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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, 624 BLSB, Philadelphia, Pennsylvania 19107. E-mail: r_baserga{at}lac.jci.tju.edu
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1245 mutant), have been previously
described (14, 24, 25, 26, 27, 28, 29, 30). The pGR96 plasmid was generated by
replacing an Xba-HindIII fragment of pGR36
encoding the 1245 receptor construct with the same fragment from the
pGR46 plasmid containing the Y950 mutant.
Cell lines and retroviral transductions
The 32D clone 3 cells (31, 32) or 32D/IRS-1 cells (13) were
transduced with a murine stem cell virus-based retroviral vector (30)
to express the wild-type IGF-IR or its various mutants, or the insulin
receptor. The cell lines expressing the wild-type receptor and the
single mutant constructs have been described previously (see above in
the plasmid section). The 32D cell line expressing a high level of IR
was described in Peruzzi et al. (23). The 32D cells
expressing both IRS-1 and mutant IGF-I receptors were obtained by
transducing the appropriate constructs into 32D/IRS-1 cells. Retroviral
transductions were carried out as described previously (30). All mixed
populations were selected in RPMI 1640 medium supplemented with 10%
FBS, 2 mM L-glutamine, 10%
conditioned medium from the murine myelomonocytic cell line WEHI-3B as
a source of IL-3, and 1 mg/ml G418.
ERK activation
32D cell lines were incubated in serum-free medium supplemented
with 0.1% BSA for 3 h, stimulated with 50 ng/ml IGF-I or insulin
for the indicated times, collected by centrifugation, and rinsed in
ice-cold PBS. Cells were lysed in 100 µl lysis buffer [50
mM HEPES (pH 7.5), 150 mM NaCl, 1.5
mM MgCl2, 1 mM EGTA, 10%
glycerol, 1% Triton X-100, 0.1 mM PMSF, 0.5 µg/ml of
aprotinin, 0.5 µg/ml of leupeptin, and 0.1 mM
Na3VO4]. Lysates (50
µg/lane) were resolved on 10% polyacrylamide gels, transferred to
nitrocellulose (Schleicher & Schuell, Inc., Keene, NH),
and immunoblotted with a phosphospecific antibody against ERK1/ERK2 as
recommended by the manufacturer (Promega Corp., Madison,
WI). To confirm equal loading of proteins, filters were stripped and
reblotted with an anti-ERK1 antibody, which recognizes both ERK1 and
ERK2 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). In
some experiments, cells were treated with the MEK inhibitor PD98059,
purchased from New England Biolabs, Inc. (Beverley, MA).
PD98059 was dissolved in dimethylsulfoxide and added to cells 30 min
before addition of growth factors to a final concentration of 50
µM and 0.1% dimethylsulfoxide.
Expression levels
Expression of the IGF-IR was assessed by Western blot using an
antibody against the 
-subunit (Santa Cruz Biotechnology, Inc.).
Survival assay
Exponentially growing cells were washed three times in HBSS,
counted, and seeded in RPMI 1640 supplemented with 10% FBS or RPMI
supplemented with 10% FBS and either 50 ng/ml IGF-I or insulin
(Life Technologies, Inc., Gaithersburg, MD). Cells were
plated at a density of 5 x 104/ml into each
well of a 24-well plate. At 0, 24, and 48 h after plating, cells
were collected, centrifuged gently, and mixed with trypan blue. The
number of viable cells in each well was determined in duplicate by
counting with a hemocytometer. The numbers given in the various
experiments indicate the numbers of viable cells.
Immunoprecipitation
Cells were starved, stimulated, and lysed as described for MAPK
activation. Proteins were immunoprecipitated from 300 µg lysate
overnight with polyclonal antibodies against Shc (Transduction Laboratories, Inc., Lexington, KY) or IRS-1 (Upstate Biotechnology, Inc., Lake Placid, NY) and were collected using
protein A-Sepharose. Immunoblotting was performed with an
antiphosphotyrosine horseradish peroxidase-conjugated antibody (PY20,
Transduction Laboratories), anti-Grb2 monoclonal antibody (Transduction
Laboratories), anti-Shc monoclonal antibody (Santa Cruz Biotechnology, Inc.), or anti-IRS-1 polyclonal antibody
(Upstate Biotechnology, Inc.).
