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Endocrinology Vol. 142, No. 3 1073-1081
Copyright © 2001 by The Endocrine Society

ARTICLES

Limited Redundancy of Survival Signals from the Type 1 Insulin-Like Growth Factor Receptor1

Magali Navarro and Renato Baserga

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

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The type 1 insulin-like growth factor receptor (IGF-IR) is effective in protecting cells from a variety of apoptotic injuries. In 32D murine hemopoietic cells, the IGF-IR sends three separate survival signals, through insulin receptor substrate-1, Shc, and mitochondrial Raf translocation. We report here that these three pathways for survival have a limited redundancy. If one of these pathways is blocked, the IGF-IR can still protect 32D cells from apoptosis induced by interleukin-3 withdrawal. However, when two of the three pathways are inactivated, the receptor is no longer capable to protect cells from apoptosis. The survival signal can use any two pathway combinations.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IT IS NOW generally acknowledged that the type 1 insulin-like growth factor receptor (IGF-IR) is a potent survival factor. The IGF-IR activated by its ligands protects many cell types from a variety of proapoptotic injuries (reviewed in Refs. 1 and 2), whereas its down-regulation causes massive apoptosis of cells, especially if the cells are grown under anchorage-independent conditions (3). The mechanism by which the IGF-IR protects cells from apoptosis has been the object of a series of investigations, culminating in a reasonable elucidation of the main pathways used by the IGF-IR against apoptotic injuries. The main pathway originates from the interaction of the IGF-IR with one of its major substrates, insulin receptor substrate-1 (IRS-1), which activates phosphatidylinositol-3 kinase (PI3K) (4, 5, 6). PI3K, in turn, activates Akt/PKB (7, 8, 9, 10). The concluding step is the phosphorylation, by Akt/PKB, of BAD (11, 12), one of the members of the Bcl-2 family of proteins. BAD is known to be a heterodimeric partner for both Bcl-XL and Bcl-2, neutralizing their antiapoptotic function and promoting cell death (13). In response to survival factors, including IGF-I (11), and activation of the Akt/PKB pathway (14), BAD is serine phosphorylated by Akt (12). It is no longer capable to heterodimerize with Bcl-XL at membrane sites, is sequestered into the cytosol, bound to 14.3.3, and inactivated as a cell death-promoting protein (15).

Although the above-mentioned pathway is generally recognized as the main pathway by which the IGF-IR exerts its antiapoptotic effect, there is substantial evidence that the IGF-IR has alternative pathways. The first clue to alternative pathways was provided by 32D cells, a murine hemopoietic cell line (16) that is IL-3 dependent for growth and undergoes apoptosis after IL-3 withdrawal. 32D cells do not express IRS-1 or IRS-2 (17, 18). Yet, 32D cells overexpressing, even modestly, the wild-type IGF-IR, survive IL-3 withdrawal, and actually grow for at least 48 h in the absence of IL-3 (19, 20, 21). 32D cells fail to grow without IL-3 when overexpressing the insulin receptor (IR), although they do grow without IL-3 when stably transfected with plasmids expressing both the IR and IRS-1 (17, 22). Overexpression of IRS-1, by itself, only offers partial protection under the same conditions (18, 23). Thus, it seems that the IGF-IR can use, for mitogenesis and/or survival, a pathway that is IRS-1 independent and is not shared with the IR. This was confirmed by the observation that the IGF-IR and the IR have similar antiapoptotic properties when overexpressed in mouse embryo fibroblasts that express substantial amounts of IRS proteins (24). However, even in mouse embryo fibroblasts, a difference could be detected when the cells were treated with inhibitors of PI3K, again suggesting that the IR depends for a survival signal on the IRS-1 pathway, whereas the IGF-IR has other pathways. One alternative pathway is the mitogen-activated protein kinase (MAPK) pathway (22, 25), originating at least in part from another major substrate of the IGF-IR, the Shc proteins (26, 27, 28, 29), and leading to Ras activation (30). Tyrosine residue 950 (Y950) is the major binding site in the IGF-IR for the Shc proteins (31, 32), and a Y950F mutation impairs the protective effect of the IGF-IR on 32D cells after IL-3 withdrawal (21, 33). Finally, a third pathway was proposed by Peruzzi et al. (22), which depends on the integrity of a serine quartet at residues 1280–1283 of the human IGF-IR (34). These serines are known to bind isoforms of the 14.3.3 protein (35, 36), and their presence promotes the mitochondrial translocation of Raf-1 (22). There is substantial evidence that 14.3.3 proteins modulate Raf activation (37, 38, 39). Targeting of Raf-1 to mitochondria also results in inhibition of apoptosis (40, 41, 42). All three pathways were shown to lead to BAD phosphorylation (22).

