de-novo acquired responsiveness to the p53 inhibitory effect, we cloned this region upstream to the mouse double minute 2 (MDM2) promoter sequence. The fusion construct p287-IRS3/MDM2-LUC was transfected, together with wt p53, into H1299 cells. The two constructs, p178-IRS3/MDM2-LUC, and pMDM2-LUC were transfected as controls. A 32.2% reduction of luciferase activity is observed on cells transfected with p287-IRS3/MDM2-LUC, whereas no effect can be demonstrated on cells transfected with p178-IRS3/MDM2-LUC (Fig. 5Go). These results have been confirmed in three independent experiments, performed using two different DNA preparations, and strongly support our hypothesis that the DNA fragment located between -287 and -178 contains a putative region, responsible for p53 inhibition. However, further experiments need to be performed to better define this putative responsive region.



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Figure 4. Inhibitory Effect of wt and Mutant p53 on mIRS-3 Promoter in H1299 Cells

The upper panel (A) shows the inhibitory effect of wt p53 on mIRS-3 promoter constructs. The p53-null human non-small cell lung cancer H1299 cells were cotransfected with equimolar amounts of pIRS3-LUC promoter constructs (left), or pIRS-1-LUC (right), in the presence of 1 µg of either wt p53 (filled bars, {blacksquare}) or the empty control vector pLxsp (open bars, {square}). The promoter activity of each constructs, normalized against the activity of the pRL vector, is expressed as folds of induction above the basal level of the LUC reporter gene, obtained with the corresponding promoterless vector (pGL3 for IRS-3, and pGL2 for IRS-1). Results represent the mean ± SD of three independent experiments, each performed in triplicate. The middle panel (B) shows the dose-response suppression of mIRS-3 promoter activity by wt p53. The -1787IRS3-LUC promoter construct was cotransfected into H1299 cells with increasing amounts of the wt p53 expression vector pLx.sp. The values of luciferase activity are expressed as a percentage of the levels seen in the absence of p53. Results represent the mean ± SD of three independent experiments, each performed in triplicate. The lower panel (C) shows the inhibitory effect of three different mutant p53 proteins on the mIRS-3 promoter activity. H1299 cells were cotransfected with the -1787IRS3-LUC promoter construct and 1 µg of three different tumor-derived mutant forms of p53: R175H, R273H, and E220S. The promoter activity has been calculated as previously described and results represent the mean ± SD of three independent experiments, each performed in triplicate. The first three bars on the left represent the promoter activity of mIRS-3 in the absence or in the presence of wt p53, or in the presence of both wt p53 and dnp53, respectively.

 


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Figure 5. Identification of the mIRS-3 Promoter Region that Mediates the p53 Inhibitory Effect by Analyzing Its Ability to Suppress an Heterologous Promoter

p53-null human non-small cell lung cancer H1299 cells were cotransfected with equimolar amounts of MDM2-LUC promoter constructs (open bars, {square}), or the two fusion constructs, p287-IRS3/MDM2-LUC (filled bars, {blacksquare}), and p178-IRS3/MDM2-LUC (striped bars, ) in which the proximal region of mIRS-3 promoter, spanning nucleotide -287 to +1, and -178 to +1, respectively, was fused upstream to the MDM-2 promoter. The promoter activity of each constructs, normalized against the activity of the pRL vector, is expressed as folds of induction above the basal level of the LUC reporter gene, obtained with the promoterless vector. Results represent the mean ± SD of three independent experiments, each performed in triplicate.

 
The inhibitory effect of p53 on mIRS-3 gene expression was also analyzed by measuring the expression level of the endogenous mIRS-3 mRNA in proliferating C2C12 myoblasts and 3T3-L1 preadipocytes, after the induction of p53 transcriptional activity by cell treatment with Dox. This potent broad-spectrum anthracycline anticancer drug induces a genotoxic damage capable to activate p53 transcription properties. The effect of Dox treatment on mIRS-3 gene expression consists in a significant reduction of mRNA level and is the same in the two cell systems analyzed (Fig. 6Go). The calculated mIRS-3 mRNA expression level in Dox-treated 3T3-L1 cells is approximately 2.8 x 107 copy number/µg of total RNA, a value almost three times lower than that observed in untreated 3T3-L1 cells (9.1 x 107 copy number/µg of total RNA). A significant reduction of the level of mIRS-3 mRNA expression has also been observed in C2C12 cells after Dox treatment, with a calculated value of 5.0 x 107 copy number/µg of total RNA. Considering the expression level in untreated C2C12 cells, equal to 13.1 x 107 copy number/µg of RNA, the occurrence of a more than 2-fold suppression can be observed (Fig. 6Go). Taken together, all of these results indicate that mIRS-3 gene may be considered a novel p53 target along the pathway activated by the occurrence of a DNA damage.



