- Email: [email protected]
Detection of EGF-induced EGF receptor degradation and tyrosine phosphorylation in intact cells
JOURNAL OF
ELSEiVIER
biochemical and biophysical methods J. B&hem.
Biophys. Methods 32 (1996) 97-108
Research article
Detection of EGF-induced EGF receptor degradation and tyrosine phosphorylation in intact cells Er-Bin Yang aqb,Dong-Fang Wang a, Peter Mack b, Li-Yao Cheng a3* ’ Department of Biochemistry, Faculty of Medicine, National Unicersi@ of Singapore, 10 Kent Ridge Crescent, Singapore 119260 b Department of Surgery. Singapore General Hospital, Outram Road, Singapore 169608 Received 30 September
1995; revised 30 January
1996; accepted 8 February
1996
Abstract In this work, a simple, sensitive, and non-isotopic assay system for the detection of EGF-induced EGF receptor degradation and tyrosine phosphorylation in intact cells is described. In this system, boiling Laemmli sample buffer was directly added to cultured Chang liver cells to stop the reactions in the cells stimulated by EGF and to make whole-cell extracts. The effects of EGF concentration and incubation time on the EGF-induced degradation and tyrosine phosphorylation of EGF receptor were successfully determined using monoclonal anti-EGF receptor, recombinant anti-phosphotyrosine peroxidase conjugate, and enhanced chemiluminescence (ECL) Western blotting assay system. Unlike other assay systems, the use of radioisotopes was avoided in this determination. The assay system is linear up to 100 pg sample protein from whole-cell extracts for the detection of EGF receptor and EGF-induced autophosphorylation. This assay may be easily adopted for identification of other growth factor receptors and phosphotyrosine-containing proteins in intact cells, using appropriate anti-growth factor receptor antibodies. Keywords: EGF receptor;
Tyrosine
phosphorylation;
ECL Western blotting
Abbreviations: BCA, bicinchoninic acid; BSA, bovine serum albumin: ECL, enhanced chemiluminescence; EGF, epidermal growth factor; EGTA, ethyleneglycolbis(g-aminoethyl ether&N, N, N’, N’-tetraacetic acid; FCS, fetal calf serum; IGF-1, insulin-like growth factor-l; MEM, minimum essential medium Eagle; PBS, phosphate-buffered saline; PDGF, platelet-derived growth factor: PMSF, phenylmethylsulfonyl fluoride: SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis * Corresponding author. Fax: (65) 7791453. 0165-022X/96/$15.00 0 1996 Elsevier Science B.V. All rights reserved PII SO1 65-022X(96)00003-6
1. Introduction It is known that growth factor receptors on the cell surface with tyrosine kinase activity. such as epidermal growth factor (EGF) receptor, insulin receptor, insulin-like growth factor- 1 (IGF- 1) receptor and platelet-derived growth factor (PDGF) receptor, play a key role in the regulation of cell growth and differentiation. The binding of growth factors to their specific receptors activates receptor tyrosine kinases which undergo self-phosphorylation and subsequently phosphorylate various cellular protein substrates on tyrosine residues to carry out signal transduction [ 1.21. It has been reported that, when EGF-EGF receptor complex is formed by EGF binding to the extracellular domain of EGF receptor on target cells, it is rapidly concentrated in clathrin-coated pits. This complex is rapidly internalized. transits through the endosomal system. and is degraded within the lysosomal compartment. These results were detected by immunohistochemical localization and counting lZ51-EGF in the cells [3,4]. It has also been reported that EGF increased the rate of disappearance of the 170 kDa EGF receptor from EGF-perfused rat liver [5]. This assay system is unable to accurately detect the process of EGF receptor degradation induced by EGF because it is very difficult to control the reaction conditions, including EGF concentration and incubation time. It was found that the addition of EGF to the plasma membrane of A431 cells, which overexpressed EGF receptor. resulted in the stimulation of membrane phosphorylation to a maximal level by 2.5 min and the phosphorylation was decreased by extending incubation time [6]. However, EGF receptor autophosphorylation and the internalization and degradation of EGF-EGF receptor complex may play a very important role in the regulation of signal transduction. Unfortunately, no rapid, safe and efficient assay system for the detection of the EGF-induced EGF receptor degradation and tyrosine phosphorylation in intact cells has been reported. In this report, we describe an assay system for the determination of the EGF-induced EGF receptor degradation and tyrosine phosphorylation in whole-cell extracts. ECL Western blotting assay system is used to directly detect EGF receptor and EGF-induced phosphotyrosine-containing proteins in intact cells. This system is very simple, rapid and safe. It is also very sensitive and does not require immunoprecipitation and radioisotope. In addition, as boiling Laemmli sample buffer for sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) is directly added to cultured cells to stop the reactions and to make whole-cell extracts, it is very easy to accurately control the reaction time.
2. Materials and methods 2. I. Materials A Chang liver cell line from normal human tissue was purchased from ATCC (Rockville, MD, USA). EGF (E32641, monoclonal anti-EGF receptor antibody (clone F4. E3 138) minimum essential medium Eagle (MEM, M7399), and bicinchoninic acid
E.-B. Yang et al. /J.