Phosphorylation of Akt and p70s6k
Cells were treated as described for MAPK activation. Lysates
were resolved on 10% polyacrylamide gels, transferred to
nitrocellulose, and immunoblotted with antibodies against
phosphorylated and total Akt using the PhosphoPlus Akt
(Ser473) Antibody Kit (New England Biolabs, Inc.). Phosphorylated p70s6k and total
p70s6k were detected using the PhosphoPlus
p70s6k Antibody Kit from New England Biolabs, Inc.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, which indicates the
names given to the mutations in the IGF-IR. All cell lines were derived
from 32D cells clone 3, previously described in several papers from our
laboratory (14, 32, 33, 34), as well as from other laboratories (31). As
controls, we used either the parental 32D cells or 32D cells transduced
with an empty vector (the same retroviral vector used for transduction
of the IGF-IR). These two cell lines behaved in exactly the same
manner. 32D cells have very low levels of either the IR or the IGF-IR.
The number of IGF-IR has been estimated at about 2.8 x
103 receptors/cell (22), which is roughly one
eighth the number of receptors in 3T3 mouse embryo fibroblasts (35). In
addition, 32D cells have neither IRS-1 nor IRS-2 (13, 14).
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), and mixed
populations were selected. The cell lines were designated in the same
way as the mutant receptors. By Western blot, we calculated that
receptor levels were well above 105
receptors/cell (not shown, but shown in Ref. 32). Our experience is
that 103 receptors/cell are sufficient for all
IGF-I-mediated functions of the IGF-IR (35). The levels of expression
are also reasonably uniform in the mixed population, as one would
expect from retroviral transduction (32). Plasmids and established cell
lines are referenced in Materials and Methods.
ERK activation
A considerable amount of information is available in the
literature on MAPK (ERK1 and ERK2) activation in cells stimulated with
IGF-I (50 ng/ml) or insulin (50 ng/ml). The primary aim of this paper
was to determine the domain(s) of the IGF-IR required for ERK
activation. As mentioned in the introduction, the IGF-IR can activate
the ERK pathway through either the Shc proteins or IRS-1 binding to
Grb2. We thought that an analysis of the relevant domains would be
facilitated in 32D cells by the absence in these cells of IRS-1.
Accordingly, the cell lines listed in Table 1 were tested in serum-free
medium for IGF-1-mediated activation of ERKs. The cell lines (growing
in IL-3-supplemented medium) were kept in serum-free medium for 3
h, at which time no cell damage was visible (see also below). Figure 1 shows the results of representative
experiments, but these experiments were repeated several times (from
3–10 times, depending on the cell line).
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None of the mutant receptors shown in Fig. 1 gave a response that was
as strong as that of the wild-type IGF-IR. However, the receptors of
the second group were all easily distinguishable from the parent cell
line and from two other mutant receptors: GR96 and the Y950/4 serine
receptor. This seems to indicate that in the absence of IRS-1, the
IGF-IR needs at least two signals to give sustained activation of ERKs.
One signal originates from Y950 (which binds Shc proteins; see
Discussion) and a second from the C-terminus, specifically
from serines 1280–1283 (binding site for 14.3.3; see also
Discussion). Some redundancy is implied by the finding that
single mutants do activate MAPK.
The IR fails to give sustained activation of ERK proteins
The previous experiments indicated that the C-terminus of the
IGF-IR may play a role in ERK activation, especially when associated
with a mutation at Y950. The C-terminus is a region of low homology
between the IGF-I and IR (37), about 44%. Furthermore, the serine
quartet at 1280–1283 is absent in the IR. We hypothesized that in the
absence of IRS-1, the IR would be defective in the activation of the
ERK pathway. Indeed, Fig. 2 shows that
32D/IR cells (overexpressing the IR) give a good, but short, activation
of ERK proteins, with a return to basal levels by 30 min. IRS-1
per se gives a very weak and short activation (Figs. 2, third panel), whereas a combination of IR and IRS-1 restores
a sustained and strong activation. These experiments with the IR
confirm previous reports by White and co-workers (13). The important
difference is that the IGF-IR does not require IRS-1 for sustained
activation of ERK in 32D cells, whereas the IR does.