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Figure 6. Analysis of the Effects of Dox-Mediated p53 Activation on mIRS-3 mRNA Expression Level in C2C12 and 3T3-L1 Cells

The upper panel (A) shows a typical agarose gel image, obtained from RT-cPCR analysis of mIRS-3 mRNA in untreated and Dox-treated C2C12 (left) and 3T3-L1 (right) cells. The cDNA fragments, corresponding to the 475-bp-long sequence of mIRS-3 gene, are visible in the upper part of each gel. The 368-bp-long of the competitor PCR products are visible in the lower part of the gels. The 257-bp-long band of the housekeeping gene GAPDH, was amplified from each sample to check for RNA integrity and used for normalization of mIRS-3 mRNA expression (lanes 5). The results of PCR, performed as negative control without adding reverse transcriptase to the reaction mixture, are visible in lanes 4. The lower panel (B) shows the analysis of the size-adjusted intensity of target bands, divided by the size-adjusted intensity of the competitor bands in either C2C12 (left) or 3T3-L1 (right) cells. The results have been plotted as function of the three different log copy number of competitor DNA/µg of total RNA (6.4 = log 2.7 x 107, 6.9 = log 8.6 x 107, 7.4 = log 2.7 x 108).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Insulin and IGF-I receptors are heterotetrameric, membrane-spanning, tyrosine protein kinases that, upon ligand binding, activate a number of shared intracellular signal transduction pathways, both during embryonic development (23, 24) and adult life (25, 26). The ligand-mediated activation of these two receptor tyrosine kinases induces the phosphorylation of IRS proteins. The phosphorylated IRS proteins, in turn, recruit SH2-domain-containing proteins, including PI 3-kinase, and trigger an insulin-stimulated protein kinase cascade, leading to glucose transport, protein synthesis, glycogen synthesis, cell proliferation, and cell survival in various cells and tissues (27). In addition to these effects, insulin and IGF-I play also a central role in suppressing programmed cell death, to support cell growth and survival (28). However, the precise mechanisms by which these two receptor tyrosine kinases (RTK) generate antiapoptotic signals are not well defined yet. Recent studies have shown that, in addition to the Ras-MAPK and PI 3-kinase pathways (29), RTKs support cell survival by regulating the function of two different types of the Bcl-2 family proteins. RTK-mediated cell survival is, in fact, regulated by the ratio of expressed amounts of antiapoptotic Bcl-2 family members, such as Bcl-2, and Bcl-XL, and proapoptotic Bcl-2 family members, including Bad, Bax, and Bik (30). Recently it has been reported that IRS proteins are involved in this process. IRS-1 and IRS-2 are able to suppress apoptotic cell death by regulating the phosphorylation of Bcl-2 (31). Several reports indicate that IRS-1 can mediate many antiapoptotic signals originated by growth factors such as insulin (32), IGFs (33, 34), and IL-4 (35), and the role of IRS proteins in regulation of cell proliferation is also confirmed by knockout experiments. Homozygous IRS-1-deficient mice are severely retarded in embryonal and postnatal growth (1, 2), whereas mice lacking both IRS-1 and IRS-2 are lethal (36). These observations are in agreement with the effects of the recently discovered Drosophila melanogaster homolog of vertebrate IRS-1–4 (37). This gene, named CHICO, plays an essential role in the control of cell size and growth of flies, and it also participates to the complex mechanisms that control life span in flies (38). Taken together, all these observations indicate that, both in mammals and in Drosophila, IRS proteins are critical for development, cell proliferation, and regulation of apoptosis.