Biochem. Biophys. Methods 32 (I 9961 97- 108
99
(BCA) protein assay kit (BCA-1) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Enhanced chemiluminescence (ECL) Western blotting analysis system (RPN 2108) and Hyperfilm-ECL (RPN 2103) were obtained from Amersham (Buckinghamshire, UK). Recombinant RC20 anti-phosphotyrosine peroxidase conjugate (E 120H) was purchased from Affiniti (Nottingham, UK). Nitrocellulose membrane (401196) was purchased from Schleicher and Schuell (Dassel, Germany). 2.2. Cell culture Chang liver cells from normal human tissue were cultured in MEM containing 100 units/ml penicillin, 100 ,ug/ml streptomycin, and 10% heat-inactivated fetal calf serum (FCS) in an atmosphere of 95% sir/5% CO, at 37°C. Confluent cells were trypsinized and transferred either to a 12-well plate (lo5 cells/well) for detecting the effects of EGF on EGF receptor and EGF-induced phosphotyrosine-containing proteins in intact cells or to a 260 ml tissue culture flask for extracting EGF receptor and EGF-induced phosphotyrosine-containing proteins from the cultured cells. 2.3. Extraction of EGF receptor and EGF-induced from cultured cells
phosphoerosine-containing
proteins
Confluent cells in a 260 ml tissue culture flask were washed twice with 20 ml of 50 mM Hepes, pH 7.5, containing 150 mM NaCl, 1 mM ethyleneglycolbis@aminoethyl ether)-N, N, N’, N’-tetraacetic acid (EGTA), and 10% glycerol, scraped into 20 ml of the same buffer using a rubber policeman, and centrifuged at 600 X g for 10 min at 4°C. The cells were lysed in solubilization solution (10’ cells/ml) containing 50 mM Hepes, pH 7.5, 1% Triton X-100, 10% glycerol, 1.5 mM MgCl?, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 20 pg/ml leupeptin, and homogenized in a glass Dounce homogenizer. The insoluble material was removed by centrifugation at 40000 X g for 30 min at 4°C. Protein concentration in the supernatant was estimated with BCA protein assay kit as described by Smith et al. [7]. The EGF-induced phosphotyrosine-containing proteins were obtained from the cells which had been pretreated with 100 ng/ml EGF at 37°C for 5 min using the same method described above. All extracts were stored at -70°C prior to analysis. 2.4. Identification
of EGF receptor in intact cells
In order to directly identify EGF receptor in intact cells, confluent cells in a 12-well plate were washed twice with 1 ml of serum-free medium containing 0.1% bovine serum albumin (BSA) and 2 mM glutamine. They were then incubated in 0.5 ml of the same medium with or without EGF for the indicated time or with various EGF concentrations for 2 h at 37°C. After draining off the reaction solution, 100 ~1 of boiling Laemmli sample buffer containing 125 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, and 10% 2-mercaptoethanol was immediately added to the cells to stop the reactions and to make whole-cell extracts which were subsequently transferred to a microfuge tube and boiled for 5 min. 30 ~1 of the extracts was applied to 7.5% SDS-PAGE 181. Proteins separated
by 7.5% SDS-PAGE were electrophoretically transferred to nitrocellulose membrane. A semi-dry electroblotting method in a AE-6675 Horiblot apparatus (Atto Corporation, Tokyo, Japan) was used [9.10]. Non-specific binding sites on the blot were blocked by incubating with I% BSA in washing buffer containing 10 mM Tris-HCI. pH 7.5, 100 mM NaCI. and 0. I% Tween 20 at room temperature for I h. The blot was then incubated with monoclonal anti-EGF receptor antibody in washing buffer (I : 2000) at room temperature for I h. The blot was washed for 10 min with three changes of washing buffer and then incubated with anti-mouse IgG peroxidase conjugate in washing buffer (I : 2000) at room temperature for I h. After washing three times with washing buffer. EGF receptor binding with anti-EGF receptor antibody was identified by ECL Western blotting assay system according to the manufacture’s instruction. An equal volume of ECL detection solution I was mixed with detection solution 2 before use. This mixture was directly added to the blot which was subsequently incubated for I min at room temperature and immediately wrapped in SaranWrap. The signals on the blot were visualised by exposing to Hyperfilm-ECL for 1.5-30 s and scanned by a densitometer (Shimadzu. CS-9000. Japan) at 595 nm. 2.5. Idrnt~ficutiot~
of’ EGF-itlduced plto.sl’ltot?~ositte-c.otltuitlitlg proteins in intact cells
Confluent cells in a I?-well plate were washed twice with 1 ml of serum-free medium containing 0. I % BSA and 2 mM glutamine and incubated with or without 100 ng/ml EGF in the same medium for the indicated time or with various EGF concentrations for 5 min at 37°C. Whole-cell extracts were made by adding 100 ~1 of boiling Laemmli sample buffer to each well. transferred to an Eppendorf tube, and boiled for 5 min. 30 ~1 of the extracts was loaded onto 7.5% SDS-PAGE. Separated proteins on the gel were electrophoretically transferred to nitrocellulose membrane. After non-specific binding sites were blocked with I% BSA in washing buffer for I h, the blot was incubated with recombinant RC20 anti-phosphotyrosine peroxidase conjugate (1 : 2000) in washing buffer for I h at room temperature. After washing three times with washing buffer for IO min each, the signals of phosphotyrosine-containing proteins on the blot were visualised by ECL Western blotting assay system as described above.
3. Results
It is known that the binding of EGF to its receptor stimulates endocytosis and the receptor degradation within lysosomes using perfused liver system [I I]. In our experiment, after Chang liver cells were incubated with 100 ng/ml EGF for a varying period of time at 37°C. whole-cell extracts were immediately made by directly adding the boiling Laemmli sample buffer to the plate and analysed by ECL Western blotting with anti-EGF receptor antibody or anti-phosphotyrosine antibody. As shown in Fig. 1, the EGF-induced degradation of the 170 kDa EGF receptor was increased by extending the
E.-B. Yang et al. /J.
Biochem. Biophy.
A
Methods 32 (1996)
97-108
101
Time (min)
MW (kDa)
0 15 30 60 120 240 &”
.
/.
c 170 kDa EGF receptor
1601 1161 669 56-
B 1oo*t h .tE: E a, E 8
80 -““, 6o 40-
\
200 -0
40
80
incubation
120
160
Time
200
2’4
(min)
Fig. I. Time course of EGF-induced EGF receptor degradation in intact cells. After confluent cells were incubated with 100 ng/ml EGF for the indicated time, boiling Laemmli sample buffer was immediately added to the cells to make whole-cell extracts which were subjected to 7.5% SDS-PAGE and analysed by ECL Western blotting assay system using monoclonal anti-EGF receptor antibody as described in Section 2: Materials and methods. (A) ECL Western blotting assay. (B) densitometric scanning of the 170 kDa EGF receptor at 595 nm.
incubation time. It was also shown that the tyrosine phosphorylation of the 170 kDa EGF receptor was initially increased by extending the incubation time (Fig. 2). However, the maximum level was attained by 10 min, after which phosphorylation started to decrease. The EGF-induced phosphotyrosine-containing proteins with molecular masses of 150, 116, 85, 64, 56,49 and 45 kDa were also identified in intact Chang liver cells by this assay system. 3.2. Effects of EGF concentration rylation in intact cells
on EGF receptor degradation
and tyrosine phospho-
Using the present assay system, it was found that the degradation and autophosphorylation of EGF receptor induced by EGF in intact cells were dose-dependent. When Chang liver cells were incubated with various concentrations of EGF for 2 h at 37”C, it was found that the degradation of EGF receptor was enhanced with increasing EGF concentration (Fig. 3). The concentration resulting in 50% degradation of EGF receptor was about 6 ng/ml. The autophosphorylation of EGF receptor on tyrosine residues was also increased with increasing EGF concentration in cultured cells which were incubated
102
A Time
MW (kDa)
2
'
5
(min) 10
30
60 120 170kOa
180116-
EGF
* c receptor *
_
6658-
B loor!,
I / ‘; 80
..
60 A 40 1
0
/I
0
--j L--L.,
20
40
60
80
GOA 120
Incubation Time (min) Fig. 2. Time course of EGF-induced EGF receptor tyrosine phosphorylation in intact cells. Boiling Laemmli sample buffer was directly added to cultured cells which had been preincubated with 100 ng/ml EGF for the indicated time. Whole-cell extracts were applied to 7.5% SDS-PAGE and analysed by ECL Western blotting assay system using recombinant RCX anti-phosphotyrosine peroxidase conjugate as described in Section 2: Materials and Methoda. (A) ECL Western blotting assay. (B) densitometric scanning of the 170 kDa EGF receptor at 595 nm.
with EGF for 5 min (Fig. 4). In this case, the concentration autophosphorylation of EGF receptor was approx. 12 ng/ml.
of EGF, resulting
3.3. Sen.sitil,itJ of’ ECL Western blotting us.wy s?stem ,for the Identification
in 50%
qf
EGF
receptor arid EGF-induced phosphot~rosine-L.ontaiilirlR proteirls
The results in Figs. 5 and 6 showed that ECL Western blotting assay system using monoclonal anti-EGF receptor and anti-phosphotyrosine antibodies for the identification of EGF receptor and EGF-induced phosphotyrosine-containing proteins with various protein concentrations was very sensitive and appeared to be linear. Using the assay system, 20 ~g sample protein from whole-cell extracts was sufficient for the detection of EGF receptor and phosphotyrosine-containing proteins. The amounts of the 170 kDa EGF receptor, a native EGF receptor [51, and its tyrosine phosphorylation were proportional to the amount of loaded protein up to 100 pg by using ECL Western blotting assay system as shown in Figs. 5 and 6. respectively. In order to compare the
E.-B. Yang et al/J.
Biochem. Biophys. Methods 32 (19961 97-108
EGF Concentration
103
(ng/ml)
EGF Concentration (ng/ml) Fig. 3. Effect of EGF concentration on EGF-induced EGF receptor degradation in intact cells. After confluent cells were incubated with the indicated concentrations of EGF for 2 h, boiling Laemmli sample buffer was added to make whole-cell extracts which were then analyzed by ECL Western blotting system using monoclonal anti-EGF receptor antibody for the determination of EGF receptor degradation. (A) ECL Western blotting assay, (B) densitometric scanning of the 170 kDa EGF receptor at 595 nm.
sensitivity between ECL Western blotting assay system and radioisotope-labelled antibody, 1 &i ‘251-anti-mouse IgG antibody was used to detect EGF receptor or EGF-induced phosphotyrosine-containing proteins after the blots were incubated with anti-EGF receptor antibody or anti-phosphotyrosine antibody. It was found that the signals were very weak although the blots were exposed to X-ray film over 4 days (data not shown as signals were too week to be shown on the picture).
4. Discussion At present, autoradiography with immunoprecipitation is the main method for the determination of EGF receptor and EGF-induced phosphotyrosine-containing proteins [ 12,131. In this method, EGF receptor or phosphotyrosine-containing proteins in wholecell extracts or the plasma membrane are immunoprecipitated, subjected to Western blot analysis using anti-EGF rece tor or anti-phos hotyrosine antibody. The immunological D P reaction is visualised using I2 I-protein A or I2 I-second antibody followed by autoradio-
(ng/ml)
EGF Concentration 0
0.39
1.56 6.25
25
100
MW (kDa) ,170 kDa EGF receptor
B 100
.l
__/’ ..,’
80
__-. _*’
60.
,. ,’
,
’
40-
0
IO
20
30
40
50
60
70
EGF Concentration Fig. 4. Effect of EGF concentration on EGF-induced
80
90
100
(ng/ml)
EGF receptor tyrosine phosphorylation in intact cella. For
detecting the effect of EGF concentration on ECF receptor tyrosine phosphorylation, after confluent cells were incubated with various concentrations of EGF for 5 min. boiling Laemmli buffer was immediately added to make whole-cell extracts. These extracts were subjected to 7.5% SDS-PAGE
and analyzed by ECL Western
blotting system using recombinant
conjugate.
RC20
anti-phosphotyrobine
peroxidase
(A)
ECL
Western
blotting a&say. (B) densitometric scanning of the I70 kDa EGF receptor at 595 nm.
graphy. Although a sensitive material like “51-protein A or “‘l-second antibody is used, immunoprecipitation is often required to eliminate the non-specific binding and to increase the sensitivity further because the concentration of EGF receptor or phosphotyrosine-containing proteins is usually very low in the cells. 3’P incorporation is another method commonly used for the identification of phosphotyrosine-containing proteins. In this method. ” P-labelled phosphotyrosine-containing proteins are immunoprecipitated. separated on SDS-PAGE, and autoradiographed. This method has serious drawbacks when used for routine application. A very high concentration (about 0.5 mCi/ml) of [3’P]phospllate is needed to label all phosphorylated proteins. including phosphotyrosine-containing proteins which account for only 0.05% of the acid-soluble phospho-proteins in animal cells [14]. In addition, during immunoprecipitation, some phosphorylated proteins may be digested by proteases or dephosphorylated by phosphatases which make it difficult to accurately measure EGF receptor and EGF-induced tyrosine phosphorylation in intact cells. ECL Western blotting using specific antibodies is a highly sensitive method for the identification of specific proteins. EGF receptor in the plasma membrane purified from
E.-B. Yang et al. /J.
Biochem. Biophy. Methods 32 (19961 97-108
A
Sample 10
20
40
Protein 60
105
(pg) 80
100
MW (kDa) 180116 66
-
‘“Z__
,170 kDa EGF receptor
-
Sample Protein (pg) Fig. 5. Detection of EGF receptor by ECL Western blotting using monoclonal anti-EGF receptor antibody in whole-cell extracts from Chang liver cells. (A) ECL Western blotting assay. (B) densitometric scanning of the 170 kDa EGF receptor at 595 nm.
normal rat liver tissue has been detected by ECL Western blotting using monoclonal anti-EGF receptor antibody [1.5]. In this report, we describe a simple, highly sensitive and rapid ECL Western blotting assay system for the detection of the EGF-induced EGF receptor degradation and tyrosine phosphorylation in intact cells. Chang liver cells, which were found to express EGF receptor (unpublished data), were used in the experiments. The present assay system is apparently much more sensitive than conventional methods using immmunoprecipitation and autoradiography to detect EGF receptor and phosphotyrosine-containing proteins. 20 pg protein from whole Chang liver cell extracts is sufficient for the purposes. With the same amount of sample protein, it would be difficult to detect EGF receptor and EGF-induced phosphotyrosine-containing proteins in whole-cell extracts using ‘251-anti-mouse IgG antibody. In addition, this assay system is linear up to 100 pg protein from cell extracts, so the results could be compared quantitatively. It is known that phosphatases and proteases are present in the cells. Till now, no specific inhibitor of phosphatase or protease has been found to be able to inhibit all phosphatase or protease activities. It is therefore very difficult to accurately determine tyrosine phosphorylation induced by EGF using the immunoprecipitation method although some phosphatase and protease inhibitors are added during cell lysis and the immunoprecipitation. The present method does not encounter this problem because, after the cells are incubated with EGF for the indicated time, the addition of boiling Laemmli
E.-B.
106
Yang et a/./J.
Biodwm.
Sample
A MW (kDa)
10
20
Biophn.
Mrtlmh
Protein
(pg)
40
60
32 f IYY6I
Y7- IOX
80 100 t 170 kDa EGF receptor
66 58 -
B
20
40
60
80
100
Sample Protein (pg) Fig. 6. Detection of EGF-induced anti-phosphotyrosinr
tyrosine phosphorylation by ECL Western blotting using recombinant RC20
peroxidase conjugate in whole-cell extracts from Chang liver cells. (A) ECL Western
blotting assay. (B) densitometric scanning of the 170 kDa EGF receptor at 595 nm.
sample buffer to intact cells in culture plates immediately results in denaturation of all proteins including phosphatases and proteases and stops all reactions in the cells. In addition, this assay system is used to detect not only the EGF-induced EGF receptor degradation and autophosphorylation but also other phosphotyrosine-containing proteins induced by EGF in intact cells. It is therefore a better assay system for the finding of natural substrates phosphorylated by EGF receptor tyrosine kinase. It has been found that EGF receptor was internalized by the stimulation of EGF binding. This result was detected by using immunohistochemistry and counting “51-EGF in the cells [3,4]. It was reported that EGF increased the rate of disappearance of the 170 kDa EGF receptor from EGF-perfused rat liver [5]. These methods are unable to accurately detect the process of the EGF-induced EGF receptor degradation as it is very difficult to control the reaction conditions. Using the present assay system, excellent results on the effects of EGF concentration and incubation time on the EGF-induced degradation of EGF receptor are easily obtained. We have recently adopted this system for successfully assaying the effects of other factors on the degradation of EGF receptor in intact cells (unpublished data). In conclusion, the present assay system for the determination of the EGF-induced EGF receptor degradation and tyrosine phosphorylation shows several advantages. It is
E.-B. Yang et al./.I.
Biochem. Biophys. Methods 32 (19%)
97-108
107
(1) very sensitive; (2) safe as it avoids the use of radioisotopes; (3) easy to handle; (4) very fast because immunoprecipitation and autoradiography are not necessary; and (5) accurate in detecting the effects of EGF on EGF receptor and its tyrosine phosphorylation as well as other proteins associated with EGF receptor in intact cells. This assay system is very useful for the studies of signal transduction induced by EGF. In addition, it may be adopted for the studies of other growth factor receptors using appropriate anti-growth factor receptor antibodies.
5. Simplified description of the method and its (future) applications This report describes a simple, sensitive, and non-isotopic assay system for the identification of the EGF-induced EGF receptor degradation and tyrosine phosphorylation in intact cells. EGF receptor and phosphotyrosine-containing proteins induced by EGF are identified by ECL Western blotting using monoclonal anti-EGF receptor antibody and recombinant RC20 anti-phosphotyrosine peroxidase conjugate, respectively. Since boiling Laemmli sample buffer is directly added to cultured cells to stop the reactions in the cells and to make whole-cell extracts, the effects of incubation time and EGF concentration on EGF receptor degradation and tyrosine phosphorylation in intact cells can be more accurately determined. This method may be adopted for the studies of other growth factor receptors using appropriate anti-growth factor receptor antibodies.
Acknowledgements This work was supported by grants from the National (RR9003 18) and Singapore Totalisator Board.
University
of Singapore
References 111Fantl, W.J., Johnson, Biochem.,
D.E. and Williams, 62 (1993) 453-481.
121 Ulrich, A. and Schlessinger, (1990) 203-212.
L.T., Signalling
J.. Signal transduction
by receptor
by receptors
tyrosine
with tyrosine
kinases.
Annu.
kinase activity.
Rev.
Cell, 61
[31 Wiley, H.S., Herbst, J.J.. Walsh, B.J., Lauffenburger, D.A., Rosenfeld, M.G. and Gill, G.N. The role of tyrosine kinase activity in endocytosis, compartmentation, and down-regulation of the epidermal growth factor receptor. J. Biol. Chem., 266 (1991) 11083-11094. 141 Felder, S., Miller, K., Moehren, G.. Ulrich, A., Schlessinger, J. and Hopkins, C.R., Kinase activity controls the sorting of the epidetmal growth factor receptor within the multivesicular body. Cell, 61 (1990) 623-634. [51 Cohen, S., Ushiro, H., Stoscheck, C. and Chinkers, M., A native 170,000 epidermal gowth factor receptor-kinase complex from shed plasma membrane vesicles. J. Biol. Chem., 257 (1982) 1523-1531. [61 Carpenter, Cl., King, L.Jr. and Cohen, S., Rapid enhancement of protein phosphorylation in A-431 cell membrane preparations by epidermal growth factor. J. Biol. Chem., 254 (1979) 4884-4891. 171Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallis, A.K., Gartner, F.H., Provenzano, M.D., Fujimoto,
108
[8] [9]
[IO]
[I I] [ 121
[13]
[l4] [15]
E.-B. Yung et cd. / .I. Biochrm.
BioplzFs. Merhods 32
CI9961Y7-
108
E.K.. Goeke. N.M.. Olson, B.J. and Klenk, D.C.. Measurement of protein using bicinchoninic acid. Anal. B&hem. 150 (1985) 76-85. Laemmli, U.K.. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227 (1970) 680-685. Bjerrum. O.J. and Schafer-Nielsen. C., Buffer systems and transfer parameters for semidry electroblotting with a horizontal apparatus. In Electrophoresis ‘86 (Dunn M.J.. ed.). VCH-Verlag, Weinheim, Germany. (1986) pp. 315-327. Kyhse-Andersen. J.. Semidry electroblotting-Transfer using equipment without buffer vessel, In CRC Handbook of Immunoblotting of Proteins. Vol. I. (Bjerrum, O.J. and Heegaard. N.H.H. eds.), CRC Press. Florida. USA. (1988) pp. 79-85. Dunn, W.A., Connolly, T.P. and Hubbard. A.L., Receptor-mediated endocyto& of epidermal growth factor by rat hepatocytes: receptor pathway. J. Cell Biol., 102 (1986) 24-36. Campos-Gonzalez. R. and Glenney, J.R.Jr., Tyrosine phosphorylation of mitogen-activated protein kinase in cells with tyrosine kinase-negative epidermal growth factor receptors. J. Biol. Chem., 267 (1992) 14535-14538. Lichtner, R.B.. Wiedemuth, M.. Kittmann, A.. Ulrich, A.. Schirrmacher, V. and Khazaie, K., Ligand-induced activation of epidermal growth factor receptor in intact rat mammary adenocarcinoma cells without detectable receptor phosphorylation. J. Biol. Chem.. 267 (1992) 11872-l 1880. Wang, J.Y.J., Nuclear protein tyrosine kinases. TIBS, I9 (1994) 373-376. Yang, E.B.. Mack. P. and Cheng, L.Y.. Studies of epidermal growth factor receptor in plasma membrane from rat liver and hepatoma tissues. Biochem. Mol. Biol. Int.. 33 (1994) 22 I-228.
ELSEiVIER
biochemical and biophysical methods J. B&hem.
Biophys. Methods 32 (1996) 97-108
Research article
Detection of EGF-induced EGF receptor degradation and tyrosine phosphorylation in intact cells Er-Bin Yang aqb,Dong-Fang Wang a, Peter Mack b, Li-Yao Cheng a3* ’ Department of Biochemistry, Faculty of Medicine, National Unicersi@ of Singapore, 10 Kent Ridge Crescent, Singapore 119260 b Department of Surgery. Singapore General Hospital, Outram Road, Singapore 169608 Received 30 September
1995; revised 30 January
1996; accepted 8 February
1996
Abstract In this work, a simple, sensitive, and non-isotopic assay system for the detection of EGF-induced EGF receptor degradation and tyrosine phosphorylation in intact cells is described. In this system, boiling Laemmli sample buffer was directly added to cultured Chang liver cells to stop the reactions in the cells stimulated by EGF and to make whole-cell extracts. The effects of EGF concentration and incubation time on the EGF-induced degradation and tyrosine phosphorylation of EGF receptor were successfully determined using monoclonal anti-EGF receptor, recombinant anti-phosphotyrosine peroxidase conjugate, and enhanced chemiluminescence (ECL) Western blotting assay system. Unlike other assay systems, the use of radioisotopes was avoided in this determination. The assay system is linear up to 100 pg sample protein from whole-cell extracts for the detection of EGF receptor and EGF-induced autophosphorylation. This assay may be easily adopted for identification of other growth factor receptors and phosphotyrosine-containing proteins in intact cells, using appropriate anti-growth factor receptor antibodies. Keywords: EGF receptor;
Tyrosine
phosphorylation;
ECL Western blotting
Abbreviations: BCA, bicinchoninic acid; BSA, bovine serum albumin: ECL, enhanced chemiluminescence; EGF, epidermal growth factor; EGTA, ethyleneglycolbis(g-aminoethyl ether&N, N, N’, N’-tetraacetic acid; FCS, fetal calf serum; IGF-1, insulin-like growth factor-l; MEM, minimum essential medium Eagle; PBS, phosphate-buffered saline; PDGF, platelet-derived growth factor: PMSF, phenylmethylsulfonyl fluoride: SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis * Corresponding author. Fax: (65) 7791453. 0165-022X/96/$15.00 0 1996 Elsevier Science B.V. All rights reserved PII SO1 65-022X(96)00003-6
1. Introduction It is known that growth factor receptors on the cell surface with tyrosine kinase activity. such as epidermal growth factor (EGF) receptor, insulin receptor, insulin-like growth factor- 1 (IGF- 1) receptor and platelet-derived growth factor (PDGF) receptor, play a key role in the regulation of cell growth and differentiation. The binding of growth factors to their specific receptors activates receptor tyrosine kinases which undergo self-phosphorylation and subsequently phosphorylate various cellular protein substrates on tyrosine residues to carry out signal transduction [ 1.21. It has been reported that, when EGF-EGF receptor complex is formed by EGF binding to the extracellular domain of EGF receptor on target cells, it is rapidly concentrated in clathrin-coated pits. This complex is rapidly internalized. transits through the endosomal system. and is degraded within the lysosomal compartment. These results were detected by immunohistochemical localization and counting lZ51-EGF in the cells [3,4]. It has also been reported that EGF increased the rate of disappearance of the 170 kDa EGF receptor from EGF-perfused rat liver [5]. This assay system is unable to accurately detect the process of EGF receptor degradation induced by EGF because it is very difficult to control the reaction conditions, including EGF concentration and incubation time. It was found that the addition of EGF to the plasma membrane of A431 cells, which overexpressed EGF receptor. resulted in the stimulation of membrane phosphorylation to a maximal level by 2.5 min and the phosphorylation was decreased by extending incubation time [6]. However, EGF receptor autophosphorylation and the internalization and degradation of EGF-EGF receptor complex may play a very important role in the regulation of signal transduction. Unfortunately, no rapid, safe and efficient assay system for the detection of the EGF-induced EGF receptor degradation and tyrosine phosphorylation in intact cells has been reported. In this report, we describe an assay system for the determination of the EGF-induced EGF receptor degradation and tyrosine phosphorylation in whole-cell extracts. ECL Western blotting assay system is used to directly detect EGF receptor and EGF-induced phosphotyrosine-containing proteins in intact cells. This system is very simple, rapid and safe. It is also very sensitive and does not require immunoprecipitation and radioisotope. In addition, as boiling Laemmli sample buffer for sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) is directly added to cultured cells to stop the reactions and to make whole-cell extracts, it is very easy to accurately control the reaction time.
2. Materials and methods 2. I. Materials A Chang liver cell line from normal human tissue was purchased from ATCC (Rockville, MD, USA). EGF (E32641, monoclonal anti-EGF receptor antibody (clone F4. E3 138) minimum essential medium Eagle (MEM, M7399), and bicinchoninic acid
E.-B. Yang et al. /J.
Biochem. Biophys. Methods 32 (I 9961 97- 108
99
(BCA) protein assay kit (BCA-1) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Enhanced chemiluminescence (ECL) Western blotting analysis system (RPN 2108) and Hyperfilm-ECL (RPN 2103) were obtained from Amersham (Buckinghamshire, UK). Recombinant RC20 anti-phosphotyrosine peroxidase conjugate (E 120H) was purchased from Affiniti (Nottingham, UK). Nitrocellulose membrane (401196) was purchased from Schleicher and Schuell (Dassel, Germany). 2.2. Cell culture Chang liver cells from normal human tissue were cultured in MEM containing 100 units/ml penicillin, 100 ,ug/ml streptomycin, and 10% heat-inactivated fetal calf serum (FCS) in an atmosphere of 95% sir/5% CO, at 37°C. Confluent cells were trypsinized and transferred either to a 12-well plate (lo5 cells/well) for detecting the effects of EGF on EGF receptor and EGF-induced phosphotyrosine-containing proteins in intact cells or to a 260 ml tissue culture flask for extracting EGF receptor and EGF-induced phosphotyrosine-containing proteins from the cultured cells. 2.3. Extraction of EGF receptor and EGF-induced from cultured cells
phosphoerosine-containing
proteins
Confluent cells in a 260 ml tissue culture flask were washed twice with 20 ml of 50 mM Hepes, pH 7.5, containing 150 mM NaCl, 1 mM ethyleneglycolbis@aminoethyl ether)-N, N, N’, N’-tetraacetic acid (EGTA), and 10% glycerol, scraped into 20 ml of the same buffer using a rubber policeman, and centrifuged at 600 X g for 10 min at 4°C. The cells were lysed in solubilization solution (10’ cells/ml) containing 50 mM Hepes, pH 7.5, 1% Triton X-100, 10% glycerol, 1.5 mM MgCl?, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 20 pg/ml leupeptin, and homogenized in a glass Dounce homogenizer. The insoluble material was removed by centrifugation at 40000 X g for 30 min at 4°C. Protein concentration in the supernatant was estimated with BCA protein assay kit as described by Smith et al. [7]. The EGF-induced phosphotyrosine-containing proteins were obtained from the cells which had been pretreated with 100 ng/ml EGF at 37°C for 5 min using the same method described above. All extracts were stored at -70°C prior to analysis. 2.4. Identification
of EGF receptor in intact cells
In order to directly identify EGF receptor in intact cells, confluent cells in a 12-well plate were washed twice with 1 ml of serum-free medium containing 0.1% bovine serum albumin (BSA) and 2 mM glutamine. They were then incubated in 0.5 ml of the same medium with or without EGF for the indicated time or with various EGF concentrations for 2 h at 37°C. After draining off the reaction solution, 100 ~1 of boiling Laemmli sample buffer containing 125 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, and 10% 2-mercaptoethanol was immediately added to the cells to stop the reactions and to make whole-cell extracts which were subsequently transferred to a microfuge tube and boiled for 5 min. 30 ~1 of the extracts was applied to 7.5% SDS-PAGE 181. Proteins separated
by 7.5% SDS-PAGE were electrophoretically transferred to nitrocellulose membrane. A semi-dry electroblotting method in a AE-6675 Horiblot apparatus (Atto Corporation, Tokyo, Japan) was used [9.10]. Non-specific binding sites on the blot were blocked by incubating with I% BSA in washing buffer containing 10 mM Tris-HCI. pH 7.5, 100 mM NaCI. and 0. I% Tween 20 at room temperature for I h. The blot was then incubated with monoclonal anti-EGF receptor antibody in washing buffer (I : 2000) at room temperature for I h. The blot was washed for 10 min with three changes of washing buffer and then incubated with anti-mouse IgG peroxidase conjugate in washing buffer (I : 2000) at room temperature for I h. After washing three times with washing buffer. EGF receptor binding with anti-EGF receptor antibody was identified by ECL Western blotting assay system according to the manufacture’s instruction. An equal volume of ECL detection solution I was mixed with detection solution 2 before use. This mixture was directly added to the blot which was subsequently incubated for I min at room temperature and immediately wrapped in SaranWrap. The signals on the blot were visualised by exposing to Hyperfilm-ECL for 1.5-30 s and scanned by a densitometer (Shimadzu. CS-9000. Japan) at 595 nm. 2.5. Idrnt~ficutiot~
of’ EGF-itlduced plto.sl’ltot?~ositte-c.otltuitlitlg proteins in intact cells
Confluent cells in a I?-well plate were washed twice with 1 ml of serum-free medium containing 0. I % BSA and 2 mM glutamine and incubated with or without 100 ng/ml EGF in the same medium for the indicated time or with various EGF concentrations for 5 min at 37°C. Whole-cell extracts were made by adding 100 ~1 of boiling Laemmli sample buffer to each well. transferred to an Eppendorf tube, and boiled for 5 min. 30 ~1 of the extracts was loaded onto 7.5% SDS-PAGE. Separated proteins on the gel were electrophoretically transferred to nitrocellulose membrane. After non-specific binding sites were blocked with I% BSA in washing buffer for I h, the blot was incubated with recombinant RC20 anti-phosphotyrosine peroxidase conjugate (1 : 2000) in washing buffer for I h at room temperature. After washing three times with washing buffer for IO min each, the signals of phosphotyrosine-containing proteins on the blot were visualised by ECL Western blotting assay system as described above.
3. Results
It is known that the binding of EGF to its receptor stimulates endocytosis and the receptor degradation within lysosomes using perfused liver system [I I]. In our experiment, after Chang liver cells were incubated with 100 ng/ml EGF for a varying period of time at 37°C. whole-cell extracts were immediately made by directly adding the boiling Laemmli sample buffer to the plate and analysed by ECL Western blotting with anti-EGF receptor antibody or anti-phosphotyrosine antibody. As shown in Fig. 1, the EGF-induced degradation of the 170 kDa EGF receptor was increased by extending the
E.-B. Yang et al. /J.
Biochem. Biophy.
A
Methods 32 (1996)
97-108
101
Time (min)
MW (kDa)
0 15 30 60 120 240 &”
.
/.
c 170 kDa EGF receptor
1601 1161 669 56-
B 1oo*t h .tE: E a, E 8
80 -““, 6o 40-
\
200 -0
40
80
incubation
120
160
Time
200
2’4
(min)
Fig. I. Time course of EGF-induced EGF receptor degradation in intact cells. After confluent cells were incubated with 100 ng/ml EGF for the indicated time, boiling Laemmli sample buffer was immediately added to the cells to make whole-cell extracts which were subjected to 7.5% SDS-PAGE and analysed by ECL Western blotting assay system using monoclonal anti-EGF receptor antibody as described in Section 2: Materials and methods. (A) ECL Western blotting assay. (B) densitometric scanning of the 170 kDa EGF receptor at 595 nm.
incubation time. It was also shown that the tyrosine phosphorylation of the 170 kDa EGF receptor was initially increased by extending the incubation time (Fig. 2). However, the maximum level was attained by 10 min, after which phosphorylation started to decrease. The EGF-induced phosphotyrosine-containing proteins with molecular masses of 150, 116, 85, 64, 56,49 and 45 kDa were also identified in intact Chang liver cells by this assay system. 3.2. Effects of EGF concentration rylation in intact cells
on EGF receptor degradation
and tyrosine phospho-
Using the present assay system, it was found that the degradation and autophosphorylation of EGF receptor induced by EGF in intact cells were dose-dependent. When Chang liver cells were incubated with various concentrations of EGF for 2 h at 37”C, it was found that the degradation of EGF receptor was enhanced with increasing EGF concentration (Fig. 3). The concentration resulting in 50% degradation of EGF receptor was about 6 ng/ml. The autophosphorylation of EGF receptor on tyrosine residues was also increased with increasing EGF concentration in cultured cells which were incubated
102
A Time
MW (kDa)
2
'
5
(min) 10
30
60 120 170kOa
180116-
EGF
* c receptor *
_
6658-
B loor!,
I / ‘; 80
..
60 A 40 1
0
/I
0
--j L--L.,
20
40
60
80
GOA 120
Incubation Time (min) Fig. 2. Time course of EGF-induced EGF receptor tyrosine phosphorylation in intact cells. Boiling Laemmli sample buffer was directly added to cultured cells which had been preincubated with 100 ng/ml EGF for the indicated time. Whole-cell extracts were applied to 7.5% SDS-PAGE and analysed by ECL Western blotting assay system using recombinant RCX anti-phosphotyrosine peroxidase conjugate as described in Section 2: Materials and Methoda. (A) ECL Western blotting assay. (B) densitometric scanning of the 170 kDa EGF receptor at 595 nm.
with EGF for 5 min (Fig. 4). In this case, the concentration autophosphorylation of EGF receptor was approx. 12 ng/ml.
of EGF, resulting
3.3. Sen.sitil,itJ of’ ECL Western blotting us.wy s?stem ,for the Identification
in 50%
qf
EGF
receptor arid EGF-induced phosphot~rosine-L.ontaiilirlR proteirls
The results in Figs. 5 and 6 showed that ECL Western blotting assay system using monoclonal anti-EGF receptor and anti-phosphotyrosine antibodies for the identification of EGF receptor and EGF-induced phosphotyrosine-containing proteins with various protein concentrations was very sensitive and appeared to be linear. Using the assay system, 20 ~g sample protein from whole-cell extracts was sufficient for the detection of EGF receptor and phosphotyrosine-containing proteins. The amounts of the 170 kDa EGF receptor, a native EGF receptor [51, and its tyrosine phosphorylation were proportional to the amount of loaded protein up to 100 pg by using ECL Western blotting assay system as shown in Figs. 5 and 6. respectively. In order to compare the
E.-B. Yang et al/J.
Biochem. Biophys. Methods 32 (19961 97-108
EGF Concentration
103
(ng/ml)
EGF Concentration (ng/ml) Fig. 3. Effect of EGF concentration on EGF-induced EGF receptor degradation in intact cells. After confluent cells were incubated with the indicated concentrations of EGF for 2 h, boiling Laemmli sample buffer was added to make whole-cell extracts which were then analyzed by ECL Western blotting system using monoclonal anti-EGF receptor antibody for the determination of EGF receptor degradation. (A) ECL Western blotting assay, (B) densitometric scanning of the 170 kDa EGF receptor at 595 nm.
sensitivity between ECL Western blotting assay system and radioisotope-labelled antibody, 1 &i ‘251-anti-mouse IgG antibody was used to detect EGF receptor or EGF-induced phosphotyrosine-containing proteins after the blots were incubated with anti-EGF receptor antibody or anti-phosphotyrosine antibody. It was found that the signals were very weak although the blots were exposed to X-ray film over 4 days (data not shown as signals were too week to be shown on the picture).
4. Discussion At present, autoradiography with immunoprecipitation is the main method for the determination of EGF receptor and EGF-induced phosphotyrosine-containing proteins [ 12,131. In this method, EGF receptor or phosphotyrosine-containing proteins in wholecell extracts or the plasma membrane are immunoprecipitated, subjected to Western blot analysis using anti-EGF rece tor or anti-phos hotyrosine antibody. The immunological D P reaction is visualised using I2 I-protein A or I2 I-second antibody followed by autoradio-
(ng/ml)
EGF Concentration 0
0.39
1.56 6.25
25
100
MW (kDa) ,170 kDa EGF receptor
B 100
.l
__/’ ..,’
80
__-. _*’
60.
,. ,’
,
’
40-
0
IO
20
30
40
50
60
70
EGF Concentration Fig. 4. Effect of EGF concentration on EGF-induced
80
90
100
(ng/ml)
EGF receptor tyrosine phosphorylation in intact cella. For
detecting the effect of EGF concentration on ECF receptor tyrosine phosphorylation, after confluent cells were incubated with various concentrations of EGF for 5 min. boiling Laemmli buffer was immediately added to make whole-cell extracts. These extracts were subjected to 7.5% SDS-PAGE
and analyzed by ECL Western
blotting system using recombinant
conjugate.
RC20
anti-phosphotyrobine
peroxidase
(A)
ECL
Western
blotting a&say. (B) densitometric scanning of the I70 kDa EGF receptor at 595 nm.
graphy. Although a sensitive material like “51-protein A or “‘l-second antibody is used, immunoprecipitation is often required to eliminate the non-specific binding and to increase the sensitivity further because the concentration of EGF receptor or phosphotyrosine-containing proteins is usually very low in the cells. 3’P incorporation is another method commonly used for the identification of phosphotyrosine-containing proteins. In this method. ” P-labelled phosphotyrosine-containing proteins are immunoprecipitated. separated on SDS-PAGE, and autoradiographed. This method has serious drawbacks when used for routine application. A very high concentration (about 0.5 mCi/ml) of [3’P]phospllate is needed to label all phosphorylated proteins. including phosphotyrosine-containing proteins which account for only 0.05% of the acid-soluble phospho-proteins in animal cells [14]. In addition, during immunoprecipitation, some phosphorylated proteins may be digested by proteases or dephosphorylated by phosphatases which make it difficult to accurately measure EGF receptor and EGF-induced tyrosine phosphorylation in intact cells. ECL Western blotting using specific antibodies is a highly sensitive method for the identification of specific proteins. EGF receptor in the plasma membrane purified from
E.-B. Yang et al. /J.
Biochem. Biophy. Methods 32 (19961 97-108
A
Sample 10
20
40
Protein 60
105
(pg) 80
100
MW (kDa) 180116 66
-
‘“Z__
,170 kDa EGF receptor
-
Sample Protein (pg) Fig. 5. Detection of EGF receptor by ECL Western blotting using monoclonal anti-EGF receptor antibody in whole-cell extracts from Chang liver cells. (A) ECL Western blotting assay. (B) densitometric scanning of the 170 kDa EGF receptor at 595 nm.
normal rat liver tissue has been detected by ECL Western blotting using monoclonal anti-EGF receptor antibody [1.5]. In this report, we describe a simple, highly sensitive and rapid ECL Western blotting assay system for the detection of the EGF-induced EGF receptor degradation and tyrosine phosphorylation in intact cells. Chang liver cells, which were found to express EGF receptor (unpublished data), were used in the experiments. The present assay system is apparently much more sensitive than conventional methods using immmunoprecipitation and autoradiography to detect EGF receptor and phosphotyrosine-containing proteins. 20 pg protein from whole Chang liver cell extracts is sufficient for the purposes. With the same amount of sample protein, it would be difficult to detect EGF receptor and EGF-induced phosphotyrosine-containing proteins in whole-cell extracts using ‘251-anti-mouse IgG antibody. In addition, this assay system is linear up to 100 pg protein from cell extracts, so the results could be compared quantitatively. It is known that phosphatases and proteases are present in the cells. Till now, no specific inhibitor of phosphatase or protease has been found to be able to inhibit all phosphatase or protease activities. It is therefore very difficult to accurately determine tyrosine phosphorylation induced by EGF using the immunoprecipitation method although some phosphatase and protease inhibitors are added during cell lysis and the immunoprecipitation. The present method does not encounter this problem because, after the cells are incubated with EGF for the indicated time, the addition of boiling Laemmli
E.-B.