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shows that IGF-I induces in
32D/IRS-1/GR96 cells a sustained ERK activation, similar to the signal
generated by the wild-type receptor (see Fig. 1). Indeed, the
reintroduction of IRS-1 seems to elevate the ERK response to the same
intensity of the wild-type IGF-IR in parental cells. Figure 3B, shows
that IRS-1 is detectable only in cells in which it has been
reintroduced, and that it is phosphorylated in response to IGF-I. IRS-1
is tyrosyl phosphorylated even in 32D/IRS-1/GR96 cells, indicating that
IRS-1 phosphorylation in these cells does not require a signal from
either Y950 or the C-terminus.
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, A and B, respectively. All cells grow
very well in IL-3, like the parental cell line (not shown), and all
cell lines die in 10% serum, not supplemented with either IL-3 or
IGF-I (FBS in Fig. 4). As previously reported (14, 22, 23, 32, 34, 36),
the wild-type IGF-IR protects 32D cells from apoptosis induced by IL-3
withdrawal. In fact, the wild-type IGF-IR, as shown in the
above-mentioned reports, causes 32D cells to at least double in number
in the first and second days. Again, as shown previously in both 32D
cells and mouse embryo fibroblasts (30), most of the mutant
receptors are not as effective as the wild-type receptor in inducing
growth. However, several of them can protect cells from apoptosis,
especially in the first 24 h. In surviving cells, it can be
demonstrated that the cells are viable, although they do not grow (23).
If we limit ourselves to the survival of cell lines in the first
24 h, it can be seen that three cell lines have fewer cells than
plated: the parental cells, 32D GR18, and 32D GR96. The 32D Y950–4
serine mutant barely protects cells; the number of cells remains just
above the plating level. These four cell lines are those with the
lowest MAPK activation. It seems, therefore, that sustained ERK
activation is predictive of survival in the first 24 h only, an
observation that confirms similar results with some of these and other
mutant receptors (32). The 32D Y950/4 serine mutant, that gives a weak
ERK response is a partial exception, as it protects cells in the first
24 h. It will be noted though that these cells, even at 24 h,
exhibit the least protection (with the exception of 32D GR18 and 32D
GR96, which decrease in number).
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, A and B). Only
32D/IRS-1/IR cells survive, in fact almost as well as the cells
expressing GR15 cells. IRS-1, by itself, offers only partial
protection, thus confirming previous results (14, 38), whereas the IR,
by itself, in these cells has no survival value (see
Discussion).
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summarizes the
results. Expression of IRS-1 in cells already overexpressing the IGF-IR
protects 32D cells from apoptosis, and all cell lines actually grow in
the absence of IL-3. Supplementation with IGF-I is required. It is true
that 32D/GR96/IRS-1 cells are the cells that grow most slowly, but they
do grow, indicating that, with IRS-1, the mutations in the IGF-IR
become silent for (at least a limited) survival.
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and 5). Figure 6C shows that the MEK inhibitor PD98059
inhibits ERK activation in 32D GR15 cells in a concentration-dependent
manner. We then added PD98059 to the medium at a concentration of 50
µM, and determined survival at both 24 (Fig. 6A) and
48 h (Fig. 6B). In all cases, the addition of PD98059 caused a
reduction in survival, confirming the need for ERK activation in these
cells for survival. However, it is interesting that GR15 cells are the
most resistant to the effect of PD98059, and we previously reported
(23) that inhibitors of PI3-kinase also affect GR15 cells less than
they affect 32D/IRS-1/IR cells. It confirms that the IGF-IR has another
pathway for survival, besides the IRS-1 and MAPK pathways, which is the
activation of mitochondrial Raf (23). This third pathway is dependent
on the integrity of the serine quartet and is not shared by the IR.