IRS-3 shares the same overall architecture of the IRS family and seems to be able to bind both insulin and IGF-I receptors, to engage the same downstream signaling molecules (39) and to mediate the same metabolic and mitogenic effects induced by insulin and IGF-I (40). Nevertheless, IRS-3 presents some rather peculiar features, compared with the other members of IRS family. Even if its overall structure is similar to the other IRSs, IRS-3 protein is much smaller and contains fewer phosphorylation sites (7, 41). Unlike related IRS-1 and IRS-2 that are expressed in all the major mouse tissues (liver, skeletal muscle, heart, brain, spleen, and testis), IRS-3 is characterized by a very restricted tissue distribution (7). Indeed, despite the presence of its mRNA in liver, heart, kidney, and lung, the only tissue in which the IRS-3 protein has been definitively detected is white adipose tissue (42). We have previously reported that IRS-3 gene expression is developmentally regulated in an opposite way compared with IRS-1 (7). IRS-3 mRNA is highly expressed in the early stages of mouse embryonic development, at embryonic d 7 when IRS-1 is barely detectable and its level of expression decreases during the following stages of development, from embryonic d 11–17, when IRS-1 expression is very high (7). Another peculiarity of IRS-3, compared with the other IRS proteins, relies in its different cellular localization (43). Studies performed in adipocytes, characterized by the presence of all IRS proteins except IRS-4, have demonstrated that IRS-3 is located mainly in the plasma membrane fraction of adipocytes, whereas IRS-1 and IRS-2 are located mainly in the low-density microsomes (43). Another recent observation indicate that IRS-3 protein is localized into the nucleus, where it functions as a transcriptional activator, but its specific targets still needs to be identified (44). Besides these differences in mRNA expression and subcellular localization, IRS-3 presents some peculiarities in its function and in its pattern of activation upon insulin stimulation as well. Using a rat liver-derived cell line, it has been demonstrated that, despite IRS-1, IRS-2, and IRS-3 are equally phosphorylated after 2–5 min of insulin stimulation, IRS-3 presented a prolonged time course of phosphorylation, with a sustained phosphorylation detected up to 90 min after stimulation. In contrast, IRS-1 and IRS-2 phosphorylation declined after only 5 min (45). This robust and prolonged time course for IRS-3 phosphorylation may serve additional functions in cells that may require sustained tyrosine phosphorylation of IRS proteins.

It is interesting to note that, in contrast to IRS-1 and IRS-2, IRS-3 is unable to inhibit phosphorylation of Bcl-2, and for this reason can not suppress apoptosis. Not only does IRS-3 seem to not possess the same antiapoptotic effect shown by IRS-1 and IRS-2, but it acts as a proapoptotic agent, being capable to abrogate the synergistic survival effect of IRS-1 and Bcl-2 (31). Surprisingly, IRS-3 knockout experiments did not furnish any significant insight on the role and possible function of IRS-3 (6). Indeed, mice lacking IRS-3 did not show any abnormalities with regard to growth, development, and glucose homeostasis, suggesting that IRS-3 is not essential for growth or glucose homeostasis, and hence, the specific function of this protein still remains to be identified. In the present study, we cloned the promoter of mIRS-3 gene and demonstrate that it is regulated by p53. P53 is able to suppress all our mIRS-3 promoter construct, and this effect is not dependent on a direct binding of p53 to the putative REs present on the promoter region of the mIRS-3 gene. The p53 oncosuppressor protein has been largely characterized as cell-cycle checkpoint controller and as an apoptotic inducer, involved in the control of cell proliferation and tumor progression (46). However, increasing evidences indicate its involvement also in embryonic development and in cell differentiation (10, 11).

The physical interaction between p53 and mIRS-3 promoter is based on the results of the chromatin immunoprecipitation (ChIP) experiment that we performed in proliferating C2C12 cells. The experimental design (see Materials and Methods) of ChIP was based on the hypothesis that our putative p53 REs were capable to directly bind p53. However, our efforts to demonstrate the in vitro binding of p53 to any one of the four decamers, highly related to the consensus sequence for the half-site of p53 RE, were unsuccessful. This is in agreement with the previously reported observation that p53-mediated gene suppression does not require the interaction of p53 with its specific consensus sequence, but it involves its interaction with the basal transcription machinery (47, 48). This hypothesis was originally based on the observation that p53 can repress transfected reporter constructs, containing the minimal promoter sequence required for transcription. However, the presence of atypical p53 responsive sites, responsible for p53 mediated repression of transcription, has been recently reported (49). In our study, we could not demonstrate the in vitro binding of p53 to the putative p53 REs present in the mIRS-3 promoter sequence, but we were able to identify a DNA region that could mediate p53 trans-repressive action on mIRS-3 promoter. Not only the deletion of this region abrogates the inhibitory effect of p53 on our promoter, but it is also able to suppress the activity of a heterologous promoter. In fact the fusion of this region upstream to the intronic sequence of the MDM2 gene, that contains a p53 response sequence (50), significantly reduces the stimulatory effect of p53.

