(Received for publication, January 8, 1997, and in revised form, April 29, 1997)
From the Department of Tumor Biology, Breast Cancer Basic Research Program, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030
The function of epidermal growth factor receptor (EGFR) was found to be negatively regulated in M phase in which it showed less phosphotyrosine content and reduced intrinsic kinase activity accompanied by retarded electrophoretic mobility owing to total hyperphosphorylation. Ligand-induced autophosphorylation and downstream signaling of EGFR were tightly suppressed in M phase due to a decrease in ligand binding affinity and the inability of epidermal growth factor (EGF) to induce receptor dimerization. There was no change in the number of surface-exposed EGF receptors between G0/G1 and M phases of the cell cycle. Hyperphosphorylation (due to serine and/or threonine phosphorylation) correlates with the unresponsiveness of cells to EGF-mediated stimulation of tyrosine phosphorylation in cells that express the normal or basal level of EGFR. This M phase-specific negative regulation was overcome by overexpression of EGFR, which was responsive to ligand throughout the cell cycle and revealed ligand-induced signaling in the M phase. These findings indicate that EGFR does not respond to ligand stimulation in M phase and suggest that a negative regulation of ligand-receptor interactions in M phase may control the normal function of receptor tyrosine kinase and that receptor overexpression will disrupt this cell cycle-dependent regulation of receptor tyrosine kinases.
The epidermal growth factor receptor
(EGFR)1 (1) is a member of the
c-erbB family of growth factor receptors possessing an
intrinsic protein tyrosine kinase activity. There are several lines of
evidence that implicate the enhanced activity of this growth factor
receptor with the progression of cancer. First, numerous clinically
related studies reported overexpression of EGFR in various human
cancers such as bladder (2), brain (3), breast (4), and ovary (5),
strongly suggesting that the receptor tyrosine kinase plays a role in
their progression. In addition, many cases of EGFR overexpression in
cancer cells are accompanied by the expression of transforming growth
factor , a ligand for EGFR, suggesting the involvement of an
autocrine activation mechanism in oncogenesis (6, 7). Second, the potent transforming ability of EGFR is confirmed by in vitro
or in vivo experiments. For example, it was reported that
both overexpression of EGFR (8, 9) and induction of a deletion mutant
of EGFR (10, 11) conferred an EGF-dependent and an
EGF-independent transformed phenotype on avian and mouse fibroblasts,
respectively. Indeed, v-erbB oncogene encoding a truncated
form of chicken EGFR led to erythroleukemia and fibrosarcomas in chicks
(12-14). These reports have shown that enhancement of the mitogenic
signal from receptor tyrosine kinase is sufficient to induce the
progression of cancer. Although many mitogenic signal transduction
cascades involving receptor tyrosine kinases have been defined (15,
16), the downstream mechanism of carcinogenesis still remains
unclear.
On the other hand, a number of reports have suggested that malfunction of the cell cycle control mechanism is directly involved in carcinogenesis. For example, it is well documented that an essential function of the protein products of tumor suppressor genes, such as the retinoblastoma gene and the p53 gene, whose abnormalities result in cancer progression, is to keep the cells in G0 phase (17). Recently it has been shown that the cyclin-dependent kinase (cdk) inhibitors, p21 and p16 (which block the cell cycle progression at the check point of G0 to G1 or G1 to S by inhibiting cdk activity), function as tumor suppressors (18-21). The above findings indicate that the normal cell cycle control mechanism is important for the prevention of cancer progression. Considering the fact that an essential function of receptor tyrosine kinases is to promote the progression of the cell cycle, carcinogenesis may be regarded as a result of disrupted cell cycle regulation. Because receptor tyrosine kinases are believed to promote the transition of cells from the resting state to cell division cycle, most cell cycle-related studies involving receptor tyrosine kinases have focused on the cell's entry into the cell cycle. Even though the structures and activities of numerous growth-related molecules, such as Abl (22), Src (23), p53 (24), and retinoblastoma gene product (25), have been shown to be regulated throughout the cell cycle, how receptor tyrosine kinases affect the remaining cell cycle stages or how they are regulated throughout the entire cell cycle has not been well studied.
In an attempt to understand the detailed mechanism of oncogenesis induced by enhanced tyrosine kinase activity, we investigated the relationship between the receptor tyrosine kinases and the entire cell cycle. Recently, we have reported that the structure and tyrosine kinase activity of another member of the EGFR family, ErbB-2, a protein product (185 kDa) of the neu or c-erbB-2/Her 2 proto-oncogene (26-28), is regulated in a cell cycle-specific manner, and the disruption of this regulation is suggested to be involved in cellular transformation (29). The next question we asked was about the relationship between the cell cycle-dependent regulation of receptor tyrosine kinases and ligand-induced signaling. Since a direct ligand for ErbB-2 is still unknown, despite the reports of several candidates (30-33), we have investigated the structural and functional changes of another member of this receptor tyrosine kinase family, the EGFR tyrosine kinase, and its relation to ligand stimulation during the cell cycle.
