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From the Section on Molecular and Cellular Physiology, Diabetes Branch, National Institutes of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, Maryland 20892-1770
Address all correspondence and requests for reprints to: Derek LeRoith, M.D., Ph.D., Diabetes Branch/NIDDK, Building 10, Room 8S-235A, 10 Center Drive, MSC 1770, Bethesda, Maryland 20892-1770. E-mail: derek{at}helix.nih.gov
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Abstract |
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raf-1 mitogen-activated protein (MAP) kinase kinase
(MEK) p42/p44 MAP kinase (Erk-1 and -2). Recent reports suggest
that activation of this ras/MAP kinase pathway is
involved in mitogenesis and c-fos transcription but is
not required for insulin action on metabolic processes such as glycogen
synthesis, lipogenesis, and GLUT-4-mediated glucose transport.
Previously we and others have demonstrated that substitution of both
tyrosines at positions 1250 and 1251 in the carboxy-terminal region of
the human IGF-I receptor has relatively small effects on receptor and
endogenous substrate phosphorylation but completely abrogated the
ability of these cells to form tumors in nude mice or proliferate in
response to IGF-I in culture. Replacement of the tyrosine at position
1316 also did not affect the kinase activity of the receptor with
respect to autophosphorylation or phosphorylation of endogenous
substrates but did reduce the ability of the receptor to mediate
mitogenic or tumorigenic signals. To further characterize the role of
these tyrosines in IGF-I receptor function, we have used three distinct
approaches to examine the ras/MAP kinase pathway in
IGF-I-induced mitogenesis and tumorigenesis in NIH-3T3 cells
overexpressing wild-type and mutated IGF-I receptors: 1) tyrosine
phosphorylation of the MAP kinases Erk-1 and -2; 2), mobility shifts
indicative of MAP kinase phosphorylation; and 3) in
vitro MAP kinase activation. We have also examined
IGF-I-induced phosphatidylinositol (PI) 3-kinase activation in the same
cell lines. By each method we show that the IGF-I-induced MAP kinase
phosphorylation/activation and PI 3-kinase activation, are not
different between cells overexpressing wild-type IGF-I receptors and
cells carrying IGF-I receptors having tyrosine motifs replaced at
positions 1250 and 1251. We conclude that mitogenic and tumorigenic
signals involving tyrosine residues in the C-terminal domain of the
IGF-I-receptor include pathways other than the MAP kinase and PI
3-kinase pathways. |
Introduction |
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It has been demonstrated that IGF-I induces MAP kinase phosphorylation and/or MAP kinase activation in a variety of cell lines, including fibroblasts (7, 8, 9). In many cell types transformed by diverse oncoproteins, including raf-1 and MEK, MAP kinase is constitutively activated (10, 11, 12), suggesting that the MAP kinase pathway may mediate oncogenic signaling (13). Indeed, cellular transformation by raf-1 and ras requires the activation of MAP kinase (14, 15). While in some systems the MAP kinase pathway is necessary and/or sufficient for transduction and maintenance of transformation, it remains unclear whether the activation of MAP kinase is also required for oncogenic transformation by the overexpression of IGF-I receptors.
IGF-I and insulin also augment phosphatidylinositol (PI) 3-kinase activation in fibroblasts overexpressing IGF-I receptors and insulin receptors, respectively (16, 17, 18, 19, 20, 21). In addition, PI 3-kinase inhibition studies have provided clear evidence for a role of this enzyme in the effect of insulin on mitogenesis, GLUT-4-mediated glucose transport, antilipolysis, c-fos expression, glycogen synthase kinase-3, glycogen synthesis, amino acid transport and membrane ruffling (reviewed in Refs. 3, 22, 23).
The wild-type IGF-I receptor, when overexpressed, is fully mitogenic and transforming (24, 25). It has been postulated that the carboxy(C)-terminus of the IGF-I receptor initiates a transforming signal that is in addition to and separate from the mitogenic signal (26, 27). Studies of kinase-deficient IGF-I receptor cell lines have demonstrated the requirement for tyrosine kinase activity in IGF-I-mediated cell signaling. When tyrosine 950 was mutated to phenylalanine, the resultant cell line was IGF-I receptor tyrosine kinase-deficient and unable to transmit IGF-I-mediated signals, or to transform receptor deficient (R-) cells (28). IGF-I receptor kinase-dead cell lines in which the ATP-binding site at lysine 1003 was mutated, failed to support tumor growth in nude mice (29). Studies by others have demonstrated that four serines at 1280–1283 (24), the tyrosine at 1251 (30) and the 1293–1294 region (27) of the IGF-I receptor also act as major sites involved in mediation of the IGF-I signal resulting in increased transforming activity.