We were able to confirm our results regarding the inhibitory effect of p53 on mIRS-3 promoter, obtained by transfection experiments, by analyzing the endogenous mIRS-3 gene expression after exposing C2C12 cells to Dox. The DNA damage induced by Dox treatment is responsible for a p53 activation, and we demonstrate that p53 activation is able to down-regulate mIRS-3 gene expression. Taken together, all of these results suggest that mIRS-3 gene may be included in the group of genes repressed by p53, and it can be involved in the p53-mediated cascades that regulate cell proliferation and tumorigenesis. The inhibitory effect exerted by p53 on the mIRS-3 promoter has been previously described on another gene belonging to the same signaling pathway, the IGF-I receptor gene (12). The IGF-I receptor functions as an antiapoptotic agent by enhancing cell survival and it plays a central role in mediating the trophic and differentiative actions of the IGFs, IGF-I, and IGF-II (51, 52). wt p53 is able to suppress the IGF-I receptor promoter in the postmitotic, fully differentiated cells at the level of transcription and this effect involves the interaction with TATA-binding protein, the TATA box-binding component of transcription factor IID (12). The expression of the IGF-I receptor gene is constitutively inhibited by p53 in terminally differentiated cell and, as result of this negative control, the cells remain in a postmitotic state. A rebound in gene expression has been observed in malignant states, associated with augmented cellular proliferation (53, 54). In addition, it has been demonstrated that tumor-derived mutant forms of p53 can lose this gene suppression capacity, thus leading to a de-repression of the IGF-I receptor promoter. This results in an increase of the cell-surface receptors and a marked antiapoptotic effect, mediated by circulating IGFs, with a regression of the cells toward a more undifferentiated phenotype. The observation that IGF-I receptor is overexpressed in most malignancies with p53 mutants reinforces the validity of such a mechanism of action (12). In our study, we show that among three different tumor-derived p53 mutants, two of them have lost the ability of suppressing the activity of the mIRS-3 promoter. These different behaviors is consistent with the finding that diverse p53 mutants can acquire different gain-of-function properties (55). Considering all of these observations, it can be postulated that p53 may counteract the antiapoptotic effects of IGF-I by acting at different levels of the IGF-I signaling pathway, i.e. at the level of IGF availability, by inducing the IGF binding protein 3 gene expression (56), at the cell surface, by suppressing the transcriptional activation of IGF-I receptor, and, finally, at the intracellular level, by inhibiting the IRS-3 promoter activity. We believe that the availability of the full-length IRS-3 promoter and the characterization of its regulation by p53 provides a framework for future studies addressing the role of this gene in insulin-sensitive tissues, and the complex relationship between insulin- and IGF-I-mediated signaling pathways and basic cellular functions, such as proliferation, apoptosis, and differentiation, regulated by p53.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell Cultures
Tissue culture media and reagents used for cell culture were purchased from EuroClone Ltd. (West York, UK), except for the fetal bovine serum (FBS), which was obtained from HyClone Laboratories, Inc. (Logan, UT). Human embryonic kidney cell line, HEK 293, used for the analysis of the basal promoter activity of mIRS-3 and mIRS-1 promoter constructs, were grown in DMEM supplemented with 10% FBS. The effects of p53 on the activity of both IRS-1 and IRS-3 promoters were analyzed in H1299 cells, derived from a human p53 null, non-small cell lung cancer, and cultured in Roswell Park Memorial Institute 1641 medium, supplemented with 10% FBS. C2C12 myoblasts were cultured in DMEM, supplemented with 10% FBS. The 3T3-L1 preadipocytes were purchased from American Type Culture Collection (Manassas, VA) and grown in DMEM with L-glutamine, 50 µg/ml penicillin, 100 µg/ml streptomycin, and supplemented with 10% FBS. To avoid spontaneous differentiation, C2C12 and 3T3-L1 cells were never allowed to reach confluence. To induce p53 transcriptional activity, both C2C12 and 3T3-L1 cells were grown in medium supplemented with 0.5 mM Dox (Sigma, St. Louis, MO) and incubated for 18 h.