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shows that the Shc proteins are
strongly phosphorylated by the addition of IL-3 in all cell lines
tested. In both 32D/IR and 32D GR15 cells, the 52-kDa isoform is
tyrosyl phosphorylated by the respective growth factors (Fig. 7A). Note
that the activation of Shc is associated with the coprecipitation of
Grb2 from the lysates. There is no detectable Shc phosphorylation in
the parental 32D cells stimulated with insulin. However, a weak signal
appears in parental 32D cells stimulated with IGF-I (Fig. 7B). The same
figure shows that Shc is phosphorylated, not only in GR15 cells, but
also in 32D GR46 cells, with a mutation at Y950, which is a binding
site for Shc (40, 41).
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, where the blot has been overexposed) is
definitely impaired in GR96 cells (Y950F and deletion at 1245),
indicating that full Shc phosphorylation requires two signals, one from
Y950 and one from the C-terminus. The extent of impairment can be
determined by comparing Shc phosphorylation in 32D/GR96 cells to that
in the parental 32D cells. In fact, in this figure, Shc phosphorylation
in GR96 cells is not better than that in 32D cells. Paradoxically, Shc
phosphorylation is practically abrogated if IRS-1 is introduced in GR96
cells (see Discussion).
Activation of Akt/PKB
In a previous report (32), we had shown that IGF-I caused a weak,
but reproducible, activation of Akt in 32D/GR15 cells despite the
absence of IRS-1 and the inability to detect PI3-kinase activity. We
extended these determinations to GR96 cells and to GR15 and GR96 cells
expressing IRS-1. The results of one such experiment are shown in Fig. 8. Activation of Akt/PKB by IGF-I was not
detectable in GR96 cells, although it was easily detectable in
IRS-1/GR15 cells and in IRS-1/GR96 cells. These results confirm the
reports in the literature that IRS-1 is largely responsible for the
activation of Akt/PKB (16, 17).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The main conclusions of our paper can be summarized as follows. 1) Single mutations do not seem to greatly affect the ability of the IGF-IR to activate ERK unless the receptor is a disabled one, incapable of transmitting an IGF-I signal (tyrosine kinase domain mutant). 2) In the absence of IRS-1, a receptor truncated at residue 1245 and with a mutation at Y950 is essentially incapable of giving a strong and sustained activation of ERK. 3) The serine quartet at 1280–1283 is, if not the sole, one of the domains in the C-terminus that, in combination with Y950, is required for ERK activation. 4) The reintroduction of IRS-1 in cells with defective mutant receptors (or with the IR) effectively restores the ability of the receptors to activate ERK. Thus, the IGF-IR seems to use three pathways to activate ERK: IRS-1, the Y950, and the serines 1280–1283. The fact that both Y950 and the serines have to be mutated to inhibit MAPK activation in the absence of IRS-1 indicates that there is some redundancy in this signaling.
Secondary conclusions are the following. 5) There is an imperfect correlation between ERK activation and survival after IL-3 withdrawal, confirming another IGF-IR signaling pathway for survival of 32D cells (23). 6) The IR or IRS-1 singly fails to give a sustained activation of ERK and to protect 32D cells from apoptosis. 7) A combination of IR and IRS-1 induces ERK activation and survival, especially in the presence of growth factors. 8) A MEK inhibitor affects the survival of 32D/IRS-1/IR cells more than they affect 32DGR15 cells with no IRS-1. Similar results were obtained with PI3-ki inhibitors (23). 9) Although the intensity varies, tyrosyl phosphorylation of Shc can be detected in both surviving and nonsurviving cells. Actually, IRS-1/GR96 cells (that survive after IL-3 withdrawal) show no tyrosyl phosphorylation of Shc proteins after stimulation with IGF-I. These various points will be considered separately.
By using 32D cells, we have eliminated IRS-1 (see introduction), and therefore, we have been able to identify the domains of the IGF-IR that can activate the ERK pathway in the absence of IRS-1. In retrospect, this was a fortunate decision. When IRS-1 is reintroduced in 32D cells, it rescues cells from apoptosis even when the IGF-IR is a mutant that by itself is no longer protective (GR96). Protection from apoptosis is accompanied by restoration of the mutant receptor’s ability to induce a sustained activation of ERK. Another word of caution is also necessary at this point. 32D cells, like many other hemopoietic cells, grow in suspension, so that the ERK activation by integrins and the cytoskeleton (42) must play a very modest role in this system.