Signal transduction from growth factor receptors is very complex and has a large degree of redundancy (43, 44). For the sake of clarity, we define here the three signaling pathways identified by Peruzzi et al. (22) as contributing to the antiapoptotic effect of the IGF-IR (see above): 1) the IRS-1 pathway, which could also be designated the PI3K/Akt pathway; 2) the MAPK pathway, which is strongly dependent on the Shc proteins, and involving the activation of Ras (30, 45, 46, 47, 48); and 3) the 14.3.3 pathway, which depends on the integrity of serines 1280–1283 (22, 35, 36) and results in the mitochondrial translocation of Raf-1 kinase. For simplicity, in the rest of the paper, we will refer to these pathways as the IRS-1, MAPK, and 14.3.3 pathways with the understanding that overlapping and redundancy make this nomenclature an oversimplification. In this investigation we have asked whether these three pathways are all necessary for the antiapoptotic effect of the IGF-IR. In other words, we asked how much redundancy there is in the system, and whether one pathway is sufficient or more than one pathway is necessary. For this purpose, we used different mutants of the IGF-IR that are known to affect different signaling pathways.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and retroviral transductions
The 32D cell line is a well characterized interleukin-3 (IL-3)-dependent myeloid cell line, derived from normal murine marrow (16). To generate cell lines expressing higher levels of the wild-type IGF-IR or its various mutants, 32D cells were retrovirally transduced with a murine leukemia virus-based system, as described in detail by Romano et al. (49) (see Table 1Go). The mutants of the IGF-IR have been described in previous papers from our laboratory (21, 33, 49). The 32D cells expressing IRS-1 are described in Valentinis et al. (33). The mutant Raf1 (mRaf) is the mitochondria targeted and constitutively active Raf1 described by Salomoni et al. (42) and Peruzzi et al. (22). Briefly, this mutant consists of the COOH-terminal region (amino-acids 301–648) of human Raf1 fused with the transmembrane domain of the yeast outer mitochondrial membrane protein MAS70p. The mRaf complementary DNA has been inserted in the HpaI site of the pMSCV-hph vector (49). The various mixed populations of 32D-derived cells were grown in RPMI 1640 medium, supplemented with 10% heat-inactivated FBS (Life Technologies, Inc., Gaithersburg, MD), 10% WEHI cells conditioned medium as a source of IL-3, 2 mM L glutamine, and the required antibiotic to maintain the selective pressure [G418 (Life Technologies, Inc.), 500 µg/ml; hygromycin (Calbiochem, La Jolla, CA), 500 µg/ml; puromycin (Life Technologies, Inc.), 1 µg/ml].


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  		Table 1. Cell lines

 
Survival assay
Exponentially growing cells were washed three times in RPMI 1640, then seeded at a density of 105 cells/ml in IL-3-free medium [RPMI 1640 medium, 10% heat-inactivated FBS, 2 mM L-glutamine, 0,1% BSA (Sigma, St. Louis, MO), 50 µg/ml transferrin (Sigma)] supplemented with 50 ng/ml IGF-I (Life Technologies, Inc.). In some experiments LY 2940002 (BIOMOL Research Laboratories, Inc., Plymouth Meeting, PA), or PD98059 (New England Biolabs, Inc., Beverly, MA; 25 µM) were added at the time of seeding. Cell numbers were determined in duplicate after 24 and 48 h in a hemocytometer. Only cells that excluded trypan blue were counted. All experiments were repeated several times, and SDs are given in the figures.