In this report, we describe that in addition to the regulation of intrinsic kinase activity, ligand-induced signaling of EGFR is also tightly suppressed in the M phase at its basal level expression. However, although it showed structural modification in a cell cycle-specific manner, the overexpressed EGFR is still highly responsive to the ligand to induce ligand-mediated signaling in the M phase, suggesting that EGFR overexpression may overcome the M phase-specific negative regulation.
Cell lines, A431, rat-1, MDA-MB-468, Swiss 3T3 (SW3T3), and HBL-100 were obtained from American Type Culture Collection. Her-5 cells (derived from NR-6 cells by stable transfection with the human EGFR expression vector) were provided by Dr. H.-J. Kung (Case Western Reserve University). NR-6 is a Swiss 3T3 variant that lacks EGF receptors (28, 34). Cells were grown in Dulbecco's modified Eagle's medium mixed with F-12 (1:1; Life Technologies, Inc.) supplemented with 10% fetal calf serum (FCS) at 37 °C in a humidified 5% CO2 atmosphere.
Chemicals and AntibodiesHuman recombinant EGF was
purchased from Upstate Biotechnology. Aphidicolin, Hoechst 33342, and
Protein A-agarose were supplied by Boehringer Mannheim. Nocodazole and
thymidine were bought from Sigma. IODO-GEN and BS3 were
from Pierce. Radioactive Na125I and
[-32P]ATP were obtained from Amersham Corp..
Monoclonal anti-phosphotyrosine antibody PY20 (Transduction Lab.) and
monoclonal anti-PLC-
antibody (Upstate Biotechnology) were used for
immunoblotting. Monoclonal anti-EGFR antibody RP-13 (Amersham Corp.)
and rabbit anti-PLC-
antisera (a gift from Dr. Y. Yarden, The
Weizmann Institute of Science) were used for immunoprecipitation.
Monoclonal antibody to EGFR (528) was obtained from Neo Markers, CA.
Sheep polyclonal anti-EGFR antibody (Upstate Biotechnology) and
polyclonal anti-Shc antibody (Transduction Lab.) were used for both
immunoprecipitation and immunoblotting. A monoclonal antibody to cdc2
(Santa Cruz, sc-54) and rabbit anti-mouse immunoglobulin G (Amersham
Corp.) were also used.
Cells were chemically synchronized at each cell cycle stage. To arrest cells in the G0/G1 phase, cells (50-60% confluent) were serum-starved in DMEM/F12 with 0.5% FCS for 48 h (22). After serum starvation, cells were accumulated at pre-S phase by incubation in DMEM/F12 containing 10% FCS and aphidicolin (5 µg/ml) for 24 h (35). The cells were then washed with medium and placed in DMEM/F12 containing 10% FCS and Hoechst 33342 (1 µg/ml) for 24 h to be accumulated in G2 phase (35). To synchronize cells in M phase, cells were incubated in DMEM/F12 with 10% FCS and nocodazole (0.4 µg/ml) for 12-24 h. After nocodazole treatment, about 50-90% cells had a highly rounded mitotic morphology and were collected by mechanical shake-off (23). In the case of A431 cells, because of the difficulty of shaking off, 2 × 106 cells were incubated in DMEM/F12 with 10% FCS and nocodazole in 15-cm plates for 12-24 h, a condition in which more than 80% of the cells had a mitotic shape as determined by microscopy, and then were collected by scraping.
DNA Content AnalysisFor DNA content analysis, trypsinized
cells were fixed for 30 min at 20 °C in 70% ethanol, 30%
phosphate-buffered saline (PBS) mixture. After staining with PBS
containing 50 µg/ml propidium iodide and 8 µg/ml RNase A, cells
were analyzed on an EPICS PROFILE flow cytometer (Coulter) as described
(23). The results were analyzed with the MultiCycle computer program
(Phoenix Flow System). For some experiments, rat-1 cells were blocked
in G1 phase with 4 mM thymidine in serum-free
medium for 16 h, and the block was released by removing the drug
(washing) and supplementing the medium with 10% FCS. After 9-12 h,
changes in EGFR were followed in cells (collected by a shake-off
procedure) by DNA content analysis and/or electrophoresis.
