help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2004-0668
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
145/11/5332    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sun, H.
Right arrow Articles by Baserga, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sun, H.
Right arrow Articles by Baserga, R.
Endocrinology Vol. 145, No. 11 5332-5343
Copyright © 2004 by The Endocrine Society

Deletion of the Pleckstrin and Phosphotyrosine Binding Domains of Insulin Receptor Substrate-2 Does Not Impair Its Ability to Regulate Cell Proliferation in Myeloid Cells

Hongzhi Sun and Renato Baserga

Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107

Address all correspondence and requests for reprints to: Renato Baserga, M.D., Kimmel Cancer Center, Thomas Jefferson University, 233 South 10th Street, 624 BLSB, Philadelphia, Pennsylvania 19107. E-mail: b_lupo{at}mail.jci.tju.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
32D IGF-I receptor (IR) cells are IL-3-dependent myeloid cells that can be induced to differentiate into granulocytes by IGF-I. Like the parental 32D cells, 32D IGF-IR cells do not express the insulin receptor substrate (IRS)-1 or IRS-2. We investigated the effect of ectopic expression of IRS-2 in 32D IGF-IR cells. Expression in these cells of a wild-type IRS-2 inhibits IGF-I-induced differentiation, and the cells grow indefinitely in the absence of IL-3. We also investigated the effect of a mutant IRS-2 lacking both the pleckstrin (PH) and the phosphotyrosine-binding (PTB) domains, which are known to bind to the IR. The {partial}PHPTB IRS-2 is fully as capable as the wild-type IRS-2 (and wild-type IRS-1) to stimulate the growth and inhibit the differentiation of 32D IGF-IR cells. In contrast, an IRS-1 protein lacking the same PH and PTB domains is completely inactive in blocking differentiation and stimulating IL-3-independent growth of 32D IGF-IR cells. The {partial}PHPTB IRS-2 protein is dependent for its effect on an activated IGF-IR, is cytoplasmic, binds to the ß-subunit of the IGF-IR, and requires for its action the presence of phosphatidylinositol 3-kinase binding sequences. These experiments show that the PH and PTB domains of IRS-2 (but not IRS-1) are dispensable for the IGF-I/IRS-2-mediated growth of 32D myeloid cells. Our results also indicate that IRS-2 (either wild type or {partial}PHPTB) is capable of inhibiting the differentiation of 32D cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
32D CELLS ARE murine myeloid cells that depend on IL-3 for growth and rapidly undergo apoptosis after IL-3 withdrawal (1, 2). Parental 32D cells have low IGF-I receptor (IGF-IR) levels and do not express insulin receptor substrate (IRS)-1 or IRS-2 (3, 4). When the levels of IGF-IR are increased from 2 x 103 to 17 x 103 receptors/cell, still a physiological number (5) the resulting 32D IGF-IR cells, in the absence of IL-3 and with the addition of IGF-I, grow exponentially for about 48 h. After 48 h, 32D IGF-IR cells stop growing and begin to differentiate along the granulocytic pathway (4). When 32D IGF-IR cells are stably transfected with a plasmid expressing IRS-1 (32D IGF-IR/IRS1 cells), the cells no longer differentiate (4), grow indefinitely in the absence of IL-3, and form tumors in mice (6). Ectopic expression of IRS-1 only in parental 32D cells does not result in prolonged survival in the absence of IL-3 (2, 4, 7). Thus, IRS-1 or the IGF-IR, singly, cannot protect parental 32D cells from apoptosis. In combination, they cause malignant transformation of 32D cells. In 32D IGF-IR cells, IGF-I induces phosphorylation of the 42-kDa isoform of the Shc proteins (that are very highly expressed in these cells), and a dominant-negative mutant of Shc partially inhibits differentiation (6). A reasonable explanation is that in 32D cells, the IGF-IR sends signals for both proliferation and differentiation (reviewed in Ref. 8). When IRS-1 is the predominant substrate, proliferation prevails, whereas the cells tend to differentiate if Shc is predominant. The antidifferentiation role of IRS-1 in these cells is compatible with the observation that cell types prone to differentiation often do not express IRS-1 or express very low levels (3, 9, 10, 11, 12, 13).

The ability of IGF-I to sustain the exponential growth of 32D IGF-IR cells for 48 h before differentiating is not an unusual occurrence. In hemopoietic cell lines, the induction of differentiation is often preceded by a period of vigorous cell proliferation (1, 14, 15). It has been hypothesized that in hemopoietic cells, like 32D myeloid cells, sustained cell proliferation requires not only stimulation of cell growth, but also the extinction of a differentiation program. This hypothesis has the support of a number of investigators who suggested that a block in differentiation is a requirement for the development of certain forms of leukemia. Gilliland and Tallman (16) summarized the hypothesis by proposing two classes of mutations in acute leukemia: one class of mutations confers a proliferative advantage, whereas a second class of mutations inhibits differentiation.

We recently reported that IRS-1 and IRS-2 can translocate to the nuclei of 32D IGF-IR/IRS-1 cells and mouse embryo fibroblasts (MEFs) (17, 18, 19). IRS-1 and, to a lesser extent, IRS-2 also localize to the nucleoli, in which they bind the upstream binding factor (UBF) (18, 19), a key regulator of rRNA synthesis (20, 21). The nuclear translocation of IRS-2 was observed in MEFs, which express substantial levels of IRS-2. Because parental 32D cells (3) and 32D IGF-IR cells (22) do not express IRS-2, we wanted to investigate the effect of its ectopic expression in these cells. At the same time, we also studied a mutant IRS-2, lacking the pleckstrin (PH) and the phosphotyrosine binding (PTB) domains ({partial}PHPTB IRS-2). This mutant was reported to bind the insulin receptor (23) and stimulate cell proliferation induced by IL-4 in 32D cells overexpressing the insulin receptor (24). IL-4 cooperates with IGF-I in stimulating the growth of 32D-derived cells (25). IRS-2 is often thought to be less mitogenic than IRS-1 (26) and to play a more significant role in differentiation (9, 27), and we wanted to define its action in 32D IGF-IR cells and compare it with that of IRS-1 (28, 29, 30).

We demonstrate here that wild-type IRS-2 can replace IRS-1 in inhibiting the differentiation and inducing IL-3-independent growth of 32D IGF-IR cells. The {partial}PHPTB/IRS-2 also inhibits IGF-I-mediated differentiation of 32D IGF-IR cells, which become IL-3 independent. Our results also indicate that, in myeloid cells, the predominant signal of IRS-2 is proliferative and that the PH and PTB domains of IRS-2 (but not IRS-1) are dispensable for its growth-promoting and antidifferentiation effects. Although a proliferative effect of {partial}PHPTB IRS-2 has already been reported with the insulin receptor and IL-4 (24), our results extend the effect to the IGF-IR and, in addition, localize with some precision the sequences of IRS-2 that are necessary and sufficient for the inhibition of differentiation and IL-3 independence.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Construction of plasmids
Mouse IRS-2 cDNA, kindly provided by Dr. Sciacchitano (Universitá di Roma, Rome, Italy), was excised from pCis-2 plasmid and cloned in-frame into the pMSCVpac retroviral vector. PHPTB IRS-2 was constructed and adapted by PCR to generate an XhoI-EcoRI fragment. This fragment containing the PHPTB domains of IRS-2 was subcloned into XhoI-EcoRI-cut of the pMSCVpac retroviral vector. The primers used for this procedure were the start primer (5'-GCGctcgagATGTGCTAGCGCGCCCCTGCCTGGG-3') and the end primer (5'-CGCgaattcTCACGGCACGCTGATGGGATGCGTGGC-3'). The lower case corresponds to the XhoI and EcoRI restriction sites. {delta} PHPTB IRS-2 was generated by PCR designed with appropriate restriction enzyme sites to delete the amino acids 1–300 that contain most of the PH-PTB domains and subcloned into the pMSCV retroviral vector at the XhoI and EcoRI sites. The following primers were used in this procedure: the start primer (5'-GCGctcgagACCATGAGCAAGAGTCAGTCGTCCGGGTCG-3') and the end primer (5'-GGCgaattcTCAAGGTCAAAGGGCCTCACCTTTCAC-3'). The lower case indicates the XhoI and EcoRI restriction site. To generate {partial} PHPTB IRS-1 mutant plasmid, {partial} PTB IRS-1 pMSCV plasmid (17) was cut with XhoI enzyme to remove the PH domain of IRS-1, and the XhoI-digested fragment comprising the {delta} PHPTB region of IRS-1 was then ligated into pMSCV vector and designated as {delta} PHPTB IRS-1 pMSCV.