Cloning and Sequencing of the 5' Region of the Murine IRS-3 Gene
A 4.8-kb HindIII DNA fragment, containing approximately 1.8 kb of the 5' flanking region, 1.829 bp of the coding region of the mIRS-3 gene, including the 344-bp-long intronic sequence, and approximately 1.2 kb of the 3' flanking region, was subcloned from a P1 clone, previously isolated from a mouse genomic DNA library (7). The 5' flanking region of the mIRS-3 genomic clone was inserted into the pBluescript Sk vector, and its nucleotide sequence was determined by automatic sequencing (ABI-373, PE Applied Biosystems). Computer analysis of this region, in search of putative CIS-acting elements, was performed using the Signal Scan computer program, available at the human genome mapping project web site (http://www.hgmp.mrc.ac.uk/).

Primer Extension Analysis
Primer extension analysis was performed using two different oligonucleotides complementary to the 5'-region of the mIRS-3 gene (primer P1, from nucleotides -113 to -93, and primer P2, from -192 to -174, respectively). The two oligonucleotides were end-labeled with [{gamma}-32P]ATP using T4 polynucleotide kinase (Roche Molecular Biochemicals, Basel, Switzerland). Fifteen micrograms of mouse liver total RNA, purchased from CLONTECH Laboratories, Inc. (Palo Alto, CA), were added to a mix containing 1.5 ng of the labeled primer; 250 mM Tris-HCl, pH 8.3; 200 mM KCl; 35 mM MgCl2; 2 mM deoxy (d) nucleotide triphosphate (NTP), 10 U ribonuclease inhibitor (Perkin-Elmer Corp., Boston, MA), heated at 94 C for 5 min, annealed at 56 C for 15 min, chilled on ice, and reverse transcribed in the presence of 500 U of Moloney murine leukemia virus reverse transcriptase (Gen Hunter, Nashville, TN). A reaction containing yeast RNA was performed as negative control. DNA sequence reaction was performed by the di-deoxy chain termination method (57), using a Sequenase DNA sequencing kit (Amersham Pharmacia Biotech, Buckinghamshire, UK), in the presence of the same primers used for primer elongation. Primer extension and sequencing products were resolved on a 6% urea-acrylamide gel, at the following running conditions: 60 W constant for 2 h in 1x Tris-boric acid EDTA buffer. The gel was then exposed to autoradiography at -80 C for 18 h.

Plasmid DNA Constructs
The following plasmids were used for transfection experiments: p1787-IRS3-LUC, p1487-IRS3-LUC, p1187-IRS3-LUC, p1093-IRS3-LUC, p922-IRS3-LUC, p887-IRS3-LUC, p587-IRS3-LUC, p287-IRS3-LUC, and p178-IRS3-LUC, carrying the LUC, driven by the mIRS-3 promoter sequence between -1787, -11487, -1187, -1093, -922, -887, -587, -287, -178 and +1, respectively, relative to the translation start site. For the construction of these expression vectors, all the 5' progressively deleted DNA fragments of the mIRS-3 promoter were amplified by PCR in the presence of specific chimeric primers, containing extensions of either KpnI or BglII restriction sites. PCRs were performed using Pyrococcus furiosus (Pfu) Turbo polymerase (Stratagene, La Jolla, CA) in the presence of: 1x Pfu buffer, 200 µM of each dNTP, 1.25 U of Pfu Turbo Polymerase, 25 ng template DNA, and 0.2 µM of each primer. Cycling parameters were as follows: 21 cycles of denaturing at 94 C for 1 min, annealing at 55 C for 1 min, and extension at 72 C for 1 min. PCR products were resolved on agarose gel. The expected bands were gel-purified using the Gene Clean Kit (Bio101, Carlsbad, CA), then digested with both KpnI and BglII, and ligated into the promoter-less LUC reporter plasmid pGL-3 basic (Promega Corp., Madison, WI). All the constructs generated by PCR were analyzed by direct sequencing. To better define the mIRS-3 promoter region responsible for p53 trans-repressive activity, different mIRS-3 promoter DNA fragments were tested for their ability to induce repression of heterologous promoters, known to be trans-activated by p53. Based on the luciferase assay results on H1299 cells, the p287-IRS3-LUC construct appeared to contain a putative p53-responsive DNA region. For this reason, the -287 DNA fragment was cloned on the upstream region of MDM2 promoter, previously described as one of the most strongly p53-activated promoter (50). In addition, the -178 DNA fragment was cloned in the same region of the MDM2 promoter and used as control. DNA sequences spanning, -287 to +1, and -178 to +1 of mIRS-3 promoter were amplified by PCR and cloned upstream to the p53-responsive element of the MDM2 promoter, into the pGL-3 basic vector, to generate these two fusion constructs, named p287-IRS3/MDM2-LUC and p178-IRS3/MDM2-LUC, respectively. PCRs were performed using the conditions described above, with specific primers containing extensions for KpnI restriction enzyme. PCR products were then purified, digested with KpnI and subcloned into the KpnI restriction sites of the MDM2-LUC construct. Fragments orientation was verified by PCR and by direct sequence analysis. MDM2-LUC, p287-IRS3/MDM2-LUC, and p178-IRS3/MDM2-LUC constructs were transfected into H1299 cells in the presence of wt p53. pIRS1-LUC, carrying 3369 bp of the mouse IRS-1 promoter (13), upstream of the luciferase reporter gene into the pGL-2 basic vector (Promega Corp.), was used as control for transfection experiments. To test p53 responsiveness, wt, and mutant forms of p53, inserted into a cytomegalovirus-driven expression plasmid (pLXSp, obtained by substituting the neomicine-resistance gene of the LXSN vector with the puromicin-resistance gene), were transiently transfected into H1299 cells. The following tumor-derived p53 mutants were used: Arg175His, Arg273His, and Glu220Ser. In addition, to demonstrate the specific involvement of p53 in the regulation of mIRS-3 promoter activity, the dnp53 (58) was cotransfected together with the wt p53. A plasmid that encodes for the renilla luciferase (pRL) (Promega Corp.), was cotransfected with experimental vectors (1:20 molar ratio), and used as an internal control both for transfection and luciferase assay.

