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Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
Address all correspondence and requests for reprints to: Jennifer L. Swantek, Department of Pharmacology, The University of Texas Southwestern Medical Center, Dallas, Texas 75235.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IGF-II, insulin, platelet-derived growth
factor (PDGF), epidermal growth fator (EGF), and IGF-I), separately or
in a combination that sustain the growth of W cells (as well as other
3T3 cells) (6, 11). R- cells cannot be transformed by the
SV40 large T antigen, an overexpressed constitutively activated H-ras,
or by a combination of both (6, 11), overexpression of the EGF (13) or
PDGF-ß (14) receptors, or by the BPV E5 protein (15), all of which
are able to transform W and 3T3-like cells. All growth defects of
R- cells are corrected by the stable transfection of a
wild-type (but not a mutant) IGF-IR complementary DNA (cDNA), thus
unequivocally showing that the phenotype of R- cells is
specifically due to the absence of the IGF-IR. The IGF-IR belongs to the family of transmembrane tyrosine kinase receptors that also includes receptors for PDGF, EGF, and insulin (reviewed in Ref. 16). Upon activation by its ligands, the IGF-IR autophosphorylates and transmits a signal to both insulin receptor substrate-1 and Shc (17, 18). This signal is subsequently transduced through the pathway commonly referred to as the Ras pathway (19, 20, 21, 22, 23), which ultimately reaches the nucleus resulting in a mitogenic response. A critical player in this pathway is the ERK family of mitogen-activated protein kinases (MAPKs), which include ERK-1 and ERK-2 (24, 25). The ERKs are a family of serine/threonine kinases that become activated following stimulation of cells with various growth factors including PDGF, EGF, insulin, and IGF-1 (25, 26, 27) and activation of ERKs has been shown to be necessary for the proliferation of fibroblasts (28). It has also been suggested that specific cellular responses, including cellular differentiation or induction of DNA synthesis, are determined by the duration of ERK activation, depending on the cell type (reviewed in Ref. 29).
By taking advantage of the R- and W cell system, we set out to determine the effect the absence of the IGF-1R has on the activation of ERK-2. Here, we report that growth factor-induced prolonged activation of ERK2 is dependent on a signal generated by a functional IGF-1R.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ERK activation assays
The protocol for ERK activation used in these experiments is a
modified method based on a combination of that described by Cook
et al. (33), the protocol for immune complex protein kinase
found in the Research Applications from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and protocol #10 from Transduction Laboratories, Inc. All cells used for this assay were grown in
100 mm culture dishes in DMEM (Gibco BRL, Grand Island,
NY) containing 10% FBS until 90% confluent. At least two clonal lines
expressing each receptor form were tested. The cells were then washed
three times with HBSS and 10 ml serum-free medium (SFM; DMEM,
0.1% BSA, 2.5 µM ferrous sulfate, P/S, and
L-glutamine) was added to each plate. The cells were
incubated at 37 C for 18 h. The cells were washed three times with
HBSS, and fresh SFM was added and the cells were incubated for
15 min at 37 C. The cells were then stimulated (except for cells used
for control) with purified growth factors or a combination of growth
factors, depending on the experiment (PDGF at 5 ng/ml; EGF at 20 ng/ml;
IGF-1 at 20 ng/ml; and insulin at 20 µg/ml; combinations of growth
factors were the specified growth factors at these concentrations; all
growth factors were purchased from Gibco BRL) for the
desired amounts of time. The cells were then washed with cold PBS and
were placed on ice. One milliliter of cold lysis buffer (10 mm Tris, pH
7.4, 1% Triton X-100, 0.5% Nonidet P-40, 150 mm NaCl, 20
mM sodium fluoride, 0.2 mM sodium
orthovanadate, 1.0 mM EDTA, 1.0 mM EGTA, and
0.2 mM phenylmethylsulfonyl flouride) was added to each
plate of cells. The cells were lysed at 4 C, scraped, and collected
into 1.5 ml microfuge tubes. The samples were centrifuged for 2 min. at
14,000 rpm and the supernatant was placed into fresh tubes and placed