Glutathione-S-transferase (GST) fusion proteins were constructed using PCR products corresponding to different regions of IRS-2 coding for amino acids 300–598, 590–887, and 888-1321 in IRS-2. These regions were generated using specific oligonucleotide primers containing the appropriate restriction sites (all of the start primers contain XhoI restriction site and end primers contain EcoRI restriction sites in the overhangs). Purified PCR products were then digested with XhoI and EcoRI and ligated into XhoI/EcoRI cloning sites of pGEX-5X-1 vector (Pharmacia Biotech, Inc., Uppsala, Sweden). Plasmid {delta} receptor binding domain (RBD) 2 IRS-2 containing a mouse IRS-2 with the deletion of RBD2 region was made by following a mutagenesis strategy described before (17). Briefly, two sets of primers were designed. One set was: 5'-GCG CTC GAG ATG GCT AGC GCG CCC CTG CCT GGG CCC CCC GCG-3' (upper primer); 5'-CCG CAT AGA CAG CTT GGA GCC ACA CCA CAT CGG CAC GCT GAT GGG ATG CGT GGC-3' (lower primer, the underlined sequence is complementary to IRS-2 cDNA position from 2205 to 2235). The other sets was: 5'-GCG GCC GTG CGA CTA CCC TAC GCA CCG ATG TGG TGT GGC TCC AAC CTC AAC CTG (upper primer, the underlined sequence is from 927 to 951); 5'-CCG GAA TTC TCA AAG GGC CTC ACC TTT CAC GAC-3' (lower primer). The template used in PCR was wild-type mouse IRS-2. After two round PCR, the final PCR product (the resulting deleted RBD2 mouse IRS-2 gene) was purified, digested, and inserted into the XhoI/EcoRI restriction site of plasmid pMSCV. All plasmids constructs were confirmed by DNA sequencing and protein expression to guarantee accuracy. The detailed cloning strategies are available upon request.

For biological analysis of progressively deleted {delta} PHPTB IRS-2 protein, the deletion mutants were amplified by PCR from pMSCV IRS-2 vector, digested with appropriate restriction enzymes, and inserted into pMSCVpac retroviral vector. These different deletion constructs comprised IRS-2 sequences coding for amino acids residues 394-1321, 539-1321, 735-1321, and 896-1321, respectively (residue 1321 is the C terminus of IRS-2).

Cell lines and retroviral infection
32D IGF-IR and 32D IGF-IR/IRS-1 cells have been described in detail in several papers from our laboratory (4, 6, 17). To select the cell lines with stable expression of the indicated proteins, retroviral vector stocks were generated with a transient expression system and used to transduce 32D cells and 32D-derived cells as described previously (17).

Growth and differentiation analysis
These were determined as previously described (4, 31). Briefly, 32D cells and their derivatives were collected, washed three times, and seeded at a density of 5 x 104 cells/ml in IL-3-free medium supplemented with 50 ng/ml IGF-I (RPMI 1640 medium containing only 10% heat-inactivated fetal bovine serum). Viable cells were counted by trypan blue exclusion (Life Technologies, Grand Island, NY), and Giemsa-stained cytospins were evaluated for differentiation at the indicated time after shifting from IL-3 to IGF-I. Bands and polymorphonuclear cells were considered as differentiated cells. To establish the ability for IL-3-independent growth, cells from the same experiments at d 4 were replated in fresh IL-3-free medium with IGF-I (50 ng/ml) at a density of 5 x 104 cells/ml and regrown for an additional 4 or 6 d. Cells were counted again by trypan blue exclusion and stained with Giemsa as above. Differentiation was expressed as percentage of bands and polymorphonuclear cells in the total of scored cells.

Confocal immunofluorescence
To determine the subcellular localization of the IRS proteins, we used confocal microscopy. After fixation with 3% paraformaldehyde for 25 min and washing three times with PBS at room temperature, cells on cytospins were permeabilized with 0.2% Triton X-100 in PBS for 5 min and incubated for 1 h with the appropriate primary and secondary antibodies to IRS-2 (17). Propidium iodide was used to stain the nuclei. Confocal analysis was carried out on a MRC-600 laser scanning confocal microscope (Bio-Rad Laboratories, Inc., Hercules, CA).

Western blots and immunoprecipitation
These techniques were used to determine the levels of expression and the interactions of the various proteins. Cells were lysed with lysis buffer (50 mM HEPES, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 10% glycerol, 1% Nonidet P-40, 100 mM NaF, 10 mM sodium pyrophosphate, 0.2 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aproptinin, and 10 µg/ml leupeptin). After centrifugation, protein lysates (100 µg) were separated on a 4–15% gradient gel (Bio-Rad Laboratories) and transferred to a nitrocellulose membrane. For immunoprecipitation, the lysates were incubated with the appropriate antibodies coupled with 20 µl packed protein G-Sepharose beads (Oncogene Science, Inc., San Diego, CA) at 4 C for 2 h. Immunocomplexes were separated on SDS-PAGE gels, transferred to a nitrocellulose membrane, and immunoblotted with the indicated antibodies.

Northern blots
For detection of the myeloperoxidase mRNA, the cells under the same condition used for growth analysis were collected at the indicated time, and total RNA was extracted with Rneasy minikit (Qiagen, Valencia, CA). A 15-µg portion of total RNA for each sample was run on a 1% agarose-formaldehyde gel, blotted onto a Nylon membrane (Amersham Biosciences, Piscataway, NJ), and hybridized with myeloperoxidase cDNA probe labeled with [{alpha}-32P] dCTP by the random-primed DNA labeling kit (Amersham) as described previously (32).

In vitro interaction
All GST fusion proteins with various regions of IRS-2 were expressed in BL-21 bacterial cells (Invitrogen, Carlsbad, CA) and purified onto glutathione-agarose beads using standard techniques. The beads that contained immunoimmobilized fusion protein were then incubated with cell lysates derived from R+ cells after IGF-I stimulation (10 min, 50 ng/ml). Lysates were prepared by lysis for 30 min on ice in 50 mM HEPES (pH 7.5), 1% Nonidet P-40, 1 mM EGTA, 10 mM NaF, 20 mM sodium pyrophosphate, 10 µg/ml aproptinin, and 10 µg/ml leupeptin followed by spinning at 10,000 x g for 10 min to remove insoluble material. The resulting supernatants were incubated with the immunoimmobilized GST proteins overnight at 4 C. After extensive washing with 50 mM HEPES (pH 7.5), 150 mM NaCl, and 0.1% Triton X-100, the proteins that bound to IRS-2 or control GST proteins were analyzed by SDS-PAGE followed by immunoblotting with the appropriate antibodies.