After each treatment, cells were solubilized for 30 min on ice in lysis buffer (20 mM Na2PO4, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% aprotinin, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 100 mM NaF, and 2 mM Na3VO4), and the total protein concentration was determined using Bio-Rad protein assay kit. Electrophoretically separated proteins were transferred to a nitrocellulose membrane for 1 h at 3 mA/cm2 using a semi-dry transblot system (Bio-Rad). After blocking with PBS containing 5% milk, membranes were incubated with primary antibodies for 1 h at room temperature in 3% bovine serum albumin/PBS followed by washing with 0.05% Tween 20 in PBS. After 30 min incubation with an appropriate secondary antibody conjugated with horseradish peroxidase and a subsequent washing with PBS-0.05% Tween 20, immunoblots were developed by the enhanced chemiluminescence reagent (Amersham Corp.) for 1 min and exposed to film (Hyper-film, Amersham Corp.).
For immunoprecipitations, 500 µg to 1 mg of cell lysates were incubated with 1 µg of antibody and 50 µl of 50% protein-A agarose for 1 h. After several washes with 0.05% Tween 20/PBS, immunoprecipitates were separated by electrophoresis and analyzed.
In Vitro Dephosphorylation of EGFREGFR proteins were immunoprecipitated from 100 µg of Her-5 cell lysates arrested in the G0/G1 or M phase as described above. After washing three times with PBS, the immune complexes on agarose beads were incubated in 100 µl of 50 mM Tris-HCl, pH 9.2, and 5 mM MgCl2 for 30 min at 30 °C in the absence or presence of 20 units of calf intestinal alkaline phosphatase (New England Biolabs). After washing, the proteins were eluted into gel sample loading buffer and immunoblotted with anti-EGFR polyclonal antibody after electrophoresis.
RadiolabelingThe IODO-GEN method (Pierce) was used to radioiodinate the proteins as follows. Human recombinant EGF (5 µg) or rabbit anti-mouse immunoglobulin G (50 µg) in PBS was mixed in an IODO-GEN-coated (~1 µg/tube dissolved in chloroform and air-dried) tube with Na125I (1 mCi). Following 10 min at 23 °C, tyrosine was added to a final concentration of 0.1 mg/ml, and the mixture was separated on a column of Excellulose GF-5 (Pierce). The specific activity varied between 2 and 5 × 105 cpm/ng.
Binding and Scatchard AnalysesMonolayers of cells in
24-well dishes were washed once with binding buffer (DMEM containing
0.1% bovine serum albumin and 20 mM HEPES). For ligand
binding analysis, cells were incubated with 5 ng/ml
125I-EGF in the same buffer, and unlabeled ligand, at
different concentrations, was co-incubated with the radiolabeled ligand
for 2 h at 4 °C. For Scatchard analysis, cells were incubated
with different concentrations of 125I-EGF for 2 h at
4 °C. The cells were then washed three times with ice-cold binding
buffer and lysed in 0.5 ml of 0.1 N NaOH containing 0.1%
SDS for 15 min at 37 °C, and the radioactivity was determined by
using a -counter. Nonspecific binding was determined by the addition
of 1 µg/ml unlabeled EGF together with labeled EGF under the above
incubation conditions. For some experiments, a mouse monoclonal
antibody to EGFR or a control antibody was first used to bind the cells
at 4 °C for 90 min, washed, and a radiolabeled second antibody
against the mouse immunoglobulin G was used in binding.
Cells synchronized in the G0/G1 or M phase were stimulated with or without EGF, collected in PBS by scraping, and then centrifuged. For some experiments cells were incubated with 125I-EGF (10 ng/ml) at 4 °C for 2 h. After washing with PBS twice, cells were incubated with 1 mM bis(sulfosuccinimidyl) suberate (BS3) in PBS for 30 min at 4 °C with rocking. The cells were washed with buffer containing 10 mM Tris-HCl, pH 7.5, and 150 mM NaCl and solubilized with PI/RIPA buffer (36).
In Vitro Immune Complex Kinase AssayEGFR proteins from
cell lysates were immunoprecipitated on protein-A agarose beads as
described above. After washing four times, once with PBS, twice with 50 mM Tris-HCl, pH 7.5, 0.5 M LiCl, and once with
kinase assay buffer (50 mM Tris-HCl, pH 7.5, 10 mM MnCl2), immunoprecipitates were incubated in
50 µl of kinase buffer for 20 min at room temperature with 10 µCi
of [-32P]ATP (specific activity >3000
Ci/mM, Amersham Corp.) and 10 µg of enolase (37) as an
exogenous substrate. Reactions were stopped by adding 10 µl of 6 × SDS-polyacrylamide gel sample loading buffer. After separation on a
7% SDS-polyacrylamide gel and drying, the phosphorylated proteins were
visualized by autoradiography.