Dual-Luciferase Reporter Assay
Luciferase activity of the reporter gene was evaluated in HEK 293 and H1299 cell lines, growing on 24 multiwell plates, transiently transfected with the mIRS-3 and mIRS-1 promoter constructs, using the Dual-Luciferase Reporter Assay System (Promega Corp.) as previously described (59). For each experiment, at least three independent transfections were performed using Superfect reagent (QIAGEN). Each well received 1 µg of reporter plasmid, 50 ng of the pRL plasmid, and 5 µl of Superfect reagent. For transfection experiments, performed on the p53 null H1299 cell line, 1 µg of plasmid DNA encoding wt or mutant murine p53, was cotransfected together with the reporter plasmids. After a 24-h recovery period, transfected cells were harvested and lysed in the presence of 100 µl of passive lysis buffer (Promega Corp.). The functional activity of the mIRS-3 promoter constructs was measured using the TD20/20 double injector luminometer (Turner Designs, Sunnyvale, CA). An aliquot (20 µl) of cell lysate, derived from each experiment, was subjected to Dual-Luciferase Reporter Assay. The results were expressed as folds of induction, calculated by normalizing the ratio of the firefly/renilla luminescences, obtained by transfecting the experimental vectors, with the same ratio, obtained using the corresponding promoterless control vector. In our experiments, we did not observe any effect of either wt or mutant p53 on the levels of expression of the renilla luciferase, which proved not to be affected by p53 stimulation. Paired Student’s t tests were used to compare individual points where appropriate. Values of P < 0.05 were considered to indicate statistical significance.

EMSA
To analyze the in vitro interaction between p53 and the mIRS-3 promoter region, an EMSA was performed. The mIRS-3 promoter region, containing the four decamers highly homologous to the p53 consensus sequence spanning nucleotides -1059 and -969, was used as probe. This region was subdivided into two distinct DNA fragments, Probe 1 corresponding to sequence -1065 to -1034 (5'-CCTACTAGACAAGTTCTAGTGAGCCTGTCTCAAAA-3' and its corresponding antisense), and Probe 2 from nucleotide -1002 to -951 (5'-AGAGGTTGTCTTCCAGCCTCCAATAAGCATGTGCA and its corresponding antisense). These probes have been end labeled with [{gamma}-32P]ATP, using T4 polynucleotide kinase (Roche Molecular Biochemicals). The palindromic p53 recognition site (5'-GGACATGCCCGGGCATGTCC-3') that binds p53 with high affinity was included in the experiment as a positive control (60). Approximately 0.3 ng of each probe has been added to 6 µg of nuclear proteins, extracted from C2C12 cells using the protocol previously described (61). The protein contents in nuclear extracts were measured by the Bradford procedure. To obtain supershift of p53-DNA complexes reaction 1 µg of affinity purified sheep polyclonal anti-p53 Ab-7 antibodies (Calbiochem, Darmstadt, Germany) has been added to the mixtures. The mixtures were incubated with 1 µg of poly (deoxyinosine-deoxycytidine) in a binding buffer consisting of 100 mM HEPES, pH 7.9; 5 mM dithiothreitol; 10 mM MgCl2; 0.5 mg/ml BSA; and 50% glycerol. DNA-protein complexes were resolved on a 4% polyacrylamide gel with 0.5x Tris borate-EDTA buffer, at 150 V for 2 h and 30 min at 4 C. The gel was dried and autoradiographed with intensifying screen at -80 C.