Phosphoprotein analysis
32D-derived cells were grown in IL-3-free medium with IGF-I (50 ng/ml) for 48 h. After this period, cells were washed twice, incubated with phosphate-free and 10% dialyzed fetal bovine serum RPMI 1640 medium containing 32P-inorganic phosphate (Valeant Pharmaceuticals International, Costa Mesa, CA; 1 mCi/ml) for another 7 h. The cells were then lysed and harvested for immunoprecipitation by the addition of ice-cold radioimmunoprecipitation assay buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.25% SDS, 0.15 M NaCl, 0.01 M sodium-phosphate, 2 mM EDTA, 50 mM sodium fluoride, 0.2 mM sodium vanadate, 0.5 mM phenylmethylsulfonyl fluoride, and 10 µg/ml aproptinin and leupeptin). The resulting lysate was centrifuged at 15,000 x g at 4 C for 20 min. The supernatant was incubated with the corresponding antibodies coupled with 20 µl of packed protein G-Sepharose beads (Oncogene Science) at 4 C for 2 h. Immunocomplexes were separated on SDS-PAGE gel, transferred to a nitrocellulose membrane, and visualized by autoradiography.

Antibodies used were the phosphotyrosine blots, performed with an antiphosphotyrosine horseradish peroxidase-conjugated antibody (PY20; Transduction Laboratories, Lexington, KY). Other antibodies used in this study included rabbit polyclonal anti-IRS-1 antibody (Upstate Inc., Charlottesville, VA), an antibody recognizing the N terminus of IRS-2 (Santa Cruz Biotechnologies, Santa Cruz, CA), rabbit polyclonal anti-IRS-2 antibody (Upstate), mouse monoclonal anti-Grb-2 antibody (Transduction Laboratories), a rabbit polyclonal antibody against the ß-subunit of the IGF-IR, an anti-UBF antibody (Santa Cruz Biotechnologies), anti-Shc antibody (Transduction Laboratories), and anti-phosphatidylinositol 3-kinase (PI3-K) antibody (Sigma, St. Louis, MO).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of IRS-2 and its mutants in 32D IGF-IR cells
We expressed the wild-type IRS-2, its PH/PTB domains, and its {partial} PHPTB mutant in 32D IGF-IR cells (4) by transduction with retroviral vectors. The PH/PTB mutant comprises the first 300 amino acids of IRS-2, i.e. the PH and PTB domains only, whereas the {partial}PHPTB is IRS-2 with deletion of these two domains. The expression of these IRS-2 proteins in 32D IGF-IR cells is shown in the Western blots of Fig. 1Go, using the appropriate IRS-2 antibodies (see Materials and Methods). Wild-type IRS-2 and {partial}PHPTB/IRS-2 are well expressed and can be easily distinguished by size (Fig. 1AGo), using an antibody to the C terminus of IRS-2. The PH/PTB mutant is less well expressed than wild-type IRS-2 but roughly at the same level as {partial}PHPTB IRS-2 (Fig. 1BGo), using an antibody to the amino terminus of IRS-2 (Fig. 1BGo). For both the PHPTB/IRS-2 and {partial}PHPTB/IRS-2, we used two mixed populations, obtained in separate retroviral infections. In Fig. 1CGo, we show the level of expression of {partial}PHPTB/IRS-1 (see below). For comparison, we also show the size and level of expression of wild-type IRS-1 in our standard 32D IGF-IR/IRS-1 cells (4, 6). Because three different antibodies were used, the comparisons are not strictly quantitative, but Fig. 1Go, A–C, is indicative of levels of expression of the same magnitude in the various cell lines.



View larger version (14K):
[in this window]
[in a new window]
  		FIG. 1. Expression of IRS proteins in 32D IGF-IR cells. 32D IGF-IR cells were infected with retroviruses expressing, respectively, the wild-type IRS-2, the PH/PTB domains of IRS-2, a truncated IRS-2 without the PH/PTB domains ({partial}PHPTB), and a truncated IRS-1 without the PH/PTB domains. The retroviruses are described in Materials and Methods. Mixed populations were selected, and the expression of the IRS proteins was determined in lysates of cells growing in serum plus IL-3. The expression of IRS-2 and {partial}PHPTB/IRS-2 is shown (A, 7.5% gel). The expression of PH/PTB IRS-2 is also shown (B). 1 and 2 refer to distinct mixed populations obtained in separate transductions. Parental 32D IGF-IR cells are negative for IRS-2. C, Western blot of 32D IGF-IR/IRS-1 cells and 32D IGF-IR/{partial}PHPTB IRS-1 cells. Lysates are as in A and B. The antibodies used are given in Materials and Methods.

 
Effect of IRS-2 and its mutants on growth and differentiation of 32D IGF-IR cells
We tested the growth of the original cell lines (32D IGF-IR and 32D IGF-IR/IRS1 cells) and the new mixed populations expressing the wild-type and the mutant IRS-2 proteins. As usual with 32D-derived cells, all populations grow well in IL-3 plus 10% serum and die rapidly in 10% serum without IL-3 or IGF-I (data not shown). The growth of 32D-derived cells in 10% serum supplemented with IGF-I (50 ng/ml) is given in Fig. 2AGo. Parental 32D IGF-IR cells grow for a few days, and then they stop growing, as already reported (4). Ectopic expression of IRS-1 inhibits differentiation and renders the cells IL-3-independent and capable of forming tumors in mice (6). Ectopic expression of IRS-2 has the same effect as ectopic expression of IRS-1. The cells keep growing even after the fourth day after the shift from IL-3 to IGF-I, the difference between IRS-1 and IRS-2 being marginal. Expression of the PH/PTB domains of IRS-2 has the same effect as the expression of the PH/PTB domains of IRS-1 (29, 30); the cells proliferate somewhat better than the parental 32D IGF-IR cells, but they eventually cease proliferating and differentiate. The cells expressing the {partial}PHPTB IRS-2 grow as well as the cells expressing wild-type IRS-2.



View larger version (30K):
[in this window]
[in a new window]
  		FIG. 2. Growth of 32D-derived cells in medium supplemented with IGF-I. A, The original cell lines and the mixed populations generated as described in Materials and Methods were tested for their ability to grow in medium supplemented with IGF-I (50 ng/ml). The ordinate gives the percentage increase in the number of cells over number of cells plated (100 is a doubling in number). The different cell lines and the days of growth in IGF-I are indicated. PHPTB and {partial}PHPTB refer to IRS-2 mutants. B, The experiment was repeated, but, in addition, we also determined the percentage of cells differentiating on d 4 and 6 after shifting from IL-3 to IGF-I. The mixed populations and the days are indicated. Differentiation was determined morphologically (see Fig. 3Go).

 
We repeated these experiments on a new mixed population of 32D IGF-IR {partial}PHPTB IRS-2 that had been selected separately (Fig. 2BGo). At the same time, we examined the cell lines for differentiation. Again, 32D IGF-IR and 32D IGF-IR PHPTB/IRS-2 cells do not grow after the fourth day in IGF-1 and are actually differentiating. In contrast, the 32D IGF-IR cells expressing either wild-type IRS-1 or wild-type IRS-2 or the {partial}PHPTB IRS-2 grow very well until the eighth day (with replating after d 4) and do not differentiate. This experiment was repeated a third time, with the same results (not shown). We also selected two clones of 32D IGF-IR {partial}PHPTB IRS-2, and the results were the same as with the mixed populations (not shown).