on ice. Protein amounts were quantitated using the Bio-Rad protein
assay (Bio-Rad Laboratories, Inc., Hercules, CA). We added
150 µg of total protein to 500 µl of lysis buffer and 5 µg of
anti-ERK2(C-14)AC (Santa Cruz Biotechnology, Inc., Santa
Cruz, CA) along with 50 µl Protein A/Agarose
(Calbiochem, Cambridge, MA) to each tube. The
immunoprecipitation was conducted for 20 h at 4 C on a rotating
wheel. The samples were then centrifuged for 5 min at 6000 rpm. The
pellets were washed twice with 500 µl cold lysis buffer and once with
500 µl kinase buffer (10 mM Tris, pH 7.4, 150
mM NaCl, 10 mM MgCl2 and 0.5 mM
dithiothreitol) with centrifugation at 6000 rpm for 5 min between each
wash. The pellets were then resuspended in 20 µl kinase buffer. Ten
microliters of ATP mix [83.4 µl kinase buffer, 1.2 µl 50
mM ATP, pH 7, 4 µl 2 M MgCl2, 4.4 µl 
[32P] ATP (10 mCi/ml; DuPont New England Nuclear, Boston, MA), and 7 µl 10 mg/ml myelin basic protein
(Sigma Chemical Co., St. Louis, MO)]. The kinase reaction
was allowed to proceed for 20 min at 30 C. Then 30 µl 2x Laemmli
buffer was added to each sample. The samples were boiled for 5 min and
were loaded on a 12% mini-protein ready gel (Bio-Rad Laboratories, Inc.). The gel was run in glycine buffer (3.04 g
Tris, 1 g SDS, and 14.4 g glycine/liter H2O) at
30 mA for approximately 1 h. The gel was removed from the
electrophoresis apparatus and was dried on Whatman 3MM
paper. The dried gel was exposed to Kodak X-OMAT AR film (Eastman Kodak, Rochester, NY), and bands corresponding to myelin basic
protein were excised from the gel and were counted in a scintillation
counter using Scintillation fluid.
Western blotting of ERK2
Twenty micrograms of the protein lysates from W,
R-, R+, and R-/Y950F cells
stimulated with a combination of PDGF, EGF, and IGF-1 for various
amounts of time (described above) were resolved on a 10%
polyacrylamide gel and transferred to a nitrocellulose filter.
Membranes were blocked with 5% nonfat milk in TBST buffer (10
mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1%
Tween-20) and then probed with an anti-ERK2 polyclonal antibody (Santa
Cruz Biotechology, Santa Cruz, CA). After incubation with a horseradish
peroxidase-conjugated secondary antibody (Amersham Corp.,
Arlington Heights, IL), detection was carried out with the ECL
detection kit (Amersham Corp.). The filters were exposed
to Kodak X-OMAT AR film.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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W and R- cells were starved in serum-free medium and were
subsequently stimulated with a combination of PDGF, EGF, and IGF-1
(PEI) for various periods of time as indicated in Fig. 1
, A and B. The cells were then harvested
and activation of ERK2 was tested in an in vitro kinase
assay following immunoprecipitation with an anti-ERK-2 antibody as
described in Materials and Methods. ERK2 becomes rapidly
activated in both W and R- cells, however, to a greater
degree in W cells. The peak of activation occurs at 15 min post
stimulation (hereafter referred to as transient activation) in both
cell lines. In W cells, ERK2 activity decreases by approximately 50%
between 15 and 30 min post stimulation, but it remains elevated
approximately 6-fold over basal level for a period of at least 6 h
(prolonged activation). In R- cells, ERK2 is activated
only transiently, with ERK2 activity returning to basal level within 30
min post stimulation. Thus, the IGF-1 receptor seems to be required for
prolonged activation of ERK2 in mouse embryo fibroblasts and also
increases the extent of transient ERK2 activity levels induced by EGF
and PDGF.
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, the pattern of ERK2 activity
after growth factor stimulation of R+ cells is similar to
that of W cells. (compare W and R+ cells) whereas in
R-/Y950F cells, the pattern of ERK2 activation closely
resembles that of R- cells (compare R- and
R-/Y950F cells).
To confirm our in vitro MAP kinase activation data were not
due to a lack of ERK expression in the R- and
R-/Y950F cells, we subjected the lysates of W,
R-, R+, and R-/Y950F cells used
in the in vitro kinase assays to SDS/PAGE and found (by
Western blotting) that the levels of ERK2 protein were essentially
equal in all samples (Fig. 1C). These results suggest that in these
cells prolonged activation of ERK2 requires a signal generated from a
functional IGF-1 receptor.