Chromatin Immunoprecipitation
Proliferating, subconfluent C2C12 cells were cross-linked by adding formaldehyde, directly to culture medium. DNA preparation, immunoprecipitation, and amplification were performed as described (22). Anti-p53 Ab-7 antibodies (Calbiochem) were used for immunoprecipitation experiments. A negative control without antibody as well as a positive control, were included in the immunoprecipitation and PCRs. The following primers were employed to amplify a 145-bp-long DNA fragment of the murine IRS-3 promoter: forward primer 5'-CTGAGGCCTTCCAGGCCTGC-3', and reverse primer 5'-GGAGATCATAGGCCACAAAT-3'. These primers were designed to amplify the DNA region spanning all the four potential p53 binding sites present in the mIRS-3 promoter. DNA amplification reactions were performed in a final volume of 50 µl using the following reagents: AmpliTaq Gold DNA Polymerase (Perkin-Elmer Corp.) MgCl2 (1.5 µM), all four deoxyribonucleotide triphosphates (200 µM), and oligonucleotide primers (0.2 µM). PCR was carried out for 30 cycles under the following conditions: denaturation at 95 C (10 min in the first cycle, 1 min thereafter), annealing at 55 C (1 min), extension at 72 C (1 min, but extended for 10 min in the last cycle). PCR products were resolved onto a 3% agarose gel composed of Nu Sieve and SeaKem (3:1) (FMC Bioproducts, Rockland, ME) and ethidium bromide (0.5 µg/ml) and visualized under UV light. The gel was then transferred onto Nytran Supercharge transfer membrane (Schleicher \|[amp ]\| Schuell, Keene, NH) and hybridized with an oligonucleotide probe, internal to the amplified region. The oligonucleotide probe used (5'-AGAGGGATCGCTCTTGAGGA-3') was end-labeled with [{gamma}32P]-dATP (Amersham Pharmacia Biotech) using the T4 polynucleotide kinase, according to the method previously described (62).