We examined the cell lines morphologically for differentiation, and this is summarized in Fig. 3Go. 32D IGF-IR and 32D IGF-IR/PHPTB IRS-2 differentiate into granulocytes (Fig. 3Go, A and B), confirming (for the original cell line) the findings of Valentinis et al. (4). 32D IGF-IR cells expressing wild-type IRS-2 or {partial}PHPTB IRS-2 do not differentiate (Fig. 3Go, C and D). Figure 3EGo gives the negative control for differentiation, the 32D IGF-IR/IRS-1 cells (6). Actually, a small fraction of 32D IGF-IR/{partial}PHPTB/IRS-2 cells do differentiate in the early days (Fig. 2BGo), but they eventually disappear. This result was confirmed by observation of smears of cells stained by the Giemsa method (not shown). We suspect that the presence of a fraction of differentiating 32D IGF-IR/{partial}PHPTB/IRS-2 cells may be due to the fact that this is a mixed population (see below and Discussion).



View larger version (31K):
[in this window]
[in a new window]
  		FIG. 3. Morphological differentiation of 32D-derived cells. The different cell lines were stained with Giemsa on d 4 after shifting from IL-3 to IGF-I, and representative fields are shown. A, 32D IGF-IR cells; B, 32D IGF-R/PHPTB IRS-2; C, 32D IGF-R cells expressing wild-type IRS-2; D, 32D IGF-IR cells expressing {partial}PHPTB IRS-2; E, 32D IGF-IR/IRS-1 cells. Differentiating cells (granulocytes) are detectable only in A and B.

 
Confirmation of truncated expression, absence of IRS-1 induction, and dependence on IGF-I
The ability of wild-type IRS-2 to promote IL-3-independent growth of 32D IGF-IR cells is interesting in itself (see Discussion), but we also wanted to confirm that the effect of the {partial}PH/PTB IRS-2 on the proliferation of 32D IGF-IR cells was a real one and not due to other coincidental causes. We monitored again the status of IRS-2 and confirmed (Fig. 4AGo) that the {partial}PHPTB IRS-2 is shorter than the wild type. The absence of the PHPTB domains in the plasmid had been previously confirmed by direct sequencing (data not shown). Then we ruled out the possibility that these cells had reactivated spontaneously the expression of IRS-1. We could not detect IRS-1 expression in 32D IGF-IR cells expressing either the wild-type or the {partial} PHPTB IRS-2 (Fig. 4BGo). We finally determined whether the biological effect of the {partial}PHPTB mutant was IGF-I dependent. Figure 4CGo shows that, in 10% serum but in the absence of IGF-I, the 32D IGF-IR/{partial}PHPTB/IRS-2 cells die rapidly, like all the other 32D-derived cells. This last experiment indicates that the biological effects of the {partial}PHPTB IRS-2 on 32D IGF-IR cells is strictly IGF-I dependent and indicates that the effect requires IGF-IR signaling.



View larger version (24K):
[in this window]
[in a new window]
  		FIG. 4. Characteristics of 32D IGF-IR/{partial}PHPTB/IRS-2 cells. A, The IRS-2 expressed in these cells is shorter than the wild-type IRS-2. Western blot is from lysates, after immunoprecipitation with an antibody to IRS-2 and also developed with anti-IRS-2 antibody (C terminus, upper panel) or anti-IRS-1 antibody (lower panel). Lane 1, 32D IGF-IR cells (no IRS-1 or IRTS-2); lane 2, cells expressing wild-type IRS-2; lane 3, cells expressing the IRS-2 lacking the PH/PTB domains. None of the cell populations express IRS-1. B, Growth of 32D-derived cells in 10% serum, without IL-3 or IGF-I. The cells lines are indicated. The number of cells is expressed as percentage decrease in cell number over initial plating.

 
Status of the {partial}PHPTB IRS-2 in 32D IGF-IR cells
In subsequent experiments, we focused on the {partial}PHPTB mutant of IRS-2 and its effect on the proliferation and differentiation of 32D IGF-IR cells because it has the same biological effects as wild-type IRS-2. Sun et al. (20) reported that IRS-2 can translocate to the nuclei of mouse embryo fibroblasts stimulated with IGF-I. Figure 5Go shows a confocal microscopy picture of {partial}PHPTB IRS-2 in 32D IGF-IR cells. The cells were stained with propidium iodide (red) and with an antibody to IRS-2 (green). The merged picture shows that the nuclei remain red and that the mutant IRS-2 is localized to the cytoplasm. Although we cannot rule out that small amounts may be nuclear, the mutant IRS-2, at variance with wild-type IRS-1 and IRS-2, is not present in detectable amounts in the nuclei of cells.



View larger version (43K):
[in this window]
[in a new window]
  		FIG. 5. Cytoplasmic localization of {partial}PHPTB IRS-2 in 32D IGF-IR cells. Confocal microscopy pictures, in which the nuclei were stained with propidium iodide (PI, red, A) and with an antibody to IRS-2 (green, B). The merged picture confirms the cytoplasmic localization of the mutant IRS-2. The cells had been previously stimulated with IGF-I (50 ng/ml). This picture was taken 24 h after shifting the cells to IGF-I. The same results (not shown) were obtained at 8 and 16 h.

 
Figure 6GoA shows that the {partial}PHPTB/IRS-2 is tyrosyl phosphorylated when the cells are stimulated with IGF-I. Quiescent cells were stimulated with IGF-I, lysates were immunoprecipitated with an antibody to IRS-2, and the blot was first developed with an antiphosphotyrosine antibody. The induction of phosphorylation is slow because it is detectable only after 6 h but is reproducible. In Fig. 6BGo, we show that {partial}PHPTB IRS-2 interacts with Grb2 (see also below). In Fig. 6CGo, we show that stimulation with IGF-I induces tyrosyl phosphorylation of wild-type IRS-2 in 1 h (probably even earlier but definitely earlier than with the mutant IRS-2). We could not detect any interaction of the mutant IRS-2 with Shc (not shown).



View larger version (25K):
[in this window]
[in a new window]
  		FIG. 6. Status of {partial}PHPTB IRS-2 in 32D IGF-IR cells. A, 32D IGF-IR {partial}PHPTB IRS-2 cells were stimulated with IGF-I (50 ng/ml) for the indicated times, and the lysates were immunoprecipitated with an antibody to IRS-2. Upper row, Blot with an antiphosphotyrosine antibody. Lower row, Blot with an anti-IRS-2 antibody. B, 32D IGF-IR, 32D IGF-IR/IRS-1 and 32D IGF-IR{partial}PHPTB/IRS-2 cells were stimulated with IGF-I for 24 h. Lysates were immunoprecipitated with an antibody to IRS-2 (C terminus). Blots were developed with an antibody to Grb2. C, Lysates from 32D IGF-IR IRS-2 cells. The cells were unstimulated (left lane) or stimulated with IGF-I for 1 h (right lane). IP, Immunoprecipitate; WB, Western blot.