Western blots for IGF-IR, IRS-1, and mitochondrial RAf expression
The levels of IGF-IR and IRS-1 expression were monitored by Western blot as previously described (24, 33, 49). Briefly, 100 µg whole cell lysates from exponentially growing cells were resolved by SDS-PAGE and transferred to a nitrocellulose filter. The filter was then immunoblotted with either a polyclonal antibody against the IGF-IR {alpha}-subunit (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or a polyclonal antibody against the IRS-1 carboxyl-terminus (Upstate Biotechnology, Inc., Lake Placid, NY). The blots were developed using the enhanced chemiluminescence system (Amersham Pharmacia Biotech, Arlington Heights, IL), according to the manufacturer’s instructions. The detection of mRaf was carried out following the same procedure, using a polyclonal antibody against the carboxyl-terminus of Raf1 (Raf1 C12, Santa Cruz Biotechnology, Inc.).

Detection of phosphorylated proteins
Exponentially growing cells were washed three times in RPMI 1640 and starved for 3 h in serum-free and IL-3-free medium (RPMI 1640, 2 mM L-glutamine, 0.1% BSA, and 50 µg/ml transferrin), before stimulation with 50 ng/ml IGF-I for the indicated times. For Shc phosphorylation, 500 µg whole cell lysate were immunoprecipitated using a polyclonal antibody against Shc (Transduction Laboratories, Inc.). After resolution on SDS-PAGE and transfer on nitrocellulose filter, phosphotyrosine blots were performed with an antiphosphotyrosine horseradish peroxidase-conjugated antibody (PY20, Transduction Laboratories, Inc., Lexington, KY). Total Shc proteins were then detected using a monoclonal antibody against Shc (Santa Cruz Biotechnology, Inc.). IRS-1 phosphorylation was detected with the same procedure as Shc phosphorylation using 1 mg whole cell lysate immunoprecipitated with a polyclonal antibody against IRS-1 carboxyl-terminus (Upstate Biotechnology, Inc.). MAPK and AKT phosphorylation were detected on 100 µg whole cell lysate using, respectively, a monoclonal antibody against phospho-MAPK (phospho-p44/p42 Map kinase Thr202/Tyr204 E10, New England Biolabs, Inc.), and a polyclonal antibody against phospho-AKT (phospho-AKT Ser473 antibody, New England Biolabs, Inc.) following the manufacturer’s instructions. The levels of total MAPK and total AKT proteins were then determined using, respectively, a polyclonal antibody against Erk1/2 (Santa Cruz Biotechnology, Inc.), and a polyclonal antibody against AKT (AKT antibody, New England Biolabs, Inc.). The blots were developed using the enhanced chemiluminescence system.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The cell lines used in these experiments were all derived from 32D, clone 3 cells (50), which are murine hemopoietic cells with an absolute requirement for IL-3. In the parental cell line, withdrawal of IL-3 results in rapid cell death (50, 51, 52). The mechanism of cell death is apoptosis, amply documented in previous papers (19, 20, 22, 33). 32D cells do not express IRS-1 or IRS-2 (see above) and express a low number of IGF-IR (19).

The following cell lines were previously described: 32D-IGF-IR cells, which overexpress wild-type IGF-IR, and 32 D-IGF-IR/IRS-1 cells, which express both IGF-IR and IRS-1 (33). 32D-derived cell lines expressing the IGF-IR mutants were also previously described (21, 22, 33). For these studies they were transduced with either IRS-1 or the empty vector for IRS-1, thus generating new cell lines. The cell lines used are listed in Table 1Go. All 32D-derived cell lines grow very well in 10% serum supplemented with IL-3. All of them undergo apoptosis after IL-3 withdrawal, unless supplemented with IGF-I. In subsequent experiments, we will limit the presentation of data to cells in 10% serum supplemented with 50 ng/ml IGF-I.

Expression of IGF-IR and IRS-1
Figure 1AGo gives the levels of expression of the IGF-IR in the different cell lines. For the Western blot, we used an antibody to the {alpha}-subunit of the IGF-IR. Both the proreceptor and the {alpha}-subunit are visible. The proreceptors of cell lines expressing the {delta}1245 mutant receptor migrate, as expected, slightly faster than the full length proreceptors. The levels of expression vary slightly among the various cell lines. However, the levels are consistently elevated. By comparison with cell lines with known receptor numbers (not shown), one can calculate that these cell lines express approximately from 5 x 104–105 receptors/cell.