Quantitation of the Expression of mIRS-3 Gene by RT-Competitive PCR
A 325-bp-long DNA fragment at the 3' end of the coding sequence of mIRS-3 has been chosen to generate the DNA competitor, used as an exogenous internal standard in the competitive PCR, according to the method previously described (63). Briefly, one of the primers used for the amplification of the target DNA sequence, was designed as a chimeric primer that included a 20-bp-long sequence located 150 bp upstream to the sequence recognized by the 3' end of the same primer. In addition, the forward and reverse primers contained the sequence of the BamHI and HindIIII enzyme recognition sites, respectively, to allow the cloning into the pBluescript vector. The target DNA sequence, corresponding to the 3' region of mIRS-3 gene, was, then, amplified from the cDNA obtained by the cells, using the following specific primer pair (forward primer: 5'-TGGGCGGGAACTACATCACCAT-3' and reverse primer: 5'-CCCAAGCTTGGGGAACTTGATGCTGGCATA-3'). A quantitative analysis of mIRS-3 gene expression was performed by reverse transcription competitive PCR (RT-cPCR), using total RNA, obtained from C2C12 and 3T3-L1 cells, treated or not with Dox, to induce p53 transcriptional activity. cDNA was synthesized in a total volume of 20 µl of reaction mixture containing 1 µg of RNA, 0.5 µM each of random hexamers and oligo(deoxythymidine), 1x RT reaction buffer (QIAGEN, Milan, Italy), 0.5 mM of each dNTP, 10 U of ribonuclease inhibitor (Perkin-Elmer Corp.), and 4 U (1 µl) of Omniscript RT enzyme (QIAGEN). Samples were incubated at 37 C for 2 h, and the reaction was terminated by heating at 93 C for 5 min, followed by rapid chilling on ice. Preliminary series of six competitive PCRs, using 10-fold dilutions of DNA competitor (ranging from 1 x 10-3 µg to 1 x 10-8 µg equal to 2.7 x 103 to 2.7 x 108 copy numbers), in the presence of a constant amount (2 µl) of the first-strand cDNA obtained from untreated, and Dox-treated cells, were performed in a total volume of 50 µl. PCR mixture contained 0.4 µM of each primer set, 0.2 mM of each dNTPs and 1 U of Ampli Taq Gold (Perkin-Elmer Corp.). To better quantitate the mIRS-3 mRNA expression level, a second round of RT-cPCRs was performed using only three dilutions of competitor DNA, namely 1 x 10-4 µg, 1 x 10-5 µg, plus the intermediate dilution corresponding to 1 x 10-4.5 µg. In this second set of RT-cPCRs we excluded the competitor DNA concentrations that, in the first series of RT-cPCR, resulted to high or to low for the competition to occur. The housekeeping GAPDH gene mRNA was amplified using the following primers: forward, 5'-ATCATCCCTGCATCCACTGG-3' and reverse 5'-TGGGAGTTGCTGTTGAAGTC-3', to check the integrity of RNA and normalize the results. cPCR comprised 35 cycles for mIRS-3 and 26 cycles for GAPDH, each one of them composed of denaturing at 94 C for 1 min, annealing at 58 C for 1 min, and extension at 58 C for 1 min. At least six RT-cPCR experiments were performed starting from two different RNA preparations. Negative controls without cDNA were also run at the beginning and at the end of each single PCR experiment to check for contamination by PCR products from previous PCRs. PCR products, obtained from the second set of RT-cPCRs, were resolved onto a 2% agarose gel, composed of Nu Sieve and SeaKem (3:1) (BioWhittaker, Inc., Rockland, ME) and ethidium bromide (0.5 µg/ml) and visualized under UV light. The length of the amplified mIRS-3 cDNA (475 bp) was easily distinguishable from the length of the internal control, used as a competitor (368 bp), and amplified in the same reaction with the same set of primers. Because the primers used in the amplification were identical, the cDNA and the internal control were amplified in a reaction in which a competition took place for the available PCR substrates. The images of the gels were photographed and imported into the NIH image computerized densitometry program (NIH Image, version 1.57) for quantitative analysis of each PCR product. Data from densitometry were analyzed using Microsoft Corp. Excel software for Power Macintosh. Molar ratios were calculated by dividing the density of the bands by the length of the corresponding DNA molecules. To determine the number of target cDNA copies, the molar ratio intensities of each competitor band and the corresponding target band were plotted, against three different competitor DNA concentrations (10, 31.6, and 100 pg/µg of total RNA), expressed as log copy number/µg of total RNA. For each experiment a standard curve was calculated, using linear regression, and the equivalence point (log molar ratio = 0) was determined to calculate the copy numbers of mIRS-3 mRNA.


    ACKNOWLEDGMENTS
 
We are grateful to Roberto Dominici and Carlo Santolamazza for excellent technical assistance.


    FOOTNOTES
 
This work was supported by a grant from the Italian "Ministero dell’Università e della Ricerca Scientifica e Tecnologica" (MURST), and by the Institute of Neurobiology and Molecular Medicine, CNR. This sequence data has been submitted to the DDBJ/EMBL/GeneBank databases under accession no. AF367626.

Abbreviations: ChIP, Chromatin-cross-linked immunoprecipitation assay; d, deoxy; dnp53, dominant negative p53; Dox, doxorubicin; FBS, fetal bovine serum; HEK, human embryonic kidney; IRS, insulin receptor substrate; LUC, luciferase reporter gene; MDM2, mouse double minute 2; mIRS-3, mouse insulin receptor substrate-3; NTP, nucleotide triphosphate; Pfu, Pyrococcus furiosus; PI 3-K, phosphatidylinositol 3-kinase; pRL, renilla luciferase; RE, responsive element; RT-cPCR, RT-competitive PCR; RTK, receptor tyrosine kinase; SH2, Src homology 2; TSS, transcription starting site; wt, wild-type.

Received for publication July 13, 2001. Accepted for publication March 14, 2002.


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