 
The {partial} PHPTB IRS-1 fails to inhibit the differentiation of 32D IGF-IR cells
Because wild-type IRS-1 blocks the differentiation and promotes the malignant transformation of 32D IGF-IR cells, the question we had at this point was whether the removal of the PHPTB domains from IRS-1 would also generate an active mutant capable of stimulating the growth of 32D IGF-IR cells. We obtained a mixed population expressing the {partial}PHPTB IRS-1 in 32D IGF-IR cells. The expression of {partial}PHPTB IRS-1 was shown in Fig. 1CGo, in which it is roughly at similar levels as the expression of {partial}PHPTB IRS-2. These cells were tested for growth in IGF-I (Fig. 7AGo). 32D IGF-IR cells expressing the {partial}PHPTB IRS-1 behaved essentially as the parental 32D IGF-IR cells. They grew for the first 2 d, and then growth stopped and the cells differentiated. It seems therefore that the ability of {partial}PHPTB mutants to complement the growth of 32D IGF-IR cells is true for IRS-2 but not for IRS-1. Figure 7BGo is an additional control showing the requirement for the IGF-IR for the stimulatory effect of {partial}PHPTB IRS-2. When the {partial}PHPTB IRS-2 mutant is expressed in parental 32D cells (very low levels of IGF-IR), the cells do not grow in IGF-I; in fact, they die as parental 32D cells do (18).



View larger version (28K):
[in this window]
[in a new window]
  		FIG. 7. A {partial}PHPTB IRS-1 fails to stimulate the proliferation of 32D IGF-IR cells. The {partial}PHPTB IRS-1 was introduced into 32D IGF-IR cells, and mixed populations were selected as usual. The expression of the mutant IRS-1 is shown in Fig. 1CGo, in which it is compared with the expression of wild-type IRS-1 in 32D IGF-IR IRS-1 cells (6 ). The growth and differentiation of these cells in IGF-I (50 ng/ml) is shown (A). 32D IGF-IR cells expressing {partial} PHPTB IRS-1 behave like the parental 32D IGF-IR cells: they stop growing after a few days and differentiate. B, The {partial}PHPTB IRS-2 will not prevent the death of parental 32D cells (very low IGF-IR number, no IRS-1 or IRS-2). Note the difference in the ordinates of A and B.

 
{partial}PHPTB IRS-2 inhibits the induction of myeloperoxidase (MPO) mRNA in 32D IGF-IR cells
We previously hypothesized that the IGF-IR, in 32D cells as well as in some other cells, sends two contradictory signals, one for differentiation and one for mitogenesis (see introductory part of article). We therefore tested the hypothesis that {partial}PHPTB IRS-2 simply inhibits the implementation of the differentiation program, leaving intact the mitogenic action of the IGF-IR. For this purpose, we tested 32D-derived cells for the appearance of differentiation markers. The marker examined was the mRNA for MPO, a marker of differentiation that appears early after 32D IGF-IR cells are shifted from IL-3 to IGF-I (4, 32). Figure 8GoA confirms the appearance of MPO mRNA in 32D IGF-IR shifted to IGF-I. When 32D IGF-IR cells express {partial}PHPTB IRS-2, MPO mRNA is not induced (lanes 4–6). The inhibitory effect of {partial}PHPTB IRS-2 on differentiation of 32D IGF-IR cells was confirmed morphologically (see Fig. 3Go).



View larger version (26K):
[in this window]
[in a new window]
  		FIG. 8. Expression of MPO RNA in 32D IGF-IR cells and 32D IGF-IR {partial}PHPTB IRS-2 cells. Expression of MPO mRNA (a marker of differentiation). Cells were shifted to IGF-I and Northern blots for MPO RNA were carried out at 0, 24, and 48 h later. Lanes 1–3 are 32D IGF-IR cells, lanes 4–6 are from 32D IGF-IR {partial}PHPTB/IRS-2 cells, and lanes 7–9 from 32D IGF-IR/IRS2 cells.

 
GST-fusion proteins of the {partial}PHPTB IRS-2
The next step was to determine by pull-down experiments the interactions between domains of {partial}PHPTB IRS-2 and other proteins known to interact with the IRS proteins (33). For this purpose, we prepared fusion proteins between GST and the sequences of IRS-2 given in Fig. 9Go (see Materials and Methods). We then determined the presence in the pull-down of proteins like PI3-K and Grb2 that are known to have binding sites on the IRS proteins and the IGF-IR. The results shown in Fig. 9Go indicate that the sequences between amino acids 590 and 1321 pull down the p85 subunit of PI3-K. The sequence between 590 and 888 also pulls down the ß-subunit of the IGF-IR. Grb2 interacts only with the sequences between residues 888 and 1321. The significance of these results will be considered in the Discussion.



View larger version (20K):
[in this window]
[in a new window]
  		FIG. 9. Diagram of GST fusion protein and plasmids of mutant IRS-2, and pull-down experiments for PI3-K, Grb2, and the IGF-IR. The GST-fusion proteins are represented diagrammatically (A) (for the construction, see Materials and Methods). A typical pull-down experiment is shown (B). The IRS-2 sequences 590–887 and 888-1321 interact with PI3-K. The latter sequence also interacts with Grb2. The 590–887 sequence interacts with the ß-subunit of the IGF-IR.

 
Domains of the {partial}PHPTB IRS-2 required for inhibition of differentiation
We next determined the domains of {partial}PHPTB IRS-2 required for inhibition of differentiation. For this purpose, we transfected 32D IGF-IR cells with plasmids that had been progressively shortened to express truncated IRS-2 proteins. These plasmids are described in Materials and Methods, and the sequences they comprise are indicated in Fig. 10Go. We monitored the proteins synthesized by these plasmids, and they were of the expected size (not shown). Their biological effect on the growth of 32D IGF-IR cells is also summarized in Fig. 10Go. Expression of an IRS-2 comprising the amino acids between residue 394 and the C terminus (residue 1321) results in IL-3-independent IGF-I-dependent growth, and the same can be said of an IRS-2 comprising the amino acids from residue 539 to 1321 (Fig. 10Go). Further deletions (to residues 735 or 896) abrogate the biological effect of the truncated IRS-2 proteins. This result indicates that the antidifferentiation effect of the truncated IRS-2 protein requires residues 539–735 of IRS-2. This sequence matches the data obtained with GST-fusion proteins, suggesting that the sequences for IGF-IR and PI3-K binding are a requirement for the antidifferentiation function of the truncated IRS-2.



View larger version (22K):
[in this window]
[in a new window]
  		FIG. 10. Effect of plasmids expressing truncated forms of IRS-2 on the growth of 32D IGF-IR cells. The construction of the plasmids is given in Materials and Methods. The sequences of IRS-2 in each plasmid are given in parentheses (A and B). Only the plasmids expressing the IRS-2 sequences from 394 to 1321 and from 539 to 1321 cause cell proliferation (A) and inhibition of differentiation (B).

 
These results were confirmed by making a deletion mutant in which the RBD2 sequence was deleted from the wild-type IRS-2. This is the domain that was reported to bind to the insulin receptor (23). The mutant IRS-2 with a deleted RBD2 domain was stably transfected into 32D IGF-IR cells and subsequently tested for growth and differentiation. Figure 11Go shows that this mutant loses the ability to inhibit differentiation and stimulate the proliferation of 32D IGF-IR cells. Growth is marginally better than for 32D IGF-IR cells, but the cells stop growing in IGF-1 after the fourth day. Differentiation is lower than in 32D IGF-IR cells, but it still occurs.



View larger version (30K):
[in this window]
[in a new window]
  		FIG. 11. Deletion of the RBD2 domain inactivates the biological action of {partial}PHPTB IRS-2. A mutant IRS-2 with a deletion of the RBD2 domains (see Materials and Methods) was introduced into 32D IGF-IR cells. The growth and differentiation of the parental 32D IGF-IR cells was then compared with that of the 32D IGF-IR cells expressing the RBD2 deletion mutant. The figure shows only the results after shifting the cells from IL-3 to IGF-I. As usual with 32D-derived cells, they all grow in IL-3, and they all die when both IL-3 and IGF-I are withdrawn (not shown).