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  		Figure 1. Expression of the IGF-I receptor and IRS-1 in the various cell lines. A, Expression levels of the IGF-I receptor. The cell lines (the abbreviations are explained in Table 1Go) are indicated above the respective lanes. Western blot using an antibody to the {alpha}-subunit of the IGF-I receptor. B, Levels of expression of IRS-1. The cell lines are indicated above the respective lanes. Western blot using an antibody to IRS-1. Only one parental cell line is shown (indicated as IGF-IR and transduced with an empty vector), as it has been repeatedly shown that parental 32D cells do not express IRS-1 (see text). C, Tyrosyl phosphorylation of IRS-1 in the cells expressing it. The cell lines are indicated above the lanes, each cell line before or after stimulation with IGF-I. Immunoprecipitation was performed with an antibody to IRS-1, and blotting was performed with a phosphotyrosine antibody.

 
Figure 1BGo shows the levels of expression of IRS-1. In parental cells and in all the 32D cells transduced with the empty vector, no IRS-1 is visible in Western blots (17, 22, 33). In this figure we show for simplicity only one of the cell lines transduced with the empty retroviral vector (indicated as IGF-IR), but all of the cell lines transduced with the empty vector remained negative for IRS-1. In the 32D cells transduced with IRS-1, the levels of expression are reasonably uniform, as one would expect from mixed populations transduced with retroviral vectors. In cells expressing IRS-1, we monitored its tyrosyl phosphorylation after stimulation with IGF-I. All cells responded to IGF-I with tyrosyl phosphorylation of IRS-1 (Fig. 1CGo; documented for 32-derived cells in Refs. 33 and 53).

Survival of cells
As mentioned above, parental 32D cells undergo apoptosis after IL-3 withdrawal. Figure 2Go gives cell survival for the various 32D-derived cell lines at 24 and 48 h after IL-3 withdrawal. The data presented are limited to cells in 10% serum, supplemented with IGF-I (50 ng/ml), in the absence of IL-3. All cell lines grew vigorously in IL-3-supplemented medium, and all of them died in the absence of IGF-I and IL-3 (not shown). In the absence of IRS-1 expression, only the 32D cells expressing the wild-type IGF-IR (32D-IGF-IR) survived and actually grew, confirming previous results from two different laboratories (19, 20, 22, 53). Ectopic expression of IRS-1 dramatically changed the character of these cell lines. 32D-IGF-IR/IRS-1 cells grew better than 32D-IGF-IR cells (33, 53). The cell lines expressing mutant receptors that died in the absence of IRS-1 now survived for the first 24 h and even grew somewhat. This is true also for the 32D-Y/{delta}/IRS-1 cells, although Fig. 2Go does not show it clearly, because the cells just survived, without growth. The situation changed at 48 h for 32D-Y/{delta}/IRS-1 and 32D-Y/4S/IRS-1, which now decreased in number, whereas 32D-Y950 and 32D-{delta}1245 cells expressing IRS-1 still survived and increased in number. These experiments were repeated several times with the same results. We also extended these studies to later times after IL-3 withdrawal (not shown). The same cell lines that survived at 48 h, survived (and grew) at day 4, whereas it was difficult to find live cells in those cell lines that started dying between 24 and 48 h.



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  		Figure 2. Survival of 32D-derived cells after IL-3 withdrawal. The cell lines are indicated below the abscissa (see Table 1Go for the key to the cell lines). All cell lines were grown in 10% serum supplemented with IGF-I (50 ng/ml). The results are expressed as the percent increase or decrease over the number of cells plated 24 h (A) or 48 h (B) after IL-3 withdrawal. All cell lines grow in IL-3, and all die in 10% serum, not supplemented with either IL-3 or IGF-I (not shown).

 
We constantly monitored 32D-derived cells for cell death and cell division. In growing cell lines, as previously reported (53), 95–99% of the cells incorporated bromodeoxyuridine (Brdu) over a period of 24 h, and cell death was limited to 2–3% over the same time period. In 32D IGF-IR cells (no IRS-1), this was true only for the first 48 h, as the cells began to differentiate between 2 and 3 days after IL-3 withdrawal and IGF-I supplementation (33). In the cell lines that began to die in the first 24 h or between 24 and 48 h, the number of cells incorporating Brdu was usually below 5% in the first 24 h and was zero at later times.