After in vitro immune complex kinase assay, EGF receptor bands were excised and extracted from the gel followed by hydrolyzation in 6 N constant boiling HCl for 60 min at 110 °C as described previously (38). After drying, hydrolysates were separated on thin layer chromatography plates with unlabeled phosphoamino acid standards by two-dimensional electrophoresis and then were exposed to x-ray film. The positions of phosphoamino acids were visualized by ninhydrin staining.
Since we have reported the altered
electrophoretic mobility of ErbB-2 in a cell cycle-specific manner by
means of serine and/or threonine phosphorylation with alteration of its
kinase activity (29), we first examined whether the EGFR undergoes
similar changes during the cell cycle. A human epidermoid carcinoma
cell line, A431, known for its overexpression of EGF receptors
(~2 × 106/cell), was first chemically arrested in
different stages of the cell cycle for DNA content analysis. As shown
in the upper panel of Fig. 1A,
serum starvation synchronized the cells in
G0/G1, aphidicolin treatment accumulated the
cells in pre-S phase and Hoechst in G2, and nocodazole
arrested them in M phase of the cell cycle as expected. When the EGF
receptor protein from the A431 cell lysate was visualized by
immunoblotting with anti-EGFR antibody, the 170-kDa EGF receptor band
showed a retardation of its electrophoretic mobility notably in M phase
(Fig. 1A) suggestive of cell cycle-dependent
structural alterations in the EGF receptor. Similar results were
observed in Her-5, a mouse fibroblast cell line that was made to
express the human EGF receptor by integrating a human EGFR expressing
vector into NR-6, a subline of SW3T3 (Fig. 1B), and in a
breast cancer cell line, MDA-MB-468, that overexpresses the EGFR (Fig.
1D). To see whether the electrophoretic mobility changes are
related to the level of EGFR expression, rat-1 cells, rat fibroblasts
expressing a low level of EGFR (2.8 × 104
receptors/cell), were synchronized at G0/G1 and
M phase. The electrophoretic mobility was retarded again upon
nocodazole treatment (Fig. 1C), and similar results were
obtained in HBL-100 (Fig. 1D), a mammary epithelial cell
line expressing a basal level of EGFR, indicating that the
electrophoretic mobility retardation of the EGF receptor in M phase is
independent of its expression level. It is interesting to note that the
retardation in electrophoretic mobility of EGF receptor was observed in
both the transformed cells (A431) and non-transformed cells (Her-5,
rat-1).
To address the question of whether the tyrosine kinase activity of EGFR
is also regulated in a cell cycle-specific manner, we next examined for
an alteration of phosphotyrosine content in EGFR during the cell cycle
which is thought to reflect changes in the gross functional activity of
tyrosine kinase. When the protein extracts from A431 cells in different
cell cycle stages were examined by immunoblotting with
anti-phosphotyrosine antibody, EGFR showed different levels of
phosphotyrosine content in a cell cycle-dependent manner:
phosphotyrosine levels were highest in the
G0/G1 phase, decreasing through the S and
G2 phases, and reaching their lowest level in the M phase
(Fig. 2A). We also observed similar results
with Her-5 cells that overexpress the human EGFR (Fig. 2A).
Considering these results together with those of in vitro
immune complex kinase assay which showed a reduced intrinsic kinase
activity of EGFR in the M phase (Fig. 6A, lanes 1 and
3), we concluded that the tyrosine kinase activity of EGFR
is regulated in a cell cycle-dependent manner and is least
active in the M phase.
To investigate whether these mobility shifts are due to the hyperphosphorylation of the protein which is known to occur in other proteins (23, 25, 29, 39), we next examined the effect of phosphatase on the mobility shift of EGFR. The immunoprecipitated EGFR proteins from Her-5 cells arrested in G0/G1 or M phase were treated with calf intestinal alkaline phosphatase to remove all phosphates before analysis, and the mobility retardation in the M phase was no longer observed (Fig. 2B). These results confirm that this retardation in mobility is a result of overall hyperphosphorylation of EGFR.
All the above data indicate that EGFR undergoes a structural modification that is caused by altered phosphorylation of the receptor protein in a cell cycle-specific manner and that this modification is related to functional alteration, suggesting that the structure and function of EGFR are regulated in a cell cycle-specific manner similar to ErbB-2. Our data also showed that this phenomenon is independent of the level of EGFR expression or transformation status of the cell line.