 
Effect of {partial}PHPTB IRS-2 on UBF phosphorylation
The previous experiments have shown that the {partial}PHPTB IRS-2 behaves essentially as wild-type IRS-1. IRS-1 activates UBF1, stimulates rRNA synthesis (18, 19), and inhibits the appearance of differentiation markers (32). We therefore looked at the ability of {partial}PHPTB IRS-2 to activate UBF and inhibit the appearance of differentiation markers.

Activation of UBF1 depends on its phosphorylation (20). We documented in those papers that the nucleolar localization of the IRS-1 and -2 proteins and their interaction with UBF induce its phosphorylation and increase rRNA synthesis. Both IRS-1 (6) and IRS-2 (this paper, see above) make 32D IGF-IR cells IL-3 independent, which assumes an activation of UBF. Although the {partial}PHPTB IRS-2 is cytoplasmic in location, it still may activate UBF1 by the conventional signal transduction pathway (33). We asked whether UBF was phosphorylated in 32D IGF-IR/{partial}PHPTB/IRS-2 cells growing in IGF-I. The experiment was carried out on the fourth day after shifting from IL-3 to IGF-I, and the cells were labeled for 7 h with 32P (1 mCi/ml). UBF was immunoprecipitated, run on a gel, and autoradiographed. The results of such an experiment are summarized in Fig. 12AGo. UBF is phosphorylated in 32D IGF-IR {partial}PHPTB/IRS-2 cells, as much as in 32D IGF-IR expressing either wild-type IRS-1 or wild-type IRS-2. The only two populations in which UBF phosphorylation was lower were the parental 32D IGF-IR cells and the ones expressing the PH/PTB domains of IRS-2, both of these cell lines undergoing differentiation after shifting to IGF-I (4 and this paper, see above). Figure 12BGo is a Western blot of the same cells for total UBF and Grb2. The levels of UBF are very much the same as the levels of phosphorylation, with the parental 32D IGF-IR cells and the 32D IGF-IR/PHPTB IRS-2 showing the lowest levels. These results suggest that the extent of UBF phosphorylation in these cells depends largely on protein levels, which are decreased in cells that undergo differentiation (34).



View larger version (15K):
[in this window]
[in a new window]
  		FIG. 12. Phosphorylation of UBF in selected cell populations. A, All cell lines were 32D IGF-IR, either parental (lane 1) or expressing wild-type IRS-1 (lane 2), wild-type IRS-2 (lane 3), the {partial}PHPTB IRS-2 (lane 4), or the PH/PTB domains of IRS-2 (lane 5). The cells were examined on the fourth day after shifting to IGF-I and were labeled with 32P for 7 h. UBF was immunoprecipitated, blotted, and the blot autoradiographed. B, Western blots of the same cell lines. Blots developed with antibodies to UBF and Grb2.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our results can be summarized as follows: 1) ectopic expression of IRS-2 in 32D IGF-IR cells results in cells that are IL-3 independent but IGF-I dependent for growth; 2) ectopic expression of an IRS-2 lacking the PH and PTB domains has on 32D IGF-IR cells the same effect as a wild-type IRS-2 (or wild-type IRS-1); both the wild-type and the {partial}PHPTB IRS-2 inhibit the granulocytic differentiation that occurs in 32D IGF-IR cells when IL-3 is replaced by IGF-I; 3) a similar IRS-1 construct (lacking the PH and PTB domains) is totally inactive in inhibiting IGF-I-mediated differentiation and promoting IL-3-independent proliferation of 32D IGF-IR cells; 4) a mutant IRS-2 lacking the RBD domains fails to inhibit differentiation and stimulate the growth of 32D IGF-IR cells; 5) a mutant IRS-2 comprising only the PH and PTB domain also fails to inhibit differentiation and stimulate cell proliferation; 6) {partial}PHPTB IRS-2 is tyrosyl phosphorylated by IGF-I, albeit slowly and modestly; 7) specific sequences of {partial}PHPTB IRS-2 bind the p85 subunit of PI3-K and the ß-subunit of the IGF-IR. The biological activity of {partial}PHPTB IRS-2 localizes to the same domain(s) required for PI3-K and IGF-IR binding; 8) despite the cytoplasmic localization and the modest phosphorylation, {partial}PHPTB IRS-2 causes the phosphorylation and/or stabilization of UBF1, a key regulator of rRNA synthesis (21).

Taken together, these findings demonstrate that the antidifferentiation and growth-promoting activities of IRS-2 seem to reside in sequences roughly between residues 600 and 900 that bind the p85 subunit of PI3-K and the ß-subunit of the IGF-IR. The wild-type IRS-2 and the {partial}PHPTB IRS-2 mutant have on 32D IGF-IR cells the same effects as wild-type IRS-1 (4, 30). Where IRS-1 and IRS-2 differ radically is in the ability of the respective {partial}PHPTB mutants to inhibit differentiation and stimulate growth. The unavoidable conclusion is that IRS-1 needs both the PH and PTB domains and the rest of the molecule for its growth-promoting and antidifferentiation programs, whereas IRS-2 does not need the PH and PTB domains.

We have focused on the {partial}PHPTB mutant rather than on the wild-type IRS-2 because it seems that the mutant’s biological action is peculiar to IRS-2 and, at any rate, is similar to the action of wild-type IRS-2. The most convincing evidence that the biological effect of {partial}PHPTB IRS-2 is real is that its action is IGF-I-dependent. Without IGF-I supplementation (and in the absence of IL-3), 32D IGF-IR {partial}PHPTB IRS-2 cells die like parental 32D cells. Coupled with the absence of IRS-1 or IRS-2 expression in these cells, the IGF-I dependence clearly indicates that its effect is due to its stimulation by IGF-I, a ligand that stimulates the IRS proteins in general (33). Also convincing is the fact that a similar IRS-1 mutant is totally ineffective, suggesting a specific property of the IRS-2 protein. Finally, it seems that specific sequences of IRS-2 (see below) are required for its biological effect.

There have been reports that a {partial}PHPTB IRS-2 may have biological activity. Xiao et al. (24) found that the {partial}PHPTB IRS-2 sustained the growth of 32D/IR cells in IL-4 (but not in IL-9). As we mentioned in the article introduction, IL-4 and IGF-I cooperate in promoting the growth of 32D cells (25). The difference between the {partial}PHPTB mutants of IRS-1 and IRS-2 could find an explanation in the report by Sawka-Verhelle et al. (23). Using the yeast two-hybrid system, these authors found that IRS-2 (but not IRS-1) interacted with the IR with a domain located between residues 591 and 786 of IRS-2. The binding required the tyrosine autophosphorylation of the insulin receptor. These authors proposed that this domain mediates the association between IRS-2 and the receptor. We confirmed and extended this finding to the IGF-IR. Pull-down experiments with GST fusion proteins show that the sequences between 590 and 786 interact with the ß-subunit of the IGF-IR. Interestingly, these are the sequences that we find necessary for the biological effect of {partial}PH PTB IRS-2. The 591–786 domain includes sequences that are also crucial in the interaction of IRS-2 with PI3-K (pull-down experiments in Fig. 9Go) and the biological action of truncated IRS-2 proteins (Fig. 10Go). Thus, it seems that the {partial}PHPTB IRS-2 may indeed have an antidifferentiation function based on its interactions and biological activities. Looking in more detail at the biologically active sequences in the {partial}PHPTB IRS-2 (between residues 539 and 735), we find at residue 651 (human IRS-2) a canonical PI3-K binding sequence, conserved from IRS-1 residue 611 (35, 36, 37). An accepted Grb2 binding sequence is found around human IRS-2 residue 920 (also conserved from IRS-1) (36), which is compatible with our results using GST fusion proteins. There is a second PI3-K binding motif around IRS-2 residue 980, also compatible with our GST results, but, quite obviously, this binding site is not sufficient by itself because the absence of the 651 PI3-K motif completely abrogates the biological activity of the truncated IRS-2. We have also confirmed the importance of the RBD2 domain in the biological activity of IRS-2. Deletion of this domain essentially inactivates the wild-type IRS-2. This finding, in fact, seems to indicate that, at least in 32D myeloid cells, the sequences outside the PH/PTB domains are even more important than the PH/PTB domain itself.