The results confirm that the wild-type IGF-IR protects 32D cells from apoptosis, and that even a single mutation in the receptor at critical residues (see introduction and Discussion) impairs or abrogates its protective effect. Reintroduction of IRS-1 restored the antiapoptotic effect of the IGF-IR in mutants with a single mutation. However, when the IGF-IR carried a double mutation (Y950 and {delta}1245, or Y950 and the 4-serines), then IRS-1 could not restore the protective effect, a failure that was especially evident at 48 h.

Effects of inhibitors of signal transduction
The results in Fig. 2Go are compatible with the previous finding that the IGF-IR can protect 32D cells from apoptosis through three different pathways (22) and suggest that at least two of these pathways must be conserved for the protective effect. To confirm this hypothesis, we used inhibitors of PI3K or MEK, which, respectively, block the IRS-1 and MAPK pathways (see introduction for definition of pathways). The inhibitors were tested only on the cells that survived in the first 24 h, that is, the cells expressing the wild-type IGF-IR, and the cells expressing mutant receptors and IRS-1. As usual, the cell lines were incubated in 10% serum supplemented by IGF-I. A typical experiment, at 24 h after the addition of the inhibitors, is shown in Fig. 3Go. As reported previously (22, 33), the two inhibitors (LY 294002 and PD98059) singly had a moderate effect on the survival of 32D-IGF-IR/IRS-1 cells. Cell growth was partially inhibited, but the number of cells at 24 h was still considerably greater than the number of cells plated. Cell death at 24 h was still negligible. The other three cell lines shown in Fig. 3Go, {delta}1245/IRS-1, Y950/IRS-1, and Y/4S/IRS-1, fared less well with both inhibitors. Two of the cell lines, {delta}1245/IRS-1 and Y/4S/IRS-1, decreased in number, whereas the Y950/IRS-1 barely survived. No evidence of proliferation was detectable in these cells. This trend was even more pronounced at 48 h (not shown). The combined use of both inhibitors was precluded by toxicity. These experiments confirm that at least two of the three pathways must be operative for the protective effect of the IGF-IR on 32D cells.



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  		Figure 3. Effects of inhibitors on survival of 32D-derived cells. The cell lines are indicated below the abscissa. The treatment is also indicated. LY is the PI3K inhibitor LY294002. PD is the MEK inhibitor PD98059. Survival is expressed as the percent increase or decrease over the number of cells plated.

 
Activation of Akt in 32D-derived cell lines
PI3K activity is not detectable in parental 32D cells, and ectopic expression of IRS-1 results in a strong PI3K signal (20). The experiments described above with LY294002 indirectly confirm the need for PI3K activation for one of the survival signals. To further confirm the activation of the PI3K pathway, we investigated Akt activation in the various cell lines derived from 32D cells. The results are summarized in Fig. 4AGo. All of the cell lines expressing IRS-1 showed a sharp increase in Akt phosphorylation after stimulation with IGF-I. In the absence of IRS-1, the only cell line that showed a clearly detectable increase in Akt phosphorylation was the 32D-IGF-IR cell line, as previously reported (33). A faint band was also visible after IGF-I stimulation in the {delta}1245 cell line. Interestingly, Akt activation was not detectable at all in the three cell lines with a Y950F mutation. It suggests that the IGF-IR can activate Akt by an IRS-1-independent pathway, but that this activation requires the integrity of Y950.



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  		Figure 4. IGF-I receptor signaling in 32D-derived cells. A, Phosphorylation of Akt (upper row) and total Akt (lower row) in various cells lines with or without IGF-I. B, ERK phosphorylation (upper row) and amounts (lower row) in various cell lines. The numbers indicate time in minutes after stimulation with IGF-I. C, Shc phosphorylation (upper row) and amounts (lower row) in the same cell lines as those in A and B. The antibodies used are described in Materials and Methods.

 
Activation of ERKs in 32D-derived cell lines
ERK1/2 activation is shown in Fig. 4BGo. The double mutant receptors, Y950/4S and Y950/{delta}1245, had decreased activation of ERK proteins, especially the latter one, for which no activation was detectable. Introduction of IRS-1 into these two cell lines showed a small increase in ERK activation for both, but the level of activation remained quite low compared with that in the other cell lines. This is in agreement with the generally held view that IRS-1 is less effective than Shc in activating the MAPK pathway (30). ERK activation was normal, even in the absence of IRS-1, in cells expressing receptors with a single mutation. It seems that MAPK activation depends on one of two signals, one coming from Y950 and the other from the C-terminus. The IRS-1 pathway is apparently a poor substitute for these two pathways.