M Phase-specific Suppression of Ligand-induced Signal Transduction of EGFR at Basal Level Expression but Not at High Level ExpressionWe next asked whether this cell
cycle-dependent regulation of EGFR had any effect on
ligand-receptor interaction. To address this question we compared
EGF-induced tyrosine autophosphorylation of EGFR in
G0/G1 phase with that in the M phase. When the
A431 cells were analyzed (Fig. 3A), EGFR
showed significant EGF-induced tyrosine autophosphorylation in both
phases (G0/G1 and M). It may be noted that in
Fig. 3A, we intentionally used a short exposure time to show
quantitative differences in the phosphorylation status between
unstimulated and EGF-stimulated A431 cells although the unstimulated
cells exhibited constitutive phosphorylation (Fig. 2A). We
also checked the response of EGFR to transforming growth factor in
the A431 cells and observed similar results (data not shown). Similar
results were obtained in a breast cancer cell line, MDA-MB-468, that
overexpresses the EGFR (Fig. 3B). When the tyrosine
phosphorylation level of EGF-stimulated EGFR on rat-1 cells
synchronized in either G0/G1 or M phase was
tested by immunoblotting (Fig. 3C), we observed significant
EGF-induced tyrosine autophosphorylation on EGFR in both the
G0/G1 phase and the asynchronous logarithmic growth population, but it was virtually undetected in the M phase. Similarly, EGF could not stimulate the tyrosine phosphorylation in M
phase in HBL-100 (Fig. 3D), a mammary epithelial cell line expressing a basal level of EGFR. It is apparent from the above data
that the ligand-induced autophosphorylation of EGFR is tightly suppressed in the M phase when it is expressed at basal level (rat-1
and HBL-100), but overexpressed EGFR (A431 and MDA-MB-468) can still
respond to its ligands in the M phase. However, the different genetic
backgrounds of the cell lines tested make it difficult to compare and
interpret that way. To test our results in cells of the same genetic
background, we used SW3T3 (Swiss 3T3) cells expressing low levels of
EGFR and Her-5 cells (derived from NR-6 cells by stable transfection
with the human EGFR expression vector since NR-6 is a Swiss 3T3 variant
that lacks EGF receptors), and the comparative results are shown in
Fig. 4. As we predicted, only the EGFR-transfected Her-5
cells showed EGF-induced autophosphorylation of EGFR in M phase
although the extent was less than that in the G0/G1 phase (Fig. 4B), whereas the
SW3T3 cells can be stimulated with EGF only in
G0/G1 phase but not in M phase (Fig.
4A). This gives further credibility to our interpretation
that EGF can induce tyrosine phosphorylation of EGFR in M phase in
cells that overexpress it. Since our results in M phase were based upon
those obtained from the nocodazole-treated cells, we used other
approaches to correlate the cell cycle stage and the EGF-induced
autophosphorylation of EGFR. First, the rat-1 cells were treated with
nocodazole, and the metaphase cells were collected by a shake-off
procedure, which is a standard procedure used to separate the cells in
metaphase from G2 phase cells (23). The results shown in
Fig. 5 demonstrate that the EGF-induced tyrosine
phosphorylation was minimal in the shake-off cells (i.e.
M-phase cells), but the attached cells (G2 phase) did
respond strongly to EGF (in spite of being treated with nocodazole),
clearly suggesting that the effects we observed in the shake-off cells
were not due to the nocodazole treatment per se but due to
their cell cycle stage. A similar approach was used by us earlier for
ErbB-2 (29). Second, we tested the EGF-mediated response by an
alternative procedure to synchronize rat-1 cells in M phase after a
10-h release from a G1 block using 4 mM
thymidine (16 h). The results from the Western blot using an
anti-phosphotyrosine antibody indicated an undetectable response to EGF
in M phase (Fig. 5). These results are similar to the minimal response
obtained from the nocodazole-treated shake-off cells (Fig. 5) and
suggest that the EGF-induced tyrosine phosphorylation of EGFR is
undetectable or minimal in M phase in cells that express basal level of
EGFR as confirmed by the two different approaches used. Western blot of
the same membrane using an anti-EGFR antibody detected no change in the
EGFR levels in the presence or absence of EGF (data not shown).
EGF Can Enhance Intrinsic Kinase Activity as Well as Induction of Transphosphorylation of Substrates Both in the G0/G1 Phase and in the M Phase in A431 Cells
Next, we examined whether the autophosphorylation of EGFR induced by EGF in the M phase in A431 cells has any effect on the intrinsic kinase activity of EGFR. When the intrinsic kinase activity of EGFR from A431 cells stimulated with EGF in culture was examined by in vitro immune complex kinase assay, it showed significant increase of both autophosphorylation and transphosphorylation activity in the M phase as well as in the G0/G1 phase when compared with unstimulated controls (Fig. 6A, lanes 1-4). We also tested the cell lysates from Her-5 cells and observed similar findings as in A431 cells (data not shown). On the other hand, when rat-1 cells were examined by the same procedure, EGF-induced enhancement of intrinsic kinase activity of EGFR in the M phase was not significant compared with what we observed in the G0/G1 phase (Fig. 6A, lanes 5-8). To confirm that increased phosphorylation accurately reflects enhanced intrinsic tyrosine kinase activity and not serine/threonine kinase activity, we performed phosphoamino acid analysis of the in vitro phosphorylated EGFRs shown in Fig. 6B. This result indicates that EGF-induced autophosphorylation of EGFR in the M phase in A431 cells is accompanied by enhanced intrinsic tyrosine kinase activity.