There have been contradictory reports on the biological action of IRS-2 in cells, which is sometimes toward differentiation and sometimes toward proliferation (9, 19, 27). One could hypothesize that IRS-2 sends different signals, one from the PHPTB domain and another one from the sequences binding the p85 subunit of PI3-K and the ß-subunit of the IGF-IR. This brings us to our observation that wild-type IRS-2 stimulated the growth of 32D IGF-IR cells. Our results are apparently in apparent contrast with those of Bruning et al. (26), who used IRS-1-deficient MEFs. Bruning et al. (26) reported that IRS-1 reintroduction restored the growth of these cells, whereas IRS-2 overexpression was considerably less effective. At present, we do not have any explanation on why IRS-2 can replace IRS-1 in 32D IGF-IR cells but not in MEFs

IRS-2 and its {partial}PHPTB mutant inhibit the differentiation of 32D IGF-IR cells, as effectively as wild-type IRS-1 (38). There is, however, a difference. IRS-1 definitely increases the size of 32D IGF-IR cells (7, 38), whereas IRS-2 and {partial}PHPTB IRS-2 do not seem to be as effective as IRS-1 in increasing cell size (Fig. 3Go). Another interesting aspect is that, at variance with IRS-1 and wild-type IRS-2 (18, 19), {partial}PHPTB IRS-2 inhibits differentiation and makes cells IL-3 independent without translocating to the nuclei. This is in agreement with previous findings indicating that the PTB domain is crucial for the translocation of IRS proteins to the nuclei (17, 19). Despite its cytoplasmic localization, {partial}PHPTB IRS-2 phosphorylates UBF, suggesting that its activation of cell proliferation and rRNA synthesis are regulated through the conventional signal transduction pathways (3, 33). Phosphorylation of UBF1 is a prerequisite for its activation (21, 39, 40). The decreased phosphorylation is accompanied by a decrease in UBF levels, which is not unexpected because UBF is an exclusively nucleolar protein, and the nucleolus disappears with terminal differentiation (41).

In conclusion, we have shown that both wild-type IRS-2 and a truncated IRS-2 lacking the PH and PTB domains inhibit the differentiation and sustain the IGF-I-dependent growth of 32D IGF-IR cells. The biological activity of {partial}PHPTB IRS-2 is strictly dependent on IGF-I stimulation and requires the presence of sequences that bind PI3-K and the ß-subunit of the IGF-IR. A similarly truncated IRS-1 is totally inactive in preventing the differentiation of 32D IGF-IR cells, although the wild-type IRS-1 not only inhibits differentiation but also actually transforms them into tumor cells (4, 6). We would like to suggest that the sequences of IRS-2 outside the PHPTB domains are necessary and sufficient for IGF/IRS-2 regulation of cell proliferation in myeloid cells.


   Footnotes
 
This work was supported by Grant CA89640 from the National Institutes of Health.

Abbreviations: GST, Glutathione-S-transferase; IGF-IR, IGF-I receptor; IRS, insulin receptor substrate; MEF, mouse embryo fibroblast; MPO, myeloperoxidase; PH, pleckstrin; PI3-K, phosphatidylinositol 3-kinase; PTB, phosphotyrosine binding; RBD, receptor binding domain; UBF, upstream binding factor.

Received May 26, 2004.