It is important to distinguish in studies of this type between a disabled receptor and a receptor that is signaling but has lost some of its functions. Previous studies from our (26, 43) and other laboratories (44) have shown that mutations at the ATP-binding site (lysine 1003) or at tyrosines 1131, 1135, and 1136 (tyrosine kinase domain) effectively eliminate the main functions of the IGF-IR. These two mutant receptors are not mitogenic, do not protect cells from apoptosis, are nontransforming, and cannot induce differentiation (29, 30, 32). However, there are several mutations of the IGF-IR that affect transformation, or differentiation, without affecting its mitogenicity (15). Thus, there are mutant IGF-IR that have lost the ability to transform cells, but are still capable of protecting cells from apoptosis and to induce a mitogenic response (30). As ERKs are activated by IGF-I, it is of interest to determine by mutational analysis the domains of the IGF-IR required for their sustained activation.
In the absence of IRS-1, two domains of the IGF-IR stand out, as required for ERK activation: Y950 and the serine quartet at 1280–1283. The Y950 residue of the IGF-IR is known to interact with the Shc proteins (40, 41), which, in turn, are involved in activation of the ERK pathway (1, 7, 45). The cell lines that survive show a sustained activation (at least 1 h) of ERK. Marshall (46) has already made an eloquent case for the need for sustained MAPK activation for their biological effect. Only when MAPK activation is sustained is there nuclear translocation and activation of the genes that initiate cell cycle progression and/or survival (47, 48). Mutation at Y950 by itself does not greatly affect ERK activation; a second mutation is necessary for impairment of ERK activation. A truncation of the receptor at 1245 combined with a mutation at Y950 reduce ERK activation. In the C-terminus, mutations at serines 1280–83 have the same effect as the truncation at 1245. Tentatively, we can say that the IGF-IR (in cells without IRS-1) may activate ERK both through Shc proteins and by another mechanism related to 14.3.3 proteins. Tyrosyl phosphorylation of Shc is increased by IGF-I even in 32D GR46 cells (Y950F mutation), which clearly shows that the IGF-IR has an alternative pathway in the C-terminus for Shc phosphorylation. It is only when both Y950 is mutated and the C-terminus is eliminated that Shc phosphorylation is not above the level obtained in parental cells. Despite the several reports that Y950 is the main binding site for Shc proteins (see above), mutation at this site in the IGF-IR only impairs its transforming activity. Neither mitogenicity nor protection from apoptosis is affected in cells that express IRS-1 (Ref. 30 and this report).
It is known that 14.3.3 proteins bind to the IGF-IR at serine residues located in the C-terminus, between 1272 and 1284 (49), and that this interaction depends on phosphorylation of the appropriate serines. According to Furlanetto et al. (50), the 14.3.3 ß-isoform binds to serine 1283. In addition, Craparo et al. (49) and other laboratories (51) have shown that 14.3.3 proteins also bind to IRS-1; in fact, they may bind to IRS-1 more effectively than to the IGF-IR (51). Interestingly, the IR fails to interact with 14.3.3 proteins (49, 50). The first demonstration of a functional difference between the C-terminus of the IR and that of the IGF-IR goes back to an observation by Faria et al. (52) and has been repeatedly confirmed (even in this report). We showed previously that serines 1280–1283 played a major role in IGF-IR-mediated protection from apoptosis in 32D cells. As also shown in this paper, the GR35 receptor (four-serine mutant) was still capable of activating MAPK (presumably through the intact Y950 residue). However, a MEK inhibitor abrogated its protective effect and also inhibited the mitochondrial translocation of Raf that is induced by the wild-type IGF-IR and results in the phosphorylation of BAD (23). Thus, it seems that three domains of the IGF-IR are required for MAPK activation: IRS-1 (that does not require other signals), Y950, and the serines at 1280–1283. The last two domains are apparently redundant.