To test whether the EGF-induced autophosphorylation in the M phase was
able to be transferred to a downstream signal transduction cascade in
A431 cells, EGF-induced transphosphorylation of substrates for EGFR was
examined. When the phosphotyrosine content of immunoprecipitated PLC-, an in vivo substrate for EGFR, from A431 cells in
the G0/G1 and M phases with or without EGF
stimulation were tested by immunoblotting, it was shown that EGF was
able to induce the tyrosine phosphorylation of PLC-
and to allow its
binding to EGFR in both phases (Fig. 7A).
Next, the phosphotyrosine-containing proteins from rat-1 cells were
immunoprecipitated with anti-phosphotyrosine antibody and then examined
by immunoblotting with anti-PLC-
antibody. We detected PLC-
only
in the G0/G1 phase after EGF stimulation (Fig.
7B), suggesting that the EGF-induced tyrosine
phosphorylation occurred only in the G0/G1 but
not in the M phase. We also tested another in vivo substrate
for EGFR, Shc, which is thought to mediate EGF-induced mitotic signal
by a pathway different from that of PLC-
(Fig. 7C). In
addition to the EGF-induced tyrosine phosphorylation, we also observed
EGF-induced mobility shift of Shc both in the G0/G1 and M phases in A431 cells but only in
the G0/G1 phase in rat-1 cells (Fig.
7C). When Her-5 cells were tested, results similar to those
for A431 cells were observed (data not shown). These results suggest
that EGF-induced autophosphorylation of EGFR in the M phase in A431
cells is able to trigger a downstream signal transduction cascade but
that EGF fails to induce a signal through EGFR in the M phase in rat-1
cells.
The Mechanism of Suppression of EGF-induced Autophosphorylation in the M Phase in rat-1 Cells
Our data suggest that there is an M
phase-specific negative regulation (failure to enhance tyrosine
phosphorylation and signaling in response to ligand stimulation) in
cells that have normal or low level of EGFR expression, whereas the
cells that overexpress EGFR overcome this regulation and respond to the
ligand. Since the ligand-mediated response differed between the cells
that do and do not overexpress EGF receptor, it was of interest to
study the mechanism of the M phase-specific negative regulation in
these cells. To begin with, we followed the
ligand-dependent changes in dimerization during cell cycle,
and for this purpose the A431 cells were synchronized in the
G0/G1 or M phase, treated with or without EGF,
chemically cross-linked, and then examined by immunoblotting with
anti-EGFR antibody. Dimers were detected in G0/G1 phase and M phase with EGF stimulation
(Fig. 8A), and dimerization increased with
EGF stimulation in a dose-dependent manner, and the
increase was comparable in both G0/G1 phase and
M phase (Fig. 8A, lanes 1-6). We used a similar approach in
rat-1 cells to cross-link them in the presence or absence of EGF, and
the immunoblotting with an anti-EGFR antibody detected the dimers in
the presence of EGF in G0/G1, but the dimers
were not detectable in M phase in the presence or absence of EGF (data
not shown). In addition, we used a more sensitive assay to detect the
differences in dimerization in G0/G1 phase and
M phase. Accordingly, 125I-EGF was cross-linked to rat-1
cells synchronized in G0/G1 or M phase, and the
results shown in Fig. 8B indicated a lack of dimerization in
M phase, although the dimers could be seen clearly in
G0/G1 and the bands disappeared in the presence
of excess unlabeled EGF indicating the specificity of these
results.