In previous studies, we have investigated the mitogenic and transforming potential of the human IGF-I receptors using cell lines overexpressing the wild-type receptor and compared them to cell lines overexpressing the human IGF-I receptors with replacements of the tyrosine residues in the catalytic domain (positions 1131, 1135, and 1136; 26 and in the C-terminal domain (positions 1250, 1251 and 1316; 25 . These tyrosine residues (alone or in combinations) appear to be crucial for IGF-I-induced cellular proliferation and tumor formation or maintenance. The tyrosine residues at positions 1250 and 1251 are unique to the IGF-I receptor, and when mutated to the amino acids found in the insulin receptor (phenylalanine and histidine, respectively) and overexpressed in NIH-3T3 cells, result in the loss of the ability of overexpressed IGF-I receptors to mediate signals that transform cells (25). Tyrosine 1316 is found in the homologous position of the insulin receptor, but when it is replaced in the IGF-I receptor it also causes loss of transformability of cells overexpressing IGF-I receptors. In the present study, we demonstrate that in NIH-3T3 cells expressing the human IGF-I receptor, IGF-I stimulates the activation of MAP kinase and PI 3-kinase, whereas having little effect on these activities in parental NIH-3T3 cells. We also find that cell lines expressing the IGF-I receptors with replacements of tyrosines at positions 1250 and 1251 or position 1316, will activate the MAP kinase and PI 3-kinase pathways in response to IGF-I to the same extent as found in cells expressing the wild-type IGF-I receptors. Thus, the activations of MAP kinase and PI 3-kinase in fibroblasts overexpressing tyrosine kinase functional IGF-I receptors do not correlate with the transforming capability of these cell lines. These results indicate that the mitogenic and tumorigenic potential mediated by the IGF-I receptor may use the MAP kinase- and PI 3-kinase-independent pathways.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-32P]ATP from New England Nuclear-DuPont (Wilmington,
DE), full-length myelin basic protein (MBP) from Life Technologies,
EDTA from Research Genetics (Huntsville, AL), MgCl2 from
Mallinckrodt (Paris, KY), and Protran nitrocellulose paper (0.2 µm)
from Schleicher and Schuell (Keene, NH). HEPES,
phenylmethylsulfonylfluoride (PMSF), dithiothreitol (DTT),
ß-glycerophosphate, pepstatin A, cAMP-dependent protein kinase
inhibitor (PKI), ATP, sodum orthovanadate
(Na3VO4), EGTA, aprotinin, benzamidine, and
other reagents were obtained from Sigma Chemical Co. (St. Louis, MO).
Protein A sepharose was obtained from Pharmacia (Piscataway, NJ) and
used as a 10% (wt/vol) solution in 50 mM Tris (pH 7.0).
FBS, horseradish peroxidase-conjugated antimouse and antirabbit
immunoglobulin, and the enhanced chemiluminescence system (ECL)
detection kit were purchased from Amersham Corp.(Arlinton Heights, IL).
Insulin-free BSA (fraction V) was obtained from Armour (Kankakee, IL).
Cell culture media and reagents were purchased from Biofluids, Inc.
(Rockville, MD) and Advanced Biotechnologies (Columbia, MD). Mouse
monoclonal antibody to Erk-2 and rabbit polyclonal antibodies to MAP
kinase were obtained from Zymed Laboratories (South San Francisco, CA).
Rabbit polyclonal phospho-specific MAP kinase antibody that detects
phosphorylated tyrosine 185 of mouse Erk-1 and Erk-2 was obtained from
New England Biolabs, Inc. (Beverly, MA). Polyclonal antibodies to
Erk-1(K-23), Erk-1(C-16) and Erk-2(C-14) were obtained from Santa Cruz
Biotechnology (Santa Cruz, CA). Monoclonal antiphosphotyrosine antibody
(clone 4G10) and recombinant human IGF-I were purchased from Upstate
Biotechnology, Inc. (Lake Placid, NY).
Cells and growth factors
The NIH-3T3 cells expressing wild-type and mutant IGF-I
receptors have been previously described (25, 31, 32). NWTb3 and NWTc43
are two independently derived clones expressing wild-type IGF-I
receptors. NKR-1 is a cell line expressing the kinase-inactive IGF-I
receptors with lysine 1003 replaced by arginine. The cell lines yyFHb1
and yyFHb16 are independent clones expressing IGF-I receptors with both
tyrosine residues 1250 and 1251 replaced by phenylalanine and
histidine, respectively. The cell lines yCFa12 and yCFb43 are
independent clones expressing IGF-I receptors with tyrosine 1316
replaced by phenylalanine. The cell line transfected with the
neomycin-resistance plasmid and pBPV vector without the cDNA encoding
for the IGF-I receptor is designated pNeo. Parental NIH-3T3 cells were
maintained in DMEM supplemented with 10% FBS, 2 mM
L-glutamine and antibiotics (100 U/ml penicillin, 0.25
µg/ml fungizone and 100 µg/ml streptomycin) in a humidified
atmosphere of 95% air and 5% CO2 at 37 C. Stable
transfected cell lines (pNeo, NWTc43, NWTb3, yyFHb1, yyFHb16, yCFa12,
and yCFb43 and NKR-1) were maintained in the same DMEM supplemented
with 500 µg/ml G418. For serum starvation and assays of biological
actions of IGF-I, the media was exchanged for serum-free DMEM (0.1%
insulin-free BSA, 2 mM L-glutamine, 15
mM NaOH, 20 mM HEPES, pH 7.5 and antibiotics)
for 16 h before experimental medium (serum-free DMEM ± 10
nM IGF-I) was added for the indicated time.
Western immunoblotting
For each Western blot, confluent 100 mm-diameter dishes of cells
were rinsed twice with ice-cold 1x PBS and the cells lysed in 500 µl
of Lysis Buffer (4 mM sodium pyrophosphate, 50
mM HEPES, pH 7.9, 100 mM NaCl, 10
mM EDTA, 1 mM PMSF, 10 mM sodium
fluoride, 1% Triton X-100, 2 mM
Na3VO4, 2 µg/ml leupeptin, and 2 µg/ml
aprotinin). Protein determinations were performed by the Bradford dye
method (33), using the Bio-Rad reagent and BSA as a standard. For
Western blot analysis, SDS polyacrylamide (percentage as indicated)
gels were electroblotted onto Protran nitrocellulose membranes and
nonspecific binding to the nitrocellulose was reduced by preincubating
the membrane in Blocking Buffer (3% insulin-free BSA in 1x PBST made
from 10 mM sodium phosphate, pH 7.2, 140 mM
NaCl and 0.1% Tween 20) for 60 min. Protein size was estimated using
prestained mol wt standards (Life Technologies).
The nitrocellulose blot was incubated overnight at 4 C with an anti-Erk-1 (1:2,000 dilution); anti-Erk-2 (1:2,000 dilution); anti-MAP kinase (1:2,000 dilution) antibody, or a phospho-specific MAP kinase (1:1,000 dilution) antibody. Antibodies were diluted in 1x PBST containing 1% insulin-free BSA. Blots were washed (four to five times, 15 min each wash) with 1 x PBST (containing 1% insulin-free BSA) and incubated for 60 min with the appropriate secondary antibody coupled to horseradish peroxidase. Blots were washed (four to five times, 15 min each wash) and developed with the ECL mix for 1 min. Exposures of BioMax-MR film (Kodak, Rochester, NY) to the Western blot were done for various times.
MAP kinase assays
The NIH-3T3 parental cells, pNeo, NWTc43, NWTb3, yyFHb1,
yyFHb16, yCFa12, yCFb43, and NKR-1 cell lines were cultured in 100-mm
diameter dishes until 75–85% confluent. The media was changed to
serum-free DMEM for 16 h before stimulation with 10 nM
IGF-I for 8 min. Following stimulation, cells were washed twice with
ice-cold 1x PBS and solubilized in 400 µl ice-cold Lysis Buffer
(10% glycerol, 137 mM NaCl, 25 mM
ß-glycerophosphate pH 7.3, 20 mM Tris pH 7.5, 1% Triton
X-100, 1 mM PMSF, 2 mM EDTA, 1 mM
Na3VO4, and 2 mM sodium phosphate
pH 7.0). MAP kinase activity was assayed as described by Seger et
al. (34). Briefly, lysates (400 µg) were immunoprecipitated with
the anti-Erk-2 polyclonal antibody and 10% (wt/vol). Protein A
Sepharose overnight and then washed sequentially (2x each buffer) in
ice-cold lysis buffer, followed by kinase buffer (25 mM
MgCl2, 25 mM ß-glycerophosphate pH 7.3, 20
mM HEPES pH7.6, 2 mM DTT and 0.1 mM
Na3VO4). The pellet was then incubated in assay
buffer (25 mM MgCl2, 6 µm PKI, 25
mM ß-glycerophosphate, pH 7.3, 2 mM DTT, 0.1
mM Na3VO4, 100 µM ATP
and 1.6 µM [-32P]ATP) using 2 mg/ml MBP
as the substrate. The reactions were terminated after 30 min at 30 C by
adding 5x Laemmli buffer. Samples were separated by 15% SDS-PAGE and
the gel was fixed in acetic acid/isopropanol (12.5%:25%) and exposed
briefly to Kodak X-Omat film to determine the relative locations of the
radioactive products. The radioactive products in the gels were then
quantified on a PhosphoImager (Molecular Dynamics, Sunnyvale, CA). The
results are presented as the mean ± SE for four to
five separate experiments. Nonspecific 32P incorporation
was determined in identical assays lacking the MBP substrate and was
subtracted from the assayed radioactivity before determination of the
mean incorporation into phosphorylated MBP.
Phosphatidylinositol 3-kinase activation
Phosphorylation of phosphatidylinositol was measured as
previously described (31) with modifications. Briefly, cells were grown
to confluency in 100-mm diameter dishes, washed with 1x PBS, and
incubated for 24 h in serum-free DMEM. Cells were then incubated
for 1 min in prewarmed (37 C) serum-free DMEM without or with 10
nM IGF-I. Cells were washed twice with ice-cold 1x PBS and
twice with freshly prepared Wash Buffer (20 mM Tris, pH
7.5, 100 mM NaCl, 1 mM MgCl2, 1
mM CaCl2, and 100 µM
Na3VO4). Cells were lysed in 500 µl wash
buffer containing 1% NP-40, 10% glycerol, and 0.35 mg/ml PMSF for 10
min on ice. The lysates were centrifuged for 10 min at 13,000 x
g for 4 C and supernatants frozen at -20 C. The protein
contents were measured by the method of Bradford as indicated above.
Proteins (600 µg) were immunoprecipitated overnight at 4 C with 2
µg of monoclonal antiphosphotyrosine antibody (clone 4G10) with the
addition of 30 µl protein A-sepharose for the last 4 h. The
immunoprecipitates were kept on ice and washed sequentially with
ice-cold wash buffers: once with wash buffer A (100 µM
Na3VO4 and 1% NP-40 in 1x PBS), twice with
wash buffer B (100 mM Tris, pH 7.5, 500 mM
LiCl, and 100 µM Na3VO4) and once
with wash buffer C (10 mM Tris, pH7.5, 100 mM
NaCl and 100 µM Na3VO4). The
beads were then resuspended in 40 µl of ice-cold wash buffer C.
Samples were incubated for 10 min with 10 µl of 100 mM
MnCl2, 10 µl of 2 µg/µl phosphatidylinositol, and 10
µl of 440 µM ATP containing 40 µCi
[-32P]ATP. The reaction was stopped with the addition
of 20 µl of 8N HCl and 160 µl of CHCl3/methanol (1:1).
The organic phase was extracted and chromatography carried out as
described (31) using a total of 241 ml of chromatographic fluid.
Products of the reaction were detected by autoradiography and
quantitated using NIH Image version 1.55 after the signal was
digitalized. Phosphatidylinositol 4-monophosphate was used as a marker
and visualized with iodine vapor.
Statistics
For MAP kinase, the values are expressed as percentage change
over control, where controls are unstimulated cell lysates. For PI
3-kinase, the values are expressed as fold stimulation over control,
where controls are unstimulated cell lysates.
X2-analysis was used to compare percentage
change. Tumor size in mm3 was calculated as V = a +
b2/2. Statistical differences between tumor volumes were
compared using the Student’s t test. SEM for
the experimental averages are shown.
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Results |
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).
This shifted band suggests increased phosphorylation compared with the
unstimulated cells. Similar shifts were observed in the cell lines
yyFHb1 and yyFHb16 that express mutant IGF-I receptors with tyrosine
1250 and 1251 replaced with phenylalanine and histidine, respectively.
IGF-I stimulation also induced a mobility shift of Erk-2 in the cell
lines (yCFa12 and yCFb43) expressing IGF-I receptors with tyrosine 1316
replaced with phenylalanine (data not shown). In parental NIH-3T3 (data
not shown), pNeo and NKR-1 cells, the migration of the Erk-2 band was
not different from that observed for unstimulated cells. In all cell
types, the Erk-1 (p44) band mobility was unchanged after IGF-I
stimulation (Fig. 1 and data not shown). Using the phospho-specific MAP
kinase antibody that detects phosphorylated Tyr-185 in Erk-1 and Erk-2,
we demonstrated increased tyrosine phosphorylation of both Erk-1 and
Erk-2 after IGF-I stimulation in cells overexpressing wild-type (NWTc43
and NWTb3) or double-tyrosine mutant receptors (yyFHb1 and yyFHb16)
(Fig. 2, upper panel). Tyrosine
phosphorylation of Erk-1 and Erk-2 also was seen in yCFa12 and yCFb43
cells in response to IGF-I treatment (data not shown). In contrast,
Erk-1 and Erk-2 were not phosphorylated on Tyr185 in the pNeo or NKR-1
cells (Fig. 2, upper panel), nor in the parental NIH-3T3
cells (data not shown). Reblotting this membrane with an antibody
developed against Erk-1 demonstrated that equal amounts of Erk-1 and
Erk-2 were present (Fig. 2, lower panel).
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),
whereas no response was observed in control cells (pNeo and NKR-1
cells). The patterns of stimulation were similar in two clones
expressing the double-tyrosine mutant receptor (yyFHb1 and yyFHb16) and
two clones expressing tyrosine 1316 mutant receptor (yCFa12 and yCFb43)
(data not shown). The mean enhancement of IGF-I-stimulated MAP kinase
activity is shown in Fig. 4. There is a 3-fold increase
in MAP kinase activation in cells expressing wild-type receptors
(NWTc43 and NWTb3) as compared with pNeo and NKR-1 cells
(P < 0.01). Furthermore, MAP kinase activation in
yyFHb1, yyFHb16, yCFa12, and yCFb43 cells was also 3-fold enhanced as
compared with the control cells (P < 0.01). There was
no significant difference in MAP kinase activation between NWT, yyFH,
and yCF cells.
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. In Panel A, NKR-1 cells
showed no increase in PI 3-kinase activity after IGF-I stimulation. PI
3-kinase activation in cells expressing wild-type IGF-I receptors
(NWTc43 and NWTb3) or mutant receptors (yCFa12, yCFb43, yyFHb1 and
yyFHb16) was enhanced in response to IGF-I (panels A and B). As shown
in Table 1, the fold stimulation in response to IGF-I of
PI 3-kinase activity of NWTb3 and NWTc43 cells was significantly
different from pNeo and NKR-1 cells (P < 0.05).
IGF-I-induced PI 3-kinase activation in yyFHb1 and yyFHb16 were similar
to the two cells clones expressing wild-type receptors (NWTb3 and
NWTc43).
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Discussion |
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Other investigators have postulated an ras-independent pathway for IGF-I-induced mitogenesis and transformation (36). While there is strong evidence for an activated ras requirement for optimal cell proliferation and transformation (37), more recent studies have implicated more complex signaling pathways emanating from the IGF-I receptor. Fibroblasts carrying a null-mutation of the IGF-I receptor and transfected with activated Ha-ras are only able to grow as small colonies in soft agar assays, whereas cells overexpressing wild-type IGF-I receptors form large foci in soft agar (36). These observations suggest that the IGF-I receptor mediates a transformation signal by a ras-independent pathway. An alternative mechanism by which the IGF-I receptor induces tumor growth may involve PI 3-kinase. In early work by Macara et al. (38) and Sugimoto et al. (39), it has been suggested that elevated levels of PI 3-kinase are associated with cell transformation and tumorigenesis induced by src and the middle T antigen. Kato et al. (31, 32) have described two kinase-deficient IGF-I receptor cells lines (NKR-1 and NYF3) that are unable to mediate an increase in IGF-I-stimulated PI 3-kinase activity, whereas cells expressing the wild-type IGF-I receptors do exhibit at least a 4-fold increase in IGF-I-stimulated PI 3-kinase activity. The cell lines expressing the kinase-deficient receptors were unable to form tumors when injected into nude mice, whereas cells expressing wild-type IGF-I receptors formed large tumors (29). Our current studies demonstrate that, similar to wild-type cell lines, the cell lines expressing the double tyrosine mutant (yyFH) or the tyrosine 1316 mutant (yCF) IGF-I receptors can mediate both IGF-I-induced PI 3-kinase and MAP kinase activation. However, the yyFH and yCF cell lines have reduced proliferation in response to IGF-I and fail to produce tumors when injected into nude mice. Our laboratory has recently shown that inhibition of the MAP kinase and PI 3-kinase pathways by synthetic inhibitors in differentiated PC12 cells results in decreased IGF-I-induced cellular proliferation concurrently with an increase in apoptosis (40). Thus, although activation of these pathways is necessary for IGF-I-induced mitogenesis, the two pathways together are not sufficient because in the studies presented here cells expressing mutant IGF-I receptors have functionally intact MAP kinase and PI 3-kinase pathways but markedly diminished mitogenic and tumorigenic capacities. Taken together, these data suggest that there is at least one IGF-I-inducible pathway that is important in cellular proliferation and in the development or maintenance of tumors that is in addition to PI 3-kinase and MAP kinase activation.
Other potential candidates that may mediate the IGF-I-induced tumorigenesis include the Crk family of proteins, the cellular homologs of v-crk, and the 14–3-3 family of proteins. Both groups have been shown to interact with the IGF-I receptor (41, 42). Beitner-Johnson and LeRoith (43) have shown that activated IGF-I receptors phosphorylate Crk in NIH-3T3 fibroblasts. The family of Crk proteins bears SH2 and SH3 domains, shares homology with Grb2 and Nck, and interacts with the ras-binding protein mSOS (44). 14–3-3 proteins, which apparently bind the IGF-I receptor C-terminal domain directly, also bind and activate raf (45). While both Crk and 14–3-3 seem attractive candidates because they interact with the IGF-I receptor, they have been implicated in the ras/raf/MAP kinase pathway that in this study was noted to be normally activated by the mutant receptors. Thus, their specific roles in the transforming capabilities of the overexpressed mutant IGF-I receptors in these cells requires further clarification.
In summary, we have demonstrated that in NIH-3T3 cells overexpressing the human IGF-I receptor, IGF-I has substantial effects on the phosphorylation and activation of MAP kinases and the activation of PI 3-kinase, while having little or no effect on these activities in the parental NIH-3T3 and kinase-deficient NKR-1 cells. Fibroblasts expressing IGF-I receptors with replacement of tyrosines 1250 and 1251 or tyrosine 1316 respond to IGF-I stimulation with enhanced MAP kinase and PI 3-kinase activities similar to cells expressing the wild-type IGF-I receptor. Yet, unlike the wild-type receptors, these tyrosine mutant IGF-I receptors are unable to confer mitogenic and tumorigenic potential to fibroblasts, suggesting that the MAP kinase and PI 3-kinase pathways are not sufficient for IGF-I-induced cellular proliferation and transformation. Taken together, these studies suggest that IGF-I-stimulated mitogenesis and tumorigenesis may involve one or more pathways that are distinct from the MAP kinase and PI 3-kinase signaling pathways.
Received November 26, 1996.
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