Phosphorylation of Shc proteins in 32D-derived cell lines
Shc activation of the various cell lines is shown in Fig. 4CGo. In all cell lines with a mutated Y950, Shc phosphorylation was undetectable, confirming the results of Valentinis et al. (33). It is clear that Y950 is required for Shc activation, and it is not compensated by the ectopic expression of IRS-1. The experiments shown in Fig. 4Go were repeated several times with the same results. These experiments show that there is a dissociation between Shc phosphorylation and MAPK activation in both the presence and the absence of IRS-1.

A mutant Raf-1 rescues the Y950–4S/IRS-1 cells from apoptosis
In a previous report (22) we showed that a mutant Raf could rescue from apoptosis 32D cells expressing the IGF-IR carrying a mutation at serines 1280–1283. This mutant Raf is constitutively activated and localizes to the mitochondria, where it inhibits BAD dephosphorylation and activation (22, 40, 42). We reasoned that this mutant Raf (mitRaf) should be able to rescue the double mutant Y950–4S expressing IRS-1. The mitRaf mutant was transduced into 32DY950–4S/IRS-1 cells, and a mixed population was selected. Its survival was then compared with that of the other two cell lines with the same mutation. The results of two separate experiments are shown in Fig. 5Go. As expected, the Y950/4S and the Y/950/4S/IRS-1 cells were dying within 48 h after IL-3 withdrawal and IGF-I supplementation. However, the Y950/4S/IRS-1 cells expressing mitRaf (see inset of Fig. 5Go) survived.



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  		Figure 5. Survival of cells expressing the mitochondrial Raf mutant. The three cell lines are Y950/4S, Y950/4S/IRS-1, and Y950/4S/IRS-1 cells transfected with a plasmid expressing the mitochondrial Raf mutant. Expression of the mutant Raf is shown in the inset. Survival is expressed as usual, and the cell lines are indicated on the abscissa.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results of these experiments confirm and extend the observation of Peruzzi et al. (22) that the IGF-IR uses three different pathways for protection from apoptosis. The three pathways identified by Peruzzi et al. (22) were the PI3K/IRS-1 pathway, the Shc/MAPK pathway, and the 14.3.3/mitochondrial Raf pathway (see introduction). All three pathways led to BAD phosphorylation and inactivation. The main conclusion that can be drawn from the experiments described above is that two of the three pathways are necessary and sufficient for the survival of 32D IGF-IR cells after IL-3 withdrawal. When one of the three pathways is inhibited, the cells can still survive. When two of the three pathways are blocked, the IGF-IR no longer protects from IL-3 withdrawal. Other interesting findings that should be discussed are the Akt activation in the absence of IRS-1 and the dissociation between Shc phosphorylation and MAPK activation.

It is generally accepted that there is abundant cross-talk and redundancy in signal transduction not only among different receptors, but also within a single receptor. The elegant experiments of Fambrough et al. (43) have clearly shown that signals originating from different domains of the platelet-derived growth factor-ß receptor are redundant, and their findings have led Pawson and Saxton (44) to propose a network of signal transduction. This proposal is both correct and attractive, and in a sense we have confirmed it in the experiments presented in this communication. The survival signal originating from the IGF-IR is redundant; one of the three pathways can be eliminated without affecting survival, and it really does not matter which one.

The IGF-IR depends for its basic functions on two domains: the ATP-binding site at lysine 1003, and the tyrosine kinase domain at Y1131, Y1135, and Y1136. Mutations at these sites essentially result in a receptor that is defective in all its functions (54, 55, 56). The mitogenicity of the IGF-IR in mouse embryo fibroblasts expressing IRS-1 is not affected by a single mutation at Y950 or by truncation at residue 1245 (49). These two receptors, however, have lost the ability to transform cells (49). The results are different in cells that do not express IRS-1, such as 32D cells. In these cells, single mutations at Y950 or at the serine quartet at 1280–1283 or truncation at residue 1245 all result in a severe impairment of survival after IL-3 withdrawal (Refs. 21 and 33 and this paper). In addition, these mutations affect the ability of the IGF-IR to induce differentiation of 32D cells (33). We show here that ectopic expression of IRS-1 in 32D cells expressing single mutations brings about survival. It is only when the receptor has double mutations that IRS-1 fails to protect cells from apoptosis. These results are indicative of a limited redundancy in survival signaling from the IGF-IR. Regardless of whether there are other pathways (see below), it is clear that two of three known pathways are both necessary and sufficient for IGF-IR-mediated survival of 32D cells.