Since the decrease in dimerization is likely to be related to changes
in ligand binding, it was necessary to ascertain whether EGF binding
was affected under these conditions. As expected, when
125I-EGF was displaced by different concentrations of
unlabeled EGF in A431 cells (Fig. 9A), we did
not find differences in binding in either G0/G1
or M phase. Consistent with the lack of dimerization in M phase in
rat-1 cells treated with EGF, it was found that there is a decrease in
ligand binding in M phase compared with the cells in
G0/G1 phase (Fig. 9C). The
difference in binding between G0/G1 and M
phases observed in rat-1 cells but not in A431 cells could potentially
arise from differences on their cell surfaces, in number, and/or
existence of EGF receptors. To assess the existence and level of
surface-exposed EGF receptors, we employed a binding assay of a
monoclonal antireceptor antibody to A431 and rat-1 cells. The results
shown in Fig. 9, B and E, indicated no change in
the number of antibody binding sites, which reflects the number of EGF
receptors, between G0/G1 and M phases in both A431 and rat-1 cells. Similar results were obtained by this assay in
SW3T3 and Her-5 cells (data not shown) suggesting that the number of
receptors do not change between G0/G1 and M
phases, whether the cells express low or high number of EGF receptors and whether they are transformed or not. Furthermore, we performed Scatchard analysis of 125I-EGF binding to rat-1 cells, and
the results indicate a 4-fold decrease in binding affinity in M phase,
and as expected, the number of receptors remained unaltered between
G0/G1 and M phases (Fig. 9D). These
results suggest that inhibition of dimerization and ligand binding
affinity is involved in the M phase-specific suppression mechanism of
EGF-induced autophosphorylation and transphosphorylation in cells
expressing a basal level of EGFR, and this suppression is overcome by
overexpression of EGFR.
Our study demonstrated that EGF receptor undergoes structural and functional alterations in different phases of the cell cycle. The salient features of our results suggest that EGF-induced autophosphorylation and downstream signaling of the EGFR are tightly suppressed in M phase due to a decrease in ligand binding affinity and the inability of EGF to induce receptor dimerization in cell lines expressing a low or normal level of EGFR. However, overexpression of EGFR apparently reverses this negative regulation.
Structural and Functional Changes in EGFR during Cell Cycle Are Influenced by Serine and/or Threonine PhosphorylationThe structural modifications of EGFR could be inferred by the observed retardation of its electrophoretic mobility with cell cycle progression (Fig. 1). Differential phosphorylation is known to shift the electrophoretic mobility of the non-receptor tyrosine kinases, Src and Abl, showing retarded migration in M phase (22, 23). The structural changes in ErbB-2 tyrosine kinase in M phase reported earlier by us are similar to the present results (29). The retardation of electrophoretic mobility in EGFR in M phase is due to changes in the phosphorylation status of the receptor as the retardation was no longer observed after an in vitro dephosphorylation of the receptor using a phosphatase (Fig. 2B). It is most probably due to serine and/or threonine phosphorylation of the receptor since our results do not support the involvement of tyrosine phosphorylation in this phenomenon as there was a declining trend in tyrosine phosphorylation of EGFR with cell cycle progression in cells that overexpress EGFR (Fig. 2A). Recently it has been reported that EGFR undergoes ligand-independent serine/threonine phosphorylation upon entry into the cell cycle (40). Both EGFR and ErbB-2 are reported to be phosphorylated on serine and threonine residues in vivo in a serum-dependent manner with accompanying reduction in tyrosine kinase activity without elevation of phosphotyrosine content (37, 41). Protein kinase-C is known to phosphorylate serine and threonine residues on both the EGFR (42, 43) and ErbB-2 (44). It has also been reported that the desensitization of EGFR that occurs rapidly after its binding to EGF can be accounted for, in part, by Ser-1046/7 phosphorylation on the receptor, a substrate for the calmodulin-dependent protein kinase II in vitro. (45). Cdc2, a serine/threonine kinase which is a key regulator of several growth-related proteins and is most active in the M phase (46, 47), can phosphorylate serine residues on EGFR and lead to reduction of its tyrosine kinase activity (48, 49). Our data in this report clearly show that EGFR also has a cell cycle-dependent regulatory mechanism, suggesting that such a mechanism is common among the receptor tyrosine kinases and is not unique to a particular tyrosine kinase. The suggestion that the function of the receptor tyrosine kinases that promote the cell division cycle are under negative regulation through serine/threonine hyperphosphorylation indicates the existence of a feedback regulation between tyrosine and serine/threonine phosphorylation during cell cycle.
EGF-induced Tyrosine Phosphorylation and Signaling Are Suppressed in M Phase in Cells Expressing a Basal Level of EGF Receptors but Not in Cells That Overexpress ThemA novel part of this study is related to the receptor responsiveness to the ligand being regulated in a cell cycle-specific manner under different levels of EGFR expression. Hyperphosphorylation (due to serine and/or threonine phosphorylation) correlates with the unresponsiveness of cells to EGF-mediated stimulation of tyrosine phosphorylation in cells that express the normal or basal level of EGFR. Our data clearly indicate that the M phase hyperphosphorylated EGFR (in rat-1 and HBL-100 cells) is associated with a decrease in affinity (Fig. 9), dimerization (Fig. 8), tyrosine phosphorylation (Fig. 3), and signaling (Fig. 7). Taken together, the results strongly suggest that hyperphosphorylation is the primary cause of inhibition in the cells that express low or basal level of EGFR. Consistent with our results, many reports indicated the involvement of serine or threonine phosphorylation in the negative regulation of EGFR resulting in a decrease in ligand-mediated binding affinity and tyrosine phosphorylation. For instance, protein kinase C-mediated serine/threonine phosphorylation of the EGFR resulted in the reduction of both tyrosine kinase activity and ligand binding affinity (30, 50, 51). EGF stimulation is known to induce not only tyrosine phosphorylation but also serine/threonine phosphorylation on EGFR under conditions of desensitization (52).