Stimulation with IGF-1 alone results in prolonged activation of
ERK2 in W and R+ cells
In the previous experiments, all cells were stimulated with a
combination of growth factors, namely PDGF, EGF, and IGF-1. We reasoned
that if prolonged activation of ERK2 is dependent on a signal
transmitted from a functional IGF-1R, then stimulation of W and
R+ cells with IGF-1 alone would result in prolonged
activation of ERK2. Because the peak activation of ERK2 occurred 15 min
following stimulation with PDGF, EGF, and IGF-1 in all four cell lines,
and because ERK2 activity remained elevated for at least 6 h in W
and R+ cells, we determined ERK2 activity at these two time
points in the following experiments. As shown in Fig. 2, IGF-1 stimulation of serum-starved W
and R+ cells resulted in enhanced ERK2 activity, at 15 min
post stimulation, that remained elevated for at least 6 h.
Stimulation of R- and R-/Y950F cells with
IGF-1 resulted in a modest increase in ERK2 activity at 15 min post
stimulation (see Discussion). However, ERK2 activity was at
basal level 6 h post stimulation in both cell lines.
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Activation of ERK2 in W, R-, R+, and
R-/Y950F cells in response to individual growth
factors
Because it was shown that stimulation of W and R+
cells with a combination of growth factors resulted in transient ERK2
activation that was nearly double that seen with IGF-1 alone, we
reasoned that the combination of growth factors may enhance the levels
of transient ERK2 activity over that induced by individual growth
factors. To examine the roles of PDGF, EGF, and insulin individually or
a combination of PDGF and EGF in the activation of ERK2, we stimulated
serum starved R-, W, R+, and
R-/Y950F cells with these various growth factors and
assayed for ERK2 activity as described above. As shown in Table 1, PDGF or a combination of PDGF and EGF
resulted in transient activation of ERK2 to levels half of that
observed with a combination of PDGF, EGF, and IGF-1 (compare Table 1
and Fig. 1). Prolonged ERK2 activity was not observed in any of the
cell lines following PDGF or PDGF and EGF stimulation. Stimulation of
all cell lines with EGF or insulin resulted in transient ERK2 activity
(Table 1). Surprisingly, prolonged ERK2 activity was observed following
EGF or insulin treatment, but only in cell lines containing a
mitogenically functional IGF-1R (Table 1). Thus, EGF and insulin are
able to induce prolonged ERK2 activation, but only in cells containing
a mitogenically functional IGF-1R indicating these effects are mediated
through the IGF-1R.
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R-/Y1251F cells, which are R- cells
overexpressing an IGF-1 receptor containing a mutation at tyrosine 1251
(31) and TC4 cells, which are R- cells overexpressing a
truncated IGF-1 receptor that lacks the last 108 amino acids of the
C-terminus (32), were serum-starved and subsequently stimulated with a
combination of PDGF, EGF, and IGF-1 and ERK2 activity was tested as
described above. As shown in Fig. 3, both
transient and prolonged ERK2 activity is observed in these cell lines.
Such activation is similar to W and R+ cells stimulated
with PDGF, EGF, and IGF-1 (compare with W and R+ cells,
Fig. 1A). As a further confirmation of these receptors’ ability to
activate ERK2 and to ensure that a signal generated by these receptors
was responsible for the observed activation of ERK2, serum starved
R-/Y1251F and TC4 cells were stimulated with IGF-1 alone
and ERK2 activity was assayed as described above. As shown in Fig. 3, both transient and prolonged ERK2 activity was observed following IGF-1
stimulation in these cell lines. Thus, a signal generated by the IGF-1
receptors in these cell lines is responsible for both transient and
prolonged activation of ERK2, as was the case for W and R+
cells (see above), and the segment of the C-terminus of the IGF-1
receptor which is mutated in these cell lines is not necessary for the
generation of this signal.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our results demonstrate that ERK2 becomes transiently activated in response to PDGF, EGF, and IGF-1 in both R- and W cells; however, the level of ERK2 activity in R- cells is approximately 4-fold less than in W cells. More intriguing is the observation that ERK2 activation returns to basal level in R- cells by 30 min post stimulation but remains elevated at a significant level in W cells for at least 6 h following stimulation. It has been noted previously that prolonged activation of ERK-1 results following stimulation of CCL39 cells with thrombin (35, 36), a combination of basic FGF and serotonin (35), a combination of TMP and FGF (36), and that the prolonged activation of ERK1 correlates with the induction of DNA synthesis. Likewise stimulation of PC12 cells with NGF results in prolonged activation of MAPK (37, 38, 39); however, in this case, prolonged MAPK activation was found to correlate with differentiation. Thus, prolonged activation of MAPK appears to be important for mitogenic responses as well as differentiation, depending on the type of cell and the specific stimulus. The lack of prolonged activation of ERK2 in R- cells and the fact that the introduction of a wild-type IGF-1R by stable transfection in R- cells was able to restore it indicate that prolonged ERK2 activation in these 3T3 cells is due to a signal generated from a functional IGF-1R. Also supporting this hypothesis are the results from testing ERK2 activation in a cell line overexpressing a mutant IGF-1R, R-/Y950F cells, which is unable to grow in serum-free medium supplemented with PDGF, EGF, and IGF-1 and is unable to become transformed. The activation of ERK2 in these cells resembled that seen in R- cells. Further evidence that the prolonged activation of ERK2 is mediated via IGF-1R signaling stems from the fact that stimulation of W and R+ cells with IGF-1 alone results in rapid and prolonged ERK2 activation, with prolonged ERK2 activation levels being equivalent to those seen in W and R+ cells stimulated with the combination of PDGF, EGF, and IGF-1. The fact that stimulation of R- and R-/Y950F cells with IGF-1 resulted in slight transient ERK2 activation can be attributed to cross-activation of the insulin receptor by IGF-1. This is a common occurrence (40, 41), and because insulin receptor activation also results in MAPK activation, it is not surprising that a modest transient ERK2 activation was observed.
EGF (but not PDGF) and supraphysiological concentrations of insulin are also able to induce prolonged ERK2 activation. However, this effect is only seen in cells containing a functional IGF-1R; thus, EGF and insulin-induced prolongation of ERK2 activation is probably mediated through the IGF-1R. It is known that EGF stimulation results in an increase in IGF-1 messenger RNA and IGF-1 ligand production (13, 42), therefore establishing an autocrine IGF-1 loop that would result in prolonged ERK2 activity. On the other hand, insulin, at supraphysiological concentrations (µg/ml), is known to bind to and activate IGF-1 receptors (43); thus, cross-activation of the IGF-1R by insulin is a likely mechanism for insulin-induced prolonged ERK2 activation. Interestingly, the presence of PDGF abolishes the ability of EGF to induce prolonged ERK2 activity. However, it has been shown that PDGF alters the EGFR; therefore, EGF cannot bind to the EGFR properly (44, 45), and it is unlikely that an IGF-1 autocrine loop would be established. It should also be noted that a combination of PDGF, EGF, and IGF-1 enhances transient ERK2 activation over that induced by individual growth factors because peak activation levels following W and R+ cell stimulation with PDGF, EGF, and IGF-1 separately results in transient ERK2 activation levels of approximately 7, 6, and 6.5 fold over basal levels, respectively, whereas stimulation with a combination of PDGF, EGF, and IGF-1 results in transient ERK2 activation levels that are approximately 13- to 14-fold over basal levels (in W and R+ cells).
It has been possible to separate mitogenesis and transformation at the level of the IGF-1R by demonstrating that mutant IGF-1 receptors containing mutations in the C-terminus, either a point mutation at amino acid 1251 or a truncation of the last 108 amino acids of the IGF-1R, retain the ability to transmit mitogenic signals yet fail to transform (31, 32). By testing the activation of ERK2 in R-/Y1251F and TC4 cells (corresponding to either the point mutation or the truncation noted above, respectively) in response to PDGF, EGF, and IGF-1 and to IGF-1 alone, we were able to show that ERK2 activation patterns were similar to those seen in W and R+ cells; thus, the transforming domain of the IGF-IR is not required for prolonged activation of the ERK2.
This paper demonstrates that IGF-1 is able to induce prolonged ERK2 activation and that prolonged ERK2 activation induced by other growth factors, namely EGF and insulin, is mediated through IGF-1R signaling. Combined with the growth characteristics of the cell lines used (previously described by our laboratory; 6, 11, 30, 31, 32), these data also contribute to the accumulating evidence that it is the duration of MAPK activation which is crucial for determining cellular responses, i.e. whether a cell will differentiate or proliferate (reviewed in Ref. 29), and further demonstrates the importance of the IGF-1R in this process.
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Acknowledgments |
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Footnotes |
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Received September 3, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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