Accepted for publication July 28, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Valtieri M, Tweardy DJ, Caracciolo D, Johnson K, Mavilio F, Altmann S, Santoli, D, Rovera G 1987 Cytokine dependent granulocytic differentiation. J Immunol 138:3829–3835[Abstract]
  2. Zhou-Li F, Xu S-Q, Dews M, Baserga R 1997 Co-operation of simian virus 40 T antigen and insulin receptor substrate-1 in protection from apoptosis induced by interleukin-3 withdrawal. Oncogene 15:961–970[CrossRef][Medline]
  3. Wang LM, Myers Jr MG, Sun XJ, Aaronson SA, White M, Pierce JH 1993 IRS-1: essential for insulin- and IL-4-stimulated mitogenesis in hemopoietic cells. Science 261:1591–1594[Abstract/Free Full Text]
  4. Valentinis B, Romano G, Peruzzi F, Morrione A, Prisco M, Soddu S, Cristofanelli B, Sacchi A, Baserga R 1999 Growth and differentiation signals by the insulin-like growth factor 1 receptor in hemopoietic cells are mediated through different pathways. J Biol Chem 274:12423–12430[Abstract/Free Full Text]
  5. Li YM, Schacher DH, Liu Q, Arkins S, Rebeiz N, McCusker Jr RH, Dantzer R, Kelley KW 1997 Regulation of myeloid growth and differentiation by the insulin-like growth factor receptor. Endocrinology 138:362–368[Abstract/Free Full Text]
  6. Zamorano J, Wang HY, Wang L-M, Pierce JH, Keegan AD 1996 IL-4 protects cells from apoptosis via the insulin receptor substrate pathway and a second independent signaling pathway. J Immunol 157:4926–4934[Abstract]
  7. Valentinis B, Navarro M, Zanocco-Marani T, Edmonds P, McCormick J, Morrione A, Sacchi A, Romano G, Reiss K, Baserga R 2000 Insulin receptor substrate-1, p70S6K and cell size in transformation and differentiation of hemopoietic cells. J Biol Chem 275:25451–25459[Abstract/Free Full Text]
  8. Baserga R 2000 The contradictions of the insulin-like growth factor 1 receptor. Oncogene 19:5574–5581[CrossRef][Medline]
  9. Kim B, Cheng H-L, Margolis B, Feldman EL 1998 Insulin receptor substrate 2 and Shc play different roles in insulin-like growth factor I signaling. J Biol Chem 273:34543–34550[Abstract/Free Full Text]
  10. Morrione M, Navarro M, Romano G, Dews M, Reiss K, Valentinis B, Belletti B, Baserga R 2001 The role of the insulin receptor substrate-1 in the differentiation of rat hippocampal neuronal cells. Oncogene 20:4842–4852[CrossRef][Medline]
  11. Sadowski CL, Choi TS, Le M, Wheeler TT, Wang LH, Sadowski HB 2001 Insulin induction of SOCS-2 and SOCS-3 mRNA expression in C2C12 skeletal muscle cells is mediated by Stat5. J Biol Chem 276:20703–20710[Abstract/Free Full Text]
  12. Sarbassov DD, Peterson CA 1998 Insulin receptor substrate-1 and phosphatidylinositol3-kinase regulate extracellular signal-regulated kinase-dependent and -independent signaling pathways during differentiation. Mol Endocrinol 12:1870–1878[Abstract/Free Full Text]
  13. Sun XJ, Pons S, Wang LM, Zhang Y, Yenush L, Burks D, Myers Jr MG, Glasheen E, Copeland NG, Jenkins NA, White MF 1997 The IRS-2 gene on murine chromosome 8 encodes a unique signaling adapter for insulin and cytokine action. Mol Endocrinol 11:51–262
  14. Brown G, Drayson MT, Durham J, Toellner KM, Hughes PJ, Coudhry MA, Taylor DR, Michell RH 2002 HL60 cells halted in G1 or S phase differentiate normally. Exp Cell Res 281:28–38[CrossRef][Medline]
  15. Ward AC, Smith L, de Koning JP, van Aesch Y, Touw IP 1999 Multiple signals mediate proliferation, differentiation and survival from the granulocyte-colony stimulating factor receptor in myeloid 32D cells. J Biol Chem 274:14956–14962[Abstract/Free Full Text]
  16. Gilliland DG, Tallman MS 2002 Focus on acute leukemias. Cancer Cell 1:417–420[CrossRef][Medline]
  17. Prisco M, Santini F, Baffa R, Liu M, Drakas R, Wu A, Baserga R 2002 Nuclear translocation of IRS-1 by the SV40 T antigen and the activated IGF-I receptor. J Biol Chem 277:32078–32085[Abstract/Free Full Text]
  18. Prisco M, Hongo A, Rizzo MG, Sacchi A, Baserga R 1997 The IGF-I receptor as a physiological relevant target of p53 in apoptosis caused by interleukin-3 withdrawal. Mol Cell Biol 17:1084–1092[Abstract]
  19. Tu X, Batta P, Innocent N, Prisco M, Casaburi I, Belletti B, Baserga R 2002 Nuclear translocation of insulin receptor substrate-1 by oncogenes and IGF-I: effect on ribosomal RNA synthesis. J Biol Chem 277:44357–44365[Abstract/Free Full Text]
  20. Sun H, Tu X, Prisco M, Wu A, Casiburi I, Baserga R 2003 Insulin-like growth factor I receptor signaling and nuclear translocation of insulin receptor substrates 1 and 2. Mol Endocrinol 17:472–486[Abstract/Free Full Text]
  21. Grummt I 1999 Regulation of mammalian ribosomal gene transcription by RNA polymerase I. Prog Nucleic Acids Res Mol Biol 62:109–154[Medline]
  22. Voit R, Kuhn A, Sander EE, Grummt I 1995 Activation of mammalian ribosomal gene transcription requires phosphorylation of the nucleolar transcription factor UBF. Nucleic Acids Res 23:2593–2599[Abstract/Free Full Text]
  23. Sawka-Verhelle D, Tartare-Deckert S, White MF, van Obberghen E 1996 Insulin receptor substrate-2 binds to the insulin receptor through its phosphotyrosine-binding domain and through a newly identified domain comprising amino acids 591–786. J Biol Chem 271:5980–5983[Abstract/Free Full Text]
  24. Xiao H, Yin T, Wang XY, Uchida T, Chung J, White MF, Yang YC 2002 Specificity of interleukin-2 receptor {gamma} chain superfamily cytokines is mediated by insulin receptor substrate-dependent pathway. J Biol Chem 277:8091–8098[Abstract/Free Full Text]
  25. Soon L, Flechner L, Gutkind JS, Wang LH, Baserga R, Pierce JH, Li W 1999 Insulin-like growth factor 1 synergizes with interleukin 4 for hematopietic vell proliferation independent of insulin receptor substrate expression. Mol Cell Biol 19:3816–3828[Abstract/Free Full Text]
  26. Bruning JC, Winnay J, Cheatham B, Kahn CR 1997 Differential signaling by insulin receptor substrate-1 (IRS-1) and IRS-2 in IRS-1-deficient cells. Mol Cell Biol 17:1513–1521[Abstract]
  27. Kim B, Leventhal PS, White MF, Feldman EL 1998 Differential regulation of insulin receptor substrate-2 and mitogen-activated protein kinase tyrosine phosphorylation by phosphatidylinositol 3-kinase inhibitors in SH-SY5Y human neuroblastoma cells. Endocrinology 139:4881–4889[Abstract/Free Full Text]
  28. Aguirre V, Werner ED, Giraud J, Lee JH, Shoelson SE, White MF 2002 Phosphorylation of serine307 in insulin receptor substrate-1 blocks interactions with the insulin receptor and inhibits insulin action. J Biol Chem 277:1531–1537[Abstract/Free Full Text]
  29. Yenush L, Zanella C, Uchida T, Bernal D, White MF 1998 The pleckstrin homology and phosphotyrosine binding domains of insulin receptor substrate 1 mediate inhibition of apoptosis by insulin. Mol Cell Biol 18:6784–6794[Abstract/Free Full Text]
  30. Belletti B, Prisco M, Morrione A, Valentinis B, Navarro M, Baserga R 2001 Regulation of Id2 gene expression by the IGF-I receptor requires signaling by phosphatidylinositol-3 kinase. J Biol Chem 276:13867–13874[Abstract/Free Full Text]
  31. Peruzzi F, Prisco M, Dews M, Salomoni P, Grassilli E, Romano G, Calabretta B, Baserga R 1999 Multiple signaling pathways of the IGF-I receptor in protection from apoptosis. Mol Cell Biol 19:7203–7215[Abstract/Free Full Text]
  32. Prisco M, Peruzzi F, Belletti B, Baserga R 2001 Regulation of Id gene expression by the type 1 insulin-like growth factor: roles of Stat3 and the tyrosine 950 residue of the receptor. Mol Cell Biol 21:5447–5458[Abstract/Free Full Text]
  33. White MF 1998 The IRS-signalling system: a network of docking proteins that mediate insulin action. Mol Cell Biochem 182:3–11[CrossRef][Medline]
  34. Tu X, Baffa R, Luke S, Prisco M, Baserga R 2003 Intracellular redistribution of nuclear and nucleolar proteins during differentiation of 32D murine hemopoietic cells. Exp Cell Res 288:119–130[CrossRef][Medline]
  35. Delahaye L, Mothe-Satney I, Myers MG, White MF, van Obberghen E 1998 Interaction of insulin receptor substrate-1 (IRS-1) with phosphatidylinositol 3-kinase: effect of substitution of serine for alanine in potential IRS-1 serine phosphorylation sites. Endocrinology 139:4911–4919[Abstract/Free Full Text]
  36. Sun XJ, Crimmins DL, Myers Jr MG, Miralpeix M, White MF 1993 Pleiotropic insulin signals are engaged by multisite phosphorylation of IRS-1. Mol Cell Biol 13:7418–7428[Abstract/Free Full Text]
  37. Yenush L, Fernandez R, Myers Jr MG, Grammer TC, Sun XJ, Blenis J, Pierce JH, Schlessinger J, White MF 1996 The Drosophila insulin receptor activates multiple signaling pathways but requires insulin receptor substrate proteins for DNA synthesis. Mol Cell Biol 16:2509–2517[Abstract]
  38. Prisco M, Maiorana A, Guerzoni C, Calin G, Calabretta B, Voit R, Grummt I, Baserga R 2004 The role of peccadillo and upstream binding factor in the proliferation and differentiation of murine myeloid cells. Mol Cell Biol 14:5421–5433
  39. Voit R, Hoffmann M, Grummt I 1999 Phosphorylation of G1-specific cdk-cyclin complexes activates the nucleolar transcription factor UBF. EMBO J 18:1891–1899[CrossRef][Medline]
  40. Voit R, Schafer K, Grummt I 1997 Mechanism of repression of RNA polymerase I transcription by the retinoblastoma protein. Mol Cell Biol 17:4230–4237[Abstract]
  41. Ringertz NR, Savage RE 1976 Cell hybrids. New York: Academic Press




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
145/11/5332    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles