Download Epidermal Growth Factor Receptor Protein

ICANCER RESEARCH 49. 1130-1137, March 1. 1989]
Epidermal Growth Factor Receptor Protein-Tyrosine Kinase Activity in Human
Cell Lines Established from Squamous Carcinomas of the Head and Neck1
Steve A. Maxwell, Peter G. Sacks, Jordan U. Gutterman, and Gary E. Gallick2
Departments of Tumor Biology [S. A, M., P. G. S., G. E. G.], Clinical Immunology [S. A. M., J. U. G.], and Head and Neck Surgery [P. G. S.J, The University of Texas
M. D. Anderson Hospital and Tumor Institute at Houston, Houston, Texas 77030
ABSTRACT
Two cell lines established from tumors of the head and neck area at
different clinical stages were found to differ in the expression and in the
tyrosine kinase activity of the epidermal growth factor (EGF) receptor.
Cell line 183A was derived from an early-stage tumor and cell line 1483
was derived from a tumor that had metastasized to lymph nodes. The
1483 cells displayed a higher plating efficiency and clonogenicity in soft
agar, suggesting a more tumorigenic phenotype over the 183A cells.
Analyses of EGF receptor levels by using Rl anti-EGF receptor serum
indicated that the 1483 cells expressed 5-fold more receptor than did the
183A cells. EGF receptors isolated from each cell line were active for
kinase activity in an immune complex kinase assay, using monoclonal Rl
anti-EGF receptor antibody. The autophosphorylation activity of both
receptors was stimulated by addition of EGF to isolated membrane
preparations and intact cells, although the EGF receptor of the 1483
cells was much less responsive to EGF than the receptor from 183A
cells. In addition, the 1483 receptor consistently incorporated about twice
as much phosphate as did the 183A receptor in an immune complex
kinase assay. These data suggest that the basal tyrosine kinase activity
of the EGF receptor from 1483 cells may be more active than the EGF
receptor kinase from 183 cells.
INTRODUCTION
Aberrant expression of the epidermal growth factor receptor
may be involved in the genesis and progression of several
malignant diseases. A role for elevated expression of the EGF3
receptor in tumorigenesis is exemplified by consistent obser
vations of augmented levels of the EGF receptor in several
types of malignancies. For example, amplification of the glycosylated M, 170,000 EGF receptor has been found in primary
brain tumors of glial origin (1) as well as in the epidermoid
carcinoma cell line designated A431 (2). High numbers of EGF
receptors also occur in several types of malignancies including
bladder tumors (3), breast carcinomas (4, 5), and squamous cell
carcinoma cells derived from human head and neck cancers (6,
7).
Many studies have demonstrated that the receptors for EGF
(8-11), platelet growth factor (12), insulin (13), and insulinlike growth factor 1(14) have an intrinsic kinase activity specific
for tyrosine residues. Upon binding their respective ligands, the
tyrosine kinase activity becomes stimulated severalfold as in
dicated by enhanced autophosphorylation of the receptor, in-
creased phosphorylation of exogenous substrates in vitro, and
elevated phosphorylation at the tyrosine residues of several
proteins in vivo. Constitutive activation of the tyrosine kinase
of the EGF receptor may play a role in AEV-mediated trans
formation (15-18). The \-erbB oncogene product encoded by
the AEV appears to represent a truncated EGF receptor con
taining the cytoplasmic kinase domain but lacking any sequence
corresponding to the extracellular ligand-binding domain (2,
16). Furthermore, the \-erbB protein has undergone a C-terminal modification in which 32 amino acids of the EGF recep
tor protein are replaced by 4 amino acids encoded by the
retroviral genome and as such lacks the major autophospho
rylation site of the native receptor (Tyr1173)(2). Thus, the verbB protein represents a truncated EGF receptor which may
be constitutively activated for tyrosine kinase activity (19, 20).
These examples of aberrant EGF receptor expression and
kinase activity in several malignant diseases and in AEV trans
formation suggest that defective control of the EGF receptormediated mitogenic signal may be involved in common malig
nancies of brain and head and neck areas. Although expression
of the EGF receptor has been examined in many squamous
carcinoma samples, few or no studies have been made on the
tyrosine kinase activity of the EGF receptor. To investigate the
biochemical activity of the EGF receptor in malignant lesions
of squamous epidermoid origin, we used cell lines established
from squamous carcinomas of the head and neck (21). One cell
line, designated 183A, was isolated from a poorly differentiated
squamous carcinoma of the tonsil. Another cell line was derived
from a well-differentiated squamous carcinoma of the retromolar trigone and was designated 1483. The higher plating
efficiency and clonogenicity in soft agar of the 1483 cell line
indicated that it was more tumorigenic than the 183A cell line.
Our characterization of EGF receptor expression in these squa
mous carcinoma cells has yielded important differences between
the two cell lines, not only in the expression but also in the
tyrosine protein kinase activity of the EGF receptor protein.
MATERIALS
AND METHODS
Cell Lines
Cell lines were established from tumor specimens obtained immedi
ately after surgery as previously described (21). Cell lines 183A and
1483 were recently established from squamous cell carcinomas of the
The costs of publication of this article were defrayed in part by the payment
head and neck. Line 183A was derived from a 54-year-old male patient
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
diagnosed as having a TjN0Mo tumor (greatest diameter of the primary
' This work was supported in part by the Clayton Foundation for Research to
tumor was greater than 4 cm; no positive lymph nodes, no distant
J. U. G., a Senior Clayton Foundation Investigator. This work was also aided by
métastases)that was histologically and pathologically described as a
Grant RR5511-23 to S. A. M. and Grant CA39803 to G. E. G. from the NIH.
2 To whom reprints should be addressed, at Department of Tumor Biology,
poorly differentiated squamous cell carcinoma of the tonsil. Line 1483
Box 79, M. D. Anderson Hospital and Tumor Institute, 1515 Holcombe Boule
was derived from a 66-year-old male diagnosed as having a T2N|M0
vard, Houston, TX 77030.
tumor (greatest diameter of primary tumor was 2-4 cm; single positive
* The abbreviations used are: EGF, epidermal growth factor; SDS, sodium
lymph node, no distant métastases).The lesion was identified as a welldodecyl sulfate; AEV, avian erythroblastosis; RIPA A, cell lysis buffer containing
1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, 150 min NaCl, 5 mM
differentiated squamous cell carcinoma of the retromolar trigone. A431
EDTA. 1% aprotinin, 5 mM phenylmethylsulfonyl fluoride, 10 fig/ml leupeptin,
cells, an established cell line from a human epidermoid carcinoma
1 mM sodium vanadate, 5 mM sodium pyrophosphate, 20 mM sodium phosphate,
which has been shown to overexpress the EGF receptor (2), were used
pH 7.4; RIPA B buffer, same as RIPA A but lacking SDS. sodium deoxycholate,
as control for identifying the EGF receptor in the 1483 and 183A cell
sodium pyrophosphate, and sodium vanadate; HEPES, 4-(2-hydroxyethyl)-llines.
piperazineethanesulfonic acid.
1130
Received5/16/88;revised11/1/88;accepted11/22/88.
Downloaded from cancerres.aacrjournals.org on April 29, 2017. © 1989 American Association for Cancer Research.
EGF RECEPTOR-TYROSINE
KINASE ACTIVITY IN CARCINOMA CELL LINES
Metabolic Labeling and Immunoprecipitation
For metabolic labeling, cells at approximately 70-80% confluency
were incubated overnight (16 h) in media lacking either methionine or
phosphate and were then subsequently supplemented with 0.5-1 mCi
[35S]methionine (1 nmol) (New England Nuclear and ICN) or 0.5 mCi
[32P]orthophosphate (New England Nuclear) per ml, respectively, and
2% dialyzed fetal calf serum. After incubation with radioactive medium,
cell monolayers were rinsed twice on ice with cold Ca2+-, Mg2*-free
phosphate-buffered saline. Five to 7 ml of RIPA "A" lysis buffer were
added to 32P¡-labeled
cells and 4 to 5 ml of RIPA "B" buffer were added
to [35S]methionine-labeled cell monolayers. Cells were then scraped
into the RIPA buffer and lysed in a loose-fitting Wheaton homogenizer
with 20 strokes. The lysate was incubated on ice for 5-10 min and
clarified by centrifuging at 10,000 x g for 10 min. Clarified lysates
from each cell line were adjusted to equivalent protein (1-3 mg/ml) by
the BCA protein assay method (Pierce Chemical Co., Rockford, IL),
using bovine serum albumin as a standard. Immunoprecipitation of
EGF receptor was accomplished with monoclonal antibody Rl (100
Mg/ml) purchased from Amersham Biologicals (Arlington Heights, IL).
The supernatants were incubated for l h on ice with 5 M' of Rl
antiserum/ml extract. The immune complexes were immunoprecipitated with a 15-min incubation on ice with 50 ii\ pansorbin (10% w/v
solution) for each ml of 32P-labeled extract and with 50 p\ of protein
A-Sepharose 4B (Sigma Chemical Co., St. Louis, MO; 4 mg/ml) for
each ml of 35S lysate. The immune precipitates were washed two or
three times with RIPA A, were drained, and were suspended in Laemmli
sample buffer for electrophoresis on a SDS polyacrylamide gel (22).
Autoradiography was used to visualize 32P-labeled proteins and fluorography was used to visualize 35S-labeled proteins.
Preparation of Membranes
Membranes were harvested by a modification of the procedure of
Resh and Erikson (23). Cells were washed twice with cold Ca2*-, Mg2+free phosphate-buffered saline solution and disrupted in 2 ml of hypotonic HEPES buffer (0.2 HIMMgCh, 20 mM HEPES, pH 7.0) with 50
strokes in a tight-fitting Wheaton Dounce homogenizer. After the
homogenates were incubated for 10 min on ice, EDTA was added to a
concentration of 5 mM. After further Dounce homogenization, the
lysates were centrifuged 10 min at 1,000 x g. Aprotinin and leupeptin
were then immediately added to the supernatants to final concentrations
of 2% and 20 Mg/ml, respectively. The pellet was reextracted with
hypotonie HEPES buffer and the second clarified lysate was combined
with the first extract. The extract was centrifuged at 100,000 x g for
60 min and the pelleted crude membranes resuspended in 20 mM
HEPES, pH 7.0. The membranes were washed once in 20 mM HEPES,
pH 7.0, by centrifugation at 100,000 x g for 60 min and the pellet was
gently suspended in 20 mM HEPES, pH 7.O. The crude membrane
preparation was used either immediately for the kinase assay or stored
at -70-C.
Kinase Assays
Immune Complex Kinase Assay. Immune complexes for kinase assays
were prepared as described for immunoprecipitation by lysing 35Slabeled cells in RIPA B. The protein A-Sepharose-precipitated immune
complexes were washed twice with 0.1% Triton X-100 and 150 mM
NaCl in 10 mM sodium phosphate, pH 7.4. The washed immune
precipitates were then resuspended in 0.1% Triton X-100 in 20 mM
HEPES, pH 7.0, and divided into S-M!aliquots. The aliquots were
adjusted to 100 /IM sodium vanadate and the kinase reaction was
initiated by addition of 50 n\ of 20 mM HEPES containing 5 MCi
[32P]ATP and 6 mM MnCl2. The reaction was allowed to proceed at
30°Cfor 10 min and was terminated by addition of 1 ml of cold RIPA
A. The complexes were then washed twice with RIPA A and the
phosphorylated proteins were prepared for electrophoresis according
to the procedure of Laemmli (22).
Membrane Kinase Assay. Kinase assays of membrane preparations
were performed by adjusting 30-50 Mgof membrane protein to 50 M'
in 20 mM HEPES, pH 7.0, and adding 50 ¡A
of a solution containing
20 MCi[32P]ATP (0.07 MMfinal concentration) and 6 mM MnCh in 20
mM HEPES, pH 7.0. Kinase assays were performed for 10 min on ice
and were terminated by addition of 1 ml of RIPA A. The mixture was
then disrupted in a tight-fitting Wheaton Dounce homogenizer with 20
strokes and clarified at 100,000 x g for 60 min. The supernatant was
incubated with 5 M'of Rl monoclonal anti-EGF receptor antibody for
I h and the immune complexes were collected and prepared as described
for the immunoprecipitation procedure.
Analysis of Enzyme Activity
For analysis of enzyme activity, cells were incubated for 16 h in
methiomne-free modified Eagle's medium containing 0.5-1 mCi/ml
[35S]methionine and 2% dialyzed fetal calf serum. Cell lysates were
prepared according to immune complex kinase assay. Before antiserum
was added to the lysates, 1 ml of the clarified cell extract was subjected
to protein precipitation with 20% trichloroacetic acid. The trichloroacetic acid-precipitated protein pellet was resuspended in 100 /il of RIPA
A and analyzed for specific activity by monitoring 35Scounts/min/Mg
protein. Each cell lysate from 183A and 1483 cells was normalized for
equivalent protein (2-3 mg/ml). Immune complexes were harvested
from cell lysates as described for the immune complex kinase assay,
and each precipitate was suspended in 0.1% Triton X-100 and 20 mM
HEPES, pH 7.0. The suspension was separated into two equal sets of
200 fi\. One set was further divided into 50-Ml aliquots, washed three
times in RIPA A, and suspended in sample buffer. The other set was
subjected in SU-M!portions to the kinase assay in the presence of various
concentrations of ATP (0.23 MMat 3000 MCi/nmol or 6 MMATP at
90-100 MCi/nmol) and angiotensin II (0.3-3 mM). After incubation at
30°C,the assays were immediately terminated by immersion in an ice
bath followed by pelleting of the Sepharose beads in a microfuge at
4°C.The supernatants were aspirated and "quick-frozen" in an isopropyl alcohol/dry ice bath. The frozen supernatants were then lyophilized,
resuspended in pH 3.5 pyridine acetate buffer, and spotted on thinlayer cellulose acetate plates. Phosphorylated angiotensin was separated
from labeled ATP by electrophoresis at constant voltage (400 V) for 2
h. The protein A-Sepharose pellets from the kinase assays were washed
with RIPA A and the 32P-labeled proteins were subjected to SDS
polyacrylamide gel electrophoresis and autoradiography. The 35S-labeled proteins were visualized on a separate gel by fluorography. The
[35S]methionine label was not detectable in the autoradiographic pro
cedure in the time used to expose the 32P-labeIed proteins on Kodak
XAR-5 film. Autophosphorylation and kinetic analyses of angiotensin
II phosphorylation determinations are expressed as the average of
values obtained from at least two independent experiments.
Quantitation of |35S|Methionine- and 32P-labeled Proteins
The 35S-labeled proteins were eluted from the gel slice by incubation
overnight at 37°Cin 0.5 ml of NCS tissue solubilizer (Amersham)
solution. Five ml of Atomlight fluor (New England Nuclear) were then
added and the eluate was analyzed for cpm along with a parallel blank.
Given the specific activity of cellular protein in the initial cell extract,
an estimate of the amount of EGF receptor could be made in the
immune complex kinase assay. For analyses of 32P-labeled proteins, gel
slices were incubated for 2 h at 80°Cin 1 ml of 6 N HC1. Five ml of
water were then added and the eluates were monitored for cpm along
with a parallel blank. Under these conditions, less than 0.004% of the
35S radioactivity could be detected relative to cpm obtained under
conditions using Atomlight fluor. Thus, the cpm from 35Srepresented
an insignificant contamination under the conditions used to monitor
32P. A known amount of [32P]ATP was analyzed under conditions
identical to those used for eluting 32P-labeled proteins, which allowed
a molar determination of incorporated phosphate to be made.
Quantitation of Phosphorylated Angiotensin II
The phosphorylated angiotensin II peptide was resolved from labeled
ATP by electrophoresis on a thin-layer cellulose plate in a pH 3.5
pyridine acetate buffer (pyridine:acetic acid:water; 5:50:945) at constant
voltage (400 V) for 2 h. Area corresponding to phosphorylated angio
tensin II was localized by autoradiography and eluted by incubating
overnight in 5 ml Atomlight fluor at 37°C.Moles of 32P incorporation
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EGF RECEPTOR-TYROSINE
KINASE ACTIVITY IN CARCINOMA CELL LINES
B
3
Fig. 1. Analysis of EGF receptor protein
and kinase activity of 183A and 1483 cell lines.
Protein levels were examined by metabolic
labeling with [35S]methionine (I), and kinase
activity was analyzed by immune complex ki
nase assays (A). Lane I, control serum, 183A;
Lane 2, RI anti-EGF receptor serum, 183A;
Lane 3, control serum, 1483; Lane 4, antiEGF receptor, 1483. "S- and 32P-labeled pro
teins were fractionated on individual 8% SDS
polyacrylamide gels. The "S-labeled proteins
were treated with En'hance (NEN) and local
ized by fluorography with a 20-h exposure on
Kodak XAR-5 film. The 33P-labeled proteins
were visualized by autoradiography for 4 h by
using Kodak XAR-S film and a DuPont en
hancing screen. The 3!S was not detected in
the 4 li autoradiograph procedure on Kodak
XAR-S film and DuPont enhancing screens.
Ordinate, molecular weight in thousands.
4
200-
P170-
P170-
92.5-
68-
43-
18.4-
were determined by monitoring cpm relative to a known standard of
the original [32P]ATP.
Phosphoamino Acid Analyses of 32P-labeled Proteins
Phosphoamino acid analysis was performed according to the method
of Hunter and Sefton (24). 32P-labeled EGF receptor was electroeluted
from the polyacrylamide gel fragments and precipitated with trichloroacetic acid. 32P-labeled angiotensin II was eluted in pH 3.5 pyridine
acetate (pyridine:acetic acid:water; 5:50:945) and lyophilized to a pow
der. The peptide or protein residue was suspended in 6 N HCl, sealed
in glass ampules, and heated in mineral oil at 1IOC for 2 h. After the
hydrolysis, the HCl was removed under vacuum and the residue was
suspended in pH 3.5 pyridine acetate containing 1 mg/ml each of
phosphoserine, phosphothreonine,
and phosphotyrosine markers.
Phosphoamino acids were resolved by thin-layer electrophoresis at
constant voltage (400 V) for 2 h. Phosphoamino acid markers were
visualized by staining with ninhydrin and 32P-labeled amino acids were
visualized by autoradiography.
RESULTS
Properties of Head and Neck Squamous Carcinoma Cell Lines.
The cell lines designated 183A and 1483 exhibit epithelial
morphology and grow in a cobblestone-like pattern (21). When
injected s.c. into nude mice, both lines were tumorigenic (IO7
cells/mouse) and generated tumors in all tested mice. Both cell
lines have doubling times of approximately 36 h. The higher
plating efficiency and clonogenicity in soft agar of the 1483 cell
line indicates that it exhibits a more tumorigenic phenotype
than does the 183A line. EGF at 10 ng/ml was found to
stimulate the growth of the 183A cells whereas the growth of
the 1483 cells was inhibited.4
Analysis of Steady-State Levels of EGF Receptor in Cell Lines.
Expression of the EGF receptor was examined by metabolically
labeling cells to steady state with [35S]methionine, followed by
1 Unpublished data.
immunoprecipitation with the Rl anti-EGF receptor antibody.
The R l antibodies react with the extracellular domain of the
EGF receptor but do not interfere with EGF binding to the
ligand-binding domain (25). The EGF receptor was identified
based on a relative mobility of 170,000 (Fig. \A, Lanes 2 and
4), on comigration with the EGF receptor from A431 cells (Fig.
3, I and H). and on a specific reactivity with Rl antibodies
(compare Fig. \A, Lanes 1 and 3 with Lanes 2 and 4). The
results of a 16-h [35S]methionine incorporation revealed that
1483 has a 5-fold elevated level of the EGF receptor (Fig. \A,
Lane 4) over that of 183A (Fig. IA, Lane 2) as analyzed by the
quantitation technique described in "Materials and Methods."
Quantitation on a molar basis yielded 0.144 pmol EGF receptor
for the 183A immunoprecipitate and 0.719 pmol for that of
1483. Two additional experiments yielded results similar to
those illustrated in Fig. \A. These results are in agreement with
the levels of EGF receptor previously determined by Western
immunoblotting for both 183A and 1483 cells (21).
Kinase Activity of EGF Receptor in Cell Lines. The kinase
activity of the EGF receptor from each cell line was examined,
since the 183A and 1483 cells differed in their growth response
to EGF. Rl immune complexes containing the EGF receptor
from either the 183A or 1483 cells were tested for kinase
activity. Fig. IB, Lanes 2 and 4, show that both cell lines
produce an enzymatically active EGF receptor. Phosphoamino
acid analyses of each phosphorylated EGF receptor yielded
exclusively phosphotyrosine (Fig. 2A, Lanes 1 and 2).
Response of the EGF receptor kinase activity of each cell line
to EGF stimulation was investigated by using isolated crude
membrane preparations. The EGF receptor kinase from A431
cells was stimulated approximately 4-fold in response to bind
ing of EGF to isolated membrane preparations (Fig. 3A, Lane
2), which is similar to other reported data (8, 9). The ability of
the EGF receptors from 183A and 1483 cells to be stimulated
by EGF was also examined in vitro with isolated membrane
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EGF RECEPTOR-TYROSINE
K1NASE ACTIVITY IN CARCINOMA CELL LINES
B
pserpthrPtyr-
Fig. 2. Phosphoamino
acid analyses of
phosphorylated EGF receptor (A) and the peptide substrate, angiotensin II (B). A, Lane 1,
183A; Lane 2, 1483. B, Lane 1, angiotensin II
phosphorylated by 183A receptor; Lane 2, an
giotensin II phosphorylated by 1483 receptor.
pser, phosphoserine; pthr, phosphothreonine;
ptyr, phosphotyrosine.
ptyr-
phosphopeptide -
O-
O-
with [32P]P¡
and then treating with 100 ng EGF for 10 min,
preparations. Membrane preparations of 1483 cells were di
luted approximately 5-fold to obtain a receptor level which was followed by immunoprecipitation.
The 1483 cell lysate was
equivalent to that of the 183A preparation. Immunoblotting of again diluted 5-fold to approximate an amount of receptor
membrane preparations confirmed that equivalent amounts of equivalent to that of the 183A preparation. Similar results to
receptor were present in both the 1483 and 183A membrane
those obtained in the membrane kinase assays were observed
kinase assays (data not shown). The basal level of autophosfor EGF stimulation of each cell line, indicating that differences
phorylation activity of each receptor appeared to be similar in in EGF stimulation of kinase activity was not an in vitro artifact
the isolated membrane preparations (Fig. 3B, Lanes 1 and 3). (Fig. 3C). Scanning of the autoradiograph of EGF receptors
and analyses of peak areas
However, a significant difference was consistently observed in metabolically labeled with [32P]P¡
the ability of each kinase to be stimulated by EGF. The autoindicated that EGF produced a 4.5-fold stimulation of 183A
receptor phosphorylation (Fig. 3C, Lane 2) and a 1.9-fold
phosphorylation of the EGF receptor from 183A was enhanced
6.6-fold upon addition of EGF to the membranes, as determined
increase in 1483 receptor phosphorylation (Fig. 3C, Lane 4).
by scintillation counting of radioactivity (Fig. 3B, Lane 2),
Kinetic Analyses of EGF Receptor Activity. Other possible
whereas only a 2.2-fold increase in EGF receptor activity in variations in kinase activity between the 183A and 1483 EGF
response to EGF addition was observed in the 1483 membrane
receptors were investigated by using the neuropeptide angioten
sin II. Angiotensin II has been used to study EGF receptorkinase assays (Fig. 3B, Lane 4). Stimulation of EGF kinase
activity upon addition of EGF to isolated membrane prepara
tyrosine protein kinase activity from A431 cells (26, 27) and
tions from 183 and 1483A cells was also analyzed by using the has the advantage of containing a single tyrosine residue and
peptide substrate angiotensin II. The EGF receptor in mem
no serine or threonine residues.
branes from 183A cells exhibited a 2.3-fold increase in EGFThe specificity of EGF receptor kinase activity toward angio
stimulated phosphorylation (Table 1), whereas the 1483 mem
tensin II was analyzed with the receptor isolated in the Rl
brane preparation displayed a 1.7-fold increase upon EGF
immune complex. The phosphorylation of the angiotensin II
addition (Table 1). Response of EGF receptor kinase to EGF
peptide was specific only for immune complexes containing
stimulation in vivo was studied by metabolically labeling cells either the 183A or the 1483 EGF receptor, as indicated by the
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EOF RECEPTOR-TYROSINE
KINASE ACTIVITY IN CARCINOMA CELL LINES
B
1234
P170-
92.5—
68—
43-
18.4Fig. 3. Analyses of response of EGF receptor kinase activity in 183A and 1483 cells and their derived membrane preparations to EGF stimulation. Activity was
analyzed by membrane kinase assays (A and B) and by metabolic incorporation of ["?]?, (Q. A, Lane 1, A431, untreated control; Lane 2, A431, treatment with 100
ng EGF. B, Lane 1, 183A, no EGF; Lane 2, 183A, treated with EGF; Lane 3, 1483, no EGF; Lane 4, 1483, treated with EGF. C, Lane l, 183A, no EGF; Lane 2,
183A cells treated with 100 ng/ml EGF; Lane 3, 1483, no EGF; Lane 4, 1483 cells treated with EGF. Proteins were visualized by autoradiography and exposure on
Kodak XAR-5 film with DuPont enhancing screens for 3 h (A), 16 h (B), and 12 h (C). Ordinate, molecular weight in thousands.
Table 1 Rate ofangiotensin II phosphorylation by 183 and 1483 membrane
preparations in presence and absence of EGF
Rates are expressed as fmol 1:l' incorporated into angiotensin II min ni:
membrane protein. Each value represents the mean of triplicate determinations.
(-)EGF
(+)EGF
Cell line
183A
1483
14.8
21.2
34.1
36.9
absence of detectable peptide phosphorylation in immune com
plexes obtained with control serum (data not shown). As ex
pected, phosphoamino acid analysis of phosphorylated angio
tensin II yielded exclusively phosphotyrosine in each case (Fig.
IB, Lanes 1 and 2). These data indicate that the EGF receptor
in the immune complex kinase assay covalenti) modified angio
tensin II with a phosphate residue at tyrosine.
Phosphorylation ofangiotensin II over a reaction time period
at 0.23 UM[32P]ATP and 3 mivi angiotensin II was monitored.
A sigmoidal curve was generated, suggesting the occurrence of
a biphasic reaction mechanism (Fig. 4A). A pause in activity
was observed from 1 to 10 min for the 183A receptor kinase
assay, whereafter the rate accelerated up to 15 min (Fig. 4A). A
less distiguishable pause in activity was also apparent for the
1483 receptor assay. The pause in reaction may be due to a
requirement of enzyme activation for autophosphorylation (26,
27) and/or ATP binding (28). To investigate this possibility
further, a reaction time course was performed at a higher
concentration of ATP (6 MM).If autophosphorylation and/or
activation by ATP is involved, the pause phase in activity would
be expected to be shortened. As observed in Fig. 5A, a reaction
time course at a higher ATP concentration (6 MM)still yielded
a sigmoidal curve but with a much shorter pause lasting ap
proximately 6 min. For the 183A receptor kinase, the rate of
reaction (slope of the curve) after the reaction pause greatly
increased 127-fold from 0.003 pmol/min at 0.23 MMATP to
0.38 pmol/min at 6 MMATP, whereas the reaction rate for the
1483 receptor kinase accelerated 34-fold from 0.019 pmol/min
to 0.64 pmol/min. These data suggested that the EGF receptors
from the 183A and 1483 cell lines require either autophos
phorylation or ATP binding or both for maximal activity.
Furthermore, a difference in the requirement of each receptor
for activation by ATP was evident from these changes of
reaction rate after the pause phase of the time course.
The incorporation of phosphate into the EGF receptors from
183A and 1483 cells was monitored overtime in the presence
of angiotensin II at 0.23 and 6 MMATP to further examine the
dependence of enzyme activity for autophosphorylation.
At
0.23 MMATP, the 1483 and 183A receptors autophosphorylated at different rates and to different degrees (Fig. 4B). While
the 183 receptor autophosphorylation appears to slow after 5
min, the autophosphorylation of the 1483 receptor continues
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EOF RECEPTOR-TYROSINE
KINASE
ACTIVITY
IN CARCINOMA
CELL
LINES
10
15
(mir,)
B
I
Fig. 4. Time course of EOF receptor kinase activity at 0.23 JIM ATP. A,
phosphate incorporation into angiotensin II. Concentration of peptide was 3 HIM.
Points, determined from an average of two individual assays. (•)183 receptor
assays; (A) 1483 receptor assays, li. course of autophosphorylation in the presence
Fig. 5. Time course of EGF receptor activity at 6 ^M ATP. A, phosphate
of angiotensin II. Concentration of peptide in each experiment was 0.3 HIM.(•) incorporation into angiotensin II. Concentration of angiotensin was 3 HIM.A.
183 receptor assays; (A) 1483 receptor assays. Points, average of determinations
points, determined from an average of two independent assays. (•)183 receptor
assays; (A) 1483 receptor assays. li. course of autophosphorylation in presence of
from two individual assays from two independent experiments.
angiotensin II. Concentration of peptide in each experiment was 0.3 HIM.Points,
average of determinations from two individual assays from two independent
to increase up to 15 min. The data support the observation in experiments. (•)183 receptor assays; (A) 1483 receptor assays.
Figs. 4A and 5A that a particular degree of autophosphorylation
is required for optimal activity toward the peptide substrate,
since the incorporation of phosphate into the receptors at a
level of approximately 0.35 and 0.65 mol phosphate/mol of
receptor occurred for the 183 and 1483 receptors (Fig. 4fi),
respectively, before activity for the exogenous substrate in
creases (Fig. 4A). At 6 /J.M ATP, the autophosphorylation
reaction proceeded much faster and to a higher degree, as would
be expected (Fig. SB). Under these conditions, a higher amount
of phosphate was incorporated into the 1483 receptor (0.87
mol phosphate/mol receptor) than into the 183A receptor (0.44
mol phosphate/mol receptor) after 5 min of reaction time.
Although both enzymes appear to be activated for angiotensin
II after about 6-7 min, the 1483 receptor incorporated almost
twice as much phosphate as the 183A receptor, which also
suggested that the 1483 receptor may have more activity under
these conditions.
Reciprocal plots of enzyme activity versus substrate concen
tration confirmed a higher basal activity (Vmax)for the 1483
EGF receptor over the 183 receptor (Table 2). A higher Km
value of the 1483 receptor for angiotensin suggests a difference
in affinity for angiotensin as a substrate (Table 2).
Table 2 Apparent Kmand y^, determinations for 183 and 1483 EGF receptors
obtained at 0.23 tiM A TP
Apparent Kmand Vm„
data are expressed as the average of two determinations
obtained from two independent experiments.
„¿â€ž
(fmol J2P/min/
fmol EGF-R)
Cell line
(mM)
I83A
1483
0.6
1.3
0.16
2.3
from them (6, 7) as well as in glioma (1) and breast carcinoma
cells (4, 5). These findings have led to speculation that overexpression of the EGF receptor might be involved in tumorigenicity. Evidence for this possibility has been obtained by Santon
et al. (29), who isolated variant A431 cells with different levels
of EGF receptor and showed that higher concentrations of the
EGF receptor appeared to provide the cells with a growth
advantage in vitro and in vivo. In addition, A431 cells selected
for decreased EGF receptor expression exhibited a greater
capacity to undergo terminal differentiation (30). While in
creased density of the EGF receptor appears to correlate with
tumorigenicity, as yet no biochemical differences in EGF recep
tor activity have been correlated with different levels of expres
sion.
Since the tyrosine kinase activity of the EGF receptor is
DISCUSSION
important for transmitting a mitogenic signal upon EGF bindThe EGF receptor has been found to be frequently elevated
ing, we examined the activity of the EGF receptor in the 183A
din squamous carcinoma tumor samples and cell lines derived and 1483 squamous carcinoma cell lines. We have previously
1135
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EGF RECEPTOR-TYROSINE
KINASE ACTIVITY IN CARCINOMA CELL LINES
demonstrated that 1483 cells expressed elevated amounts of
the EGF receptor as compared to the 183A cells. In this report,
we observed three important differences between the kinase
activities of the 1483 and 183A receptors: (a) the 1483 receptor
kinase was much less responsive to EGF than that observed for
183A; (b) the 1483 receptor autophosphorylated in vitro to a
greater degree than the 183A receptor; and (c) the 1483 receptor
exhibited a higher activity and a lower affinity for angiotensin
in Rl immune complexes. These differences in enzymic activity
between the two EGF receptors may explain the different
biological responses of the 183A and 1483 cells to EGF.
The lower stimulation of the EGF receptor kinase of the
1483 cells in the presence of EGF as compared to the receptor
of the 183A cells in membrane preparations may be the result
of either a difference in topology or conformation between the
two receptors in the membrane. The latter possibility is more
likely, based upon the difference in degree of in vitro autophosphorylation between these two receptors in the immune com
plex kinase assay. While the 1483 receptor underwent a similar
amount of autophosphorylation as the 183A receptor in mem
branes and intact cells, the 1483 receptor was found to undergo
greater (2-fold) phosphorylation over the 183A receptor when
assayed isolated in the Rl immune complex under both 0.23
and 6 MMATP. The greater autophosphorylation of the 1483
receptor in vitro is probably not the result of lower endogenous
phosphorylation, since both the metabolic labeling and mem
brane kinase assays suggested that a similar amount of phos
phorylation occurred for both receptors in the absence of EGF.
One possible interpretation of these results is that additional
phosphorylation sites may be exposed on the 1483 receptor in
an isolated state in vitro, whereas these sites are sequestered in
vivo. Alternatively, the higher degree of autophosphorylation
may reflect a greater activity of the 1483 receptor kinase as
compared to the 183 receptor.
The EGF receptors from 183A and 1483 cells appeared to
be dependent on autophosphorylation for optimal activity for
angiotensin II. A similar effect of autophosphorylation on the
A431 EGF receptor kinase activity has also been observed in
that preincubation of the receptor with saturating ATP concen
tration prior to the kinase assay resulted in an enhancement of
enzyme activity (26, 27). It was proposed that autophospho
rylation activated the receptor by removing an inhibitory con
straint so that exogenous substrates can have greater access to
the enzyme-active site. The higher degree and faster rate of
autophosphorylation
of the 1483 receptor may allow for a
greater activity toward the peptide substrate by increasing ac
cessibility for the active site and by increasing the affinity for
ATP. Thus, this higher degree of autophosphorylation observed
in vitro may reflect a partial activation of the 1483 receptor.
Furthermore, a higher activity of the 1483 receptor over the
183 receptor also appears evident from the greater incorpora
tion of 32P into angiotensin II, even though approximately
with receptor oligomers. The monomeric receptors possess low
ligand and reduced kinase activity, whereas the oligomeric
receptors have high EGF-binding activity and activated kinase
activity. Several recent studies have supported this model (32,
33). Subsequent EGF receptor clustering appears to be a nec
essary and sufficient signal for mitogenesis induced by EGF
(34). In light of these observations, the higher concentration of
EGF receptor in the 1483 cell membranes may favor or drive
the aggregation process resulting in enhanced kinase activation
when compared to receptor activity in 183A cells. The changes
in ligand binding after aggregation predicted by this model are
very similar to those we have observed in 1483 cells relative to
183 cells. Thus, the clustering of EGF receptor in squamous
carcinoma cells may result in partial activation of the tyrosine
kinase activity. Analysis of EGF receptor kinase activity in
membranes of variant 1483 cells expressing different levels of
the EGF receptor should provide more insight into any involve
ment of the density of the EGF receptor in increased tumorigenicity. Furthermore, analyses of additional cell lines derived
from squamous carcinomas of the head and neck, as well as
primary tumor samples, will be necessary before differences in
EGF receptor kinase activity observed here can be conclusively
correlated with the biological phenotype of the tumor.
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One mechanism proposed for EGF receptor kinase activation
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1137
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Epidermal Growth Factor Receptor Protein-Tyrosine Kinase
Activity in Human Cell Lines Established from Squamous
Carcinomas of the Head and Neck
Steve A. Maxwell, Peter G. Sacks, Jordan U. Gutterman, et al.
Cancer Res 1989;49:1130-1137.
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