We have attempted to correlate sustained ERK activation with survival after IL-3 withdrawal. Parrizas et al. (3) indicated a role for MAPK in IGF-I-mediated survival, and we have obtained similar results in 32D cells (23). To correctly evaluate these experiments, however, we should briefly point out that the restoration of IRS-1 is mandatory for the continuous growth of 32D GR15 cells in the absence of IL-3 (32). In the absence of IRS-1, there is some growth at the beginning, but then cells stop growing, including the cells that overexpress the IGF-IR (GR15). These GR15 cells grow vigorously for 2–3 days, then begin to differentiate and die (32). Therefore, protection from cell death in the absence of IRS-1 can only be evaluated in the first 24–48 h. In the present experiments, there is a good, but imperfect, correlation between MAPK activation and protection from apoptosis. As mentioned above, this is due to the fact that the IGF-IR, besides the PI3-kinase and MAPK pathways, has a third pathway for BAD phosphorylation that depends on the integrity of the serine quartet at 1280–1283.
The role of IRS-1 in these experiments cannot be overemphasized. Indeed, the IR does much better in protection from apoptosis when introduced into R- cells, that have substantial amounts of IRS-1 (33). In 32D cells, IRS-1 becomes necessary for IR full signaling and survival of cells. PI3-kinase inhibitors cause apoptosis of 32D/IRS-1/IR cells, whereas GR15 cells are more resistant (23). Shc proteins are known to activate the Grb2 to ERK pathway (8, 9), but, again, they do not seem sufficient for either ERK activation or survival. Thus, the 52-kDa isoform of Shc is activated in both GR46 and GR96, which do not survive. Paradoxically, the IRS-1/GR96 cells that survive fail to phosphorylate Shc proteins (this experiment was repeated several times). This surprising result may be explained by the observation of Pessin and co-workers of the competition of receptors for substrates (53). Perhaps, the excess of IRS-1 competes with Shc for the receptor.
In summary, our experiments indicate that the IGF-IR has at least three domains involved in the activation of ERK: Y950, the serines at 1280–1283, and the domain(s) interacting with IRS-1, one of which is the tyrosine kinase domain (54). Presumably, in a receptor with a Y950 mutation, it is the kinase domain that becomes the main partner of IRS-1 (54). At any rate, Y950 is not required for tyrosyl phosphorylation of IRS-1 (30). The pathways are not completely redundant. In the absence of IRS-1, at least two domains are necessary for ERK activation. The lack of a perfect correlation between MAPK sustained activation and survival in 32D cells can be explained by the fact that the IGF-IR has other pathways for protection from apoptosis (23).
FONDATION ARES-SERONO THE ARES-SERONO FOUNDATION International Workshop on Developmental Endocrinology Venue: The Moller Centre, Churchill College, Cambridge, UK 21st and 22nd September 2000
Scientific Committee: Michel L. Aubert, John R. G. Challis, Peter D. Gluckman, Ieuan A. Hughes, Michael Meaney, and Keith L. Parker.
Sessions: Developmental Biology, Programming of Organ Function Development, Developmental Concepts Around Growth, and Developmental Aspects of Neuroendocrine Development.
For further details, contact Cathy Stewart: Telephone, 020 8818 7247; Fax, 020 8818 7222; and e-mail, cathy.f.stewart.gb_lon01@serono.com.
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Footnotes |
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Received September 29, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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D. LeRoith Editorial: Insulin-Like Growth Factor I Receptor Signaling--Overlapping or Redundant Pathways? Endocrinology, April 1, 2000; 141(4): 1287 - 1288. [Full Text] [PDF]
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B. Belletti, M. Prisco, A. Morrione, B. Valentinis, M. Navarro, and R. Baserga Regulation of Id2 Gene Expression by the Insulin-like Growth Factor I Receptor Requires Signaling by Phosphatidylinositol 3-Kinase J. Biol. Chem., April 20, 2001; 276(17): 13867 - 13874. [Abstract] [Full Text] [PDF]
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F. Peruzzi, M. Prisco, A. Morrione, B. Valentinis, and R. Baserga Anti-apoptotic Signaling of the Insulin-like Growth Factor-I Receptor through Mitochondrial Translocation of c-Raf and Nedd4 J. Biol. Chem., July 6, 2001; 276(28): 25990 - 25996. [Abstract] [Full Text] [PDF]
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