The pathway designated here the IRS-1 pathway (through Akt/PKB) is the best recognized and is arguably the most powerful one (7, 8, 10, 11, 12). In 32D IGF-IR cells (wild-type receptor), IGF-I induces a burst of cell proliferation, which lasts about 48 h. After 48 h, the cells stop proliferating and differentiate along the granulocytic pathway (33). Ectopic expression of IRS-1 in 32D IGF-IR cells inhibits differentiation and actually transforms the cells. 32D IGF-IR/IRS-1 cells can be passaged in the absence of IL-3 and form tumors in mice (53). IRS-1 is a strong activator of the PI3K pathway (4, 5, 6). In 32D cells, ectopic expression of IRS-1 invariably results in strong activation of both PI3K (20) and Akt (33) (Fig. 4Go). It also results in a marked increase in the activation of p70S6K. The crucial role of p70S6K in the inhibition of IGF-I-mediated differentiation was confirmed by the finding that rapamycin (an inhibitor of this pathway) caused differentiation of 32D IGF-IR/IRS-1 cells (53). Thus, the pathway designated in this paper the IRS-1 pathway is certainly a very important one in IGF-I-mediated mitogenesis and survival [see also the review by Avruch (47)]. However, by itself it is not sufficient, as survival requires an additional signal from the IGF-IR.

A puzzle to be resolved is that there is modest, but reproducible, Akt activation by the wild-type receptor in the absence of IRS-1 (33). Akt activation is abolished by a Y950F mutation. The possibility that the reduced Akt activation detectable with the wild-type IGF-IR in the absence of IRS-1 may be due to Shc signaling should be considered. However, Y950 also binds to c-CrkII (57), and the Crk family of proteins is known to transmit the IGF-IR signal (58, 59). Alternatively, the modest activation of Akt by the wild-type IGF-IR may be due to its interaction with other signal transduction pathways [see review by Blakesley et al. (2)] or to integrin stimulation (60).

A MAPK pathway for IGF-I-mediated survival was described by Parrizas et al. (25) and Peruzzi et al. (22). This pathway is thought to depend at least in part on the activation of Shc proteins. The major binding site in the IGF-IR for Shc proteins is Y950 (31), and indeed, a Y950F mutation in 32D cells leads to undetectable tyrosyl phosphorylation of Shc (33). However, ERKs are still strongly activated in 32D/Y950F cells, suggesting another IGF-IR pathway for their activation. The finding that MAPK activation is completely absent from 32D cells expressing the double mutant Y950/{delta}1245 suggests a double signal, one from Y950 and one from a residue(s) in the C-terminus. These two signals have to be considered redundant, as single mutations have little effect on ERK activation. Interestingly, ectopic expression of IRS-1 seems to increase the ERK signal in all cell lines, including the double mutant cell line. In this last cell line, there is now a faint signal, presumably due to the interaction of IRS-1 with Grb2 (61), but this signal is not sufficient for survival.

The first two pathways are the generally accepted ones. The third pathway considered in this paper is dependent on the integrity of the serine quartet at 1280–1283 (22). As mentioned in the introduction, these serines are known to bind isoforms of the 14.3.3 proteins (35, 36), and their presence promotes the mitochondrial translocation of Raf-1 (22). Incidentally, the role of the IGF-IR in the mitochondrial translocation of Raf has received an independent confirmation from Nantel et al. (62), who have reported that Raf-1 and Grb10 (a substrate of the IGF-IR) can be coimmunoprecipitated from mitochondrial fractions. Activation of Raf kinase is still not well understood, although there is agreement that Raf-1 is recruited to the cellular membrane by Ras (47). A reasonable hypothesis to explain this third pathway can be based on the ambiguous effects of 14.3.3 proteins on the activation of Raf-1. Several reports (63, 64) have shown that 14.3.3 proteins can stabilize Raf-1 in both its inactive and active forms. These contradictory effects have been discussed clearly in a review by Hagemann and Rapp (65). These researchers have marshaled the evidence that inactive Raf is bound to 14.3.3 proteins at both the amino- and the carboxyl-termini (serines 259 and 621, respectively). When activated by Ras, the 14.3.3 protein at the amino-terminus (but not the carboxyl-terminus) is released, leading to a change in conformation. The activated Raf-1 is then stabilized again in its active form by binding 14.3.3 to a not yet identified serine between serine 259 and the ATP-binding site of Raf-1 (65). In support of this hypothesis is the finding that the mutant Raf that is targeted for mitochondrial translocation lacks serine 259 (42). It should be noted that mutant Raf does not protect parental 32D cells (22), indicating that it needs an activated IGF-IR. As it protects 32D cells expressing the 4-serine mutant in the absence of IRS-1 (22), it seems that IRS-1 is not required for its effect, provided another pathway is present. Regardless of the mechanism, this third pathway is also insufficient for survival by itself.

To date, we have equated the C-terminus truncation ({delta}1245) to the mutation in the 4-serine at 1280–1283. The two mutants give similar results, but a careful examination of the data will reveal that the 4-serine mutant does better in survival than {delta}1245, a difference we had noticed previously (21). It suggests that the 4-serine signal may be reinforced by another signal originating from the C-terminus. The tyrosines at 1250–1251 are possible candidates (66, 67).

There is another important issue that should be considered, and that is the distinction between survival and growth. In this report as well as in previous ones (21, 22, 23, 53), we equated mitogenicity of the receptor with a survival signal. In favor of this interpretation is our findings that by using mutants of the IGF-IR, we have never been able to dissociate mitogenicity from survival in the long term (49). However, a careful perusal of our previous data as well as those shown in Figs. 1Go and 3Go indicate that this may be an oversimplification. For instance, cells expressing the Y950F mutant do not proliferate in the first 24 h (no Brdu incorporation), but the number of cells remains roughly the same number of cells plated (in some experiments, the decrease was not significant). Taken at face value, one could say that a mutation at Y950 abrogates mitogenicity, but not survival. This interpretation would be really attractive, as one could hope to separate on the IGF-IR the mitogenic signal from its survival signal. Unfortunately, this is no longer true at 48 h, when 32D Y950F cells have rapidly decreased in number, close to a 90% loss. For the moment, we have to limit ourselves to state that when the cells do not proliferate, they eventually die. A successful separation of mitogenicity from survival may have to wait for the development of other models.

We propose the following explanation for our results. In the absence of IRS-1, MAPK activation by the IGF-IR is controlled by two signals, at Y950 and in the C-terminus (4-serine, but see above). Single mutants can still activate MAPK, although a slight decrease may be detected.

The 4-serine signal induces Raf-1 activation (30, 47) and causes its mitochondrial translocation (22). In combination with a modest activation of Akt/PKB, this results in survival. The insulin receptor cannot protect parental 32D cells, because it lacks the 4-serine signal (22) as well as IRS-1. Akt/PKB activation, in the absence of IRS-1, is controlled by Y950. The observation that LY294002 has little effect on the survival of 32D IGF-IR cells (22) also suggests that the activation of Akt by Shc is not completely PI3K dependent. Chan et al. (68) in their review pointed out that there are reports of PI3K-independent activation of Akt, and that one of the activators could be Ras. Ras, in turn, is activated by Shc (30). A simplified scheme is presented in Fig. 6Go. Summarized briefly, it suggests that for IGF-IR-mediated survival of 32D cells, one requires a double activation of MAPK, the activation of Akt, and the mitochondrial translocation of Raf-1. We cannot say at this moment whether the MAPK activation by Raf-1 is qualitatively or simply quantitatively different from the MAPK activation by Y950/Shc.



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  		Figure 6. Simplified diagram of the three pathways for IGF-I-mediated survival described in this report.

 
In conclusion, we confirmed that the IGF-IR has at least three pathways for protection of 32D cells from apoptosis induced by IL-3 withdrawal (22). Any two of these pathways are sufficient for a survival signal regardless of the combinations. The survival signal of the IGF-IR, therefore, is redundant, but its redundancy is limited.


   Footnotes
 
1 This work is supported by NIH Grants CA-78890 and AG-16291. Back

Received July 24, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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