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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
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Materials and Methods |
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). 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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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 
-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.
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Results |
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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 1
. 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 1A gives the levels of
expression of the IGF-IR in the different cell lines. For the Western
blot, we used an antibody to the -subunit of the IGF-IR. Both the
proreceptor and the -subunit are visible. The proreceptors of cell
lines expressing the 
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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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. 1C; 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 2 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//IRS-1 cells,
although Fig. 2 does not show it clearly, because the cells just
survived, without growth. The situation changed at 48 h for
32D-Y//IRS-1 and 32D-Y/4S/IRS-1, which now decreased in number,
whereas 32D-Y950 and 32D-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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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 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. 2 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. 3. 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. 3, 1245/IRS-1, Y950/IRS-1,
and Y/4S/IRS-1, fared less well with both inhibitors. Two of the cell
lines, 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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. 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 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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. The double mutant
receptors, Y950/4S and Y950/1245, 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. 4C. 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. 4 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. 5. 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. 5) survived.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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. 4). 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 Y950/1245 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 (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 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. 1 and 3 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. 6.
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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Footnotes |
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Received July 24, 2000.
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