However, when the cells overexpress EGFR, the receptor somehow escapes this negative regulation in M phase, since ligand binding, dimerization, and the ability to stimulate tyrosine phosphorylation were not very different between G0/G1 and M phases in A431 cells (Figs. 3, 8, and 9). Several possibilities exist that may allow the overexpressed receptor to escape from the M phase-specific negative regulation. Perhaps a minor fraction of the receptors that may not be detected by Western blotting (Fig. 3) escapes hyperphosphorylation and thus can respond to ligand stimulation in M phase. Alternatively, all the receptors may be hyperphosphorylated, and the hyperphosphorylated EGFR is still associated with a residual ability to respond to ligand. The residual ability of each receptor to respond to ligand (if put together) can be very high since both dimerization and tyrosine kinase activation of EGFR follow the second order kinetics with respect to EGFR concentration (53). For instance, we would expect 104-fold increase in activity when EGFR concentration is increased 100-fold (A431 cells have ~2 × 106 receptors/cells, whereas rat-1 cells have ~2.8 × 104 receptors/cell). Of course, the real situation could be more complicated than the two possibilities outlined above. The proposed mechanisms provide a plausible interpretation for the escape of EGFR from the negative regulation in M phase under conditions of overexpression. Consistent with our results Newberry and Pike (54) observed no change in the ligand binding affinity of EGF receptors with cell cycle progression in A431 cells. However, contradictory to our results, they reported a decrease in tyrosine kinase activity with increase in time after releasing A431 cells from a thymidine block, and it is not known whether this discrepancy is due to their use of a synthetic substrate to assess the tyrosine kinase activity. Similar to our results showing no changes in the number of surface-exposed EGF receptors on A431 and rat-1 cells (Fig. 9, B and E) between G0/G1 and M phases, the number of receptors were the same at all stages of the cell cycle in LIM 1215, a human colon cell line (55). Our results showing that EGF-mediated downstream signaling is enhanced in A431 cells in M phase (Fig. 7) are consistent with an upward trend of phosphatidylinositol 3-kinase activity in later stages of the cell cycle in A431 cells (54). Since both A431 and MDA-MB-468 cells are derived from human cancer tissues, we cannot eliminate the possibility that these cell lines have a specific mechanism that can inhibit phosphorylation of any specific serine and/or threonine residues, which may be important for the negative regulation in the M phase, or that they have another mechanism that can eliminate the negative regulation derived from serine/threonine phosphorylation, independent of EGFR overexpression. However, our finding that Her-5 cells, which are not cancer cells and whose EGFR expression level is intermediate between that of rat-1 cells and that of A431 cells, partially escape from this suppression suggests that overexpression itself may be sufficient to overcome this suppression. Since it has been shown that even in the absence of ligand the dimerization and enhancement of tyrosine kinase activity of ErbB-2 will occur and that such occurrence simply depends on the level of receptor expression (56), we speculate that when the EGF receptors are phosphorylated on serine/threonine residues which may suppress the dimerization, the elevation of the receptor density may overcome its suppressive effects in the presence of the ligand.
Mitogenic signals are required in the initial stages of the cell cycle (G1) for the induction of growth and proliferation, and cells at this point would be expected to be very responsive to stimulation by growth factors. Mitogenic signals are not required once the cells have crossed G1/S transition and are committed to undergo mitosis and complete the cell cycle. At this stage, growth factors are not expected to induce response to that extent seen at the initial stage. Therefore, the observation that cells with normal or low levels of EGFR do not respond to the growth factor in M phase may be a reflection of the changes in cell's need for growth signals at different stages of the cell cycle. However, overexpression of EGFR appears to deregulate this fine balance. Our results also provide a new way to understand how EGFR overexpression may contribute to development of human cancer, i.e. EGFR-overexpressing cancer cells can be constitutively activated by ligand stimulation regardless of their cell cycle stage. However, the normal cells would respond to ligand stimulation in a tight, cell cycle-dependent, regulation. Further studies on the detailed mechanism of this regulation may help us to understand better the combined role of receptor tyrosine kinases and their ligands in different cell cycle stages and also the role of receptor overexpression in cellular transformation and/or growth regulation.
We are grateful to Dr. H.-J. Kung for the
gift of the Her-5 cell line and to Dr. Y. Yarden for the anti-PLC-
antiserum.