106
Yang et a/./J.
Biodwm.
Sample
A MW (kDa)
10
20
Biophn.
Mrtlmh
Protein
(pg)
40
60
32 f IYY6I
Y7- IOX
80 100 t 170 kDa EGF receptor
66 58 -
B
20
40
60
80
100
Sample Protein (pg) Fig. 6. Detection of EGF-induced anti-phosphotyrosinr
tyrosine phosphorylation by ECL Western blotting using recombinant RC20
peroxidase conjugate in whole-cell extracts from Chang liver cells. (A) ECL Western
blotting assay. (B) densitometric scanning of the 170 kDa EGF receptor at 595 nm.
sample buffer to intact cells in culture plates immediately results in denaturation of all proteins including phosphatases and proteases and stops all reactions in the cells. In addition, this assay system is used to detect not only the EGF-induced EGF receptor degradation and autophosphorylation but also other phosphotyrosine-containing proteins induced by EGF in intact cells. It is therefore a better assay system for the finding of natural substrates phosphorylated by EGF receptor tyrosine kinase. It has been found that EGF receptor was internalized by the stimulation of EGF binding. This result was detected by using immunohistochemistry and counting “51-EGF in the cells [3,4]. It was reported that EGF increased the rate of disappearance of the 170 kDa EGF receptor from EGF-perfused rat liver [5]. These methods are unable to accurately detect the process of the EGF-induced EGF receptor degradation as it is very difficult to control the reaction conditions. Using the present assay system, excellent results on the effects of EGF concentration and incubation time on the EGF-induced degradation of EGF receptor are easily obtained. We have recently adopted this system for successfully assaying the effects of other factors on the degradation of EGF receptor in intact cells (unpublished data). In conclusion, the present assay system for the determination of the EGF-induced EGF receptor degradation and tyrosine phosphorylation shows several advantages. It is
E.-B. Yang et al./.I.
Biochem. Biophys. Methods 32 (19%)
97-108
107
(1) very sensitive; (2) safe as it avoids the use of radioisotopes; (3) easy to handle; (4) very fast because immunoprecipitation and autoradiography are not necessary; and (5) accurate in detecting the effects of EGF on EGF receptor and its tyrosine phosphorylation as well as other proteins associated with EGF receptor in intact cells. This assay system is very useful for the studies of signal transduction induced by EGF. In addition, it may be adopted for the studies of other growth factor receptors using appropriate anti-growth factor receptor antibodies.
5. Simplified description of the method and its (future) applications This report describes a simple, sensitive, and non-isotopic assay system for the identification of the EGF-induced EGF receptor degradation and tyrosine phosphorylation in intact cells. EGF receptor and phosphotyrosine-containing proteins induced by EGF are identified by ECL Western blotting using monoclonal anti-EGF receptor antibody and recombinant RC20 anti-phosphotyrosine peroxidase conjugate, respectively. Since boiling Laemmli sample buffer is directly added to cultured cells to stop the reactions in the cells and to make whole-cell extracts, the effects of incubation time and EGF concentration on EGF receptor degradation and tyrosine phosphorylation in intact cells can be more accurately determined. This method may be adopted for the studies of other growth factor receptors using appropriate anti-growth factor receptor antibodies.
Acknowledgements This work was supported by grants from the National (RR9003 18) and Singapore Totalisator Board.
University
of Singapore
References 111Fantl, W.J., Johnson, Biochem.,
D.E. and Williams, 62 (1993) 453-481.
121 Ulrich, A. and Schlessinger, (1990) 203-212.
L.T., Signalling
J.. Signal transduction
by receptor
by receptors
tyrosine
with tyrosine
kinases.
Annu.
kinase activity.
Rev.
Cell, 61
[31 Wiley, H.S., Herbst, J.J.. Walsh, B.J., Lauffenburger, D.A., Rosenfeld, M.G. and Gill, G.N. The role of tyrosine kinase activity in endocytosis, compartmentation, and down-regulation of the epidermal growth factor receptor. J. Biol. Chem., 266 (1991) 11083-11094. 141 Felder, S., Miller, K., Moehren, G.. Ulrich, A., Schlessinger, J. and Hopkins, C.R., Kinase activity controls the sorting of the epidetmal growth factor receptor within the multivesicular body. Cell, 61 (1990) 623-634. [51 Cohen, S., Ushiro, H., Stoscheck, C. and Chinkers, M., A native 170,000 epidermal gowth factor receptor-kinase complex from shed plasma membrane vesicles. J. Biol. Chem., 257 (1982) 1523-1531. [61 Carpenter, Cl., King, L.Jr. and Cohen, S., Rapid enhancement of protein phosphorylation in A-431 cell membrane preparations by epidermal growth factor. J. Biol. Chem., 254 (1979) 4884-4891. 171Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallis, A.K., Gartner, F.H., Provenzano, M.D., Fujimoto,
108
[8] [9]
[IO]
[I I] [ 121
[13]
[l4] [15]
E.-B. Yung et cd. / .I. Biochrm.
BioplzFs. Merhods 32
CI9961Y7-
108
E.K.. Goeke. N.M.. Olson, B.J. and Klenk, D.C.. Measurement of protein using bicinchoninic acid. Anal. B&hem. 150 (1985) 76-85. Laemmli, U.K.. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227 (1970) 680-685. Bjerrum. O.J. and Schafer-Nielsen. C., Buffer systems and transfer parameters for semidry electroblotting with a horizontal apparatus. In Electrophoresis ‘86 (Dunn M.J.. ed.). VCH-Verlag, Weinheim, Germany. (1986) pp. 315-327. Kyhse-Andersen. J.. Semidry electroblotting-Transfer using equipment without buffer vessel, In CRC Handbook of Immunoblotting of Proteins. Vol. I. (Bjerrum, O.J. and Heegaard. N.H.H. eds.), CRC Press. Florida. USA. (1988) pp. 79-85. Dunn, W.A., Connolly, T.P. and Hubbard. A.L., Receptor-mediated endocyto& of epidermal growth factor by rat hepatocytes: receptor pathway. J. Cell Biol., 102 (1986) 24-36. Campos-Gonzalez. R. and Glenney, J.R.Jr., Tyrosine phosphorylation of mitogen-activated protein kinase in cells with tyrosine kinase-negative epidermal growth factor receptors. J. Biol. Chem., 267 (1992) 14535-14538. Lichtner, R.B.. Wiedemuth, M.. Kittmann, A.. Ulrich, A.. Schirrmacher, V. and Khazaie, K., Ligand-induced activation of epidermal growth factor receptor in intact rat mammary adenocarcinoma cells without detectable receptor phosphorylation. J. Biol. Chem.. 267 (1992) 11872-l 1880. Wang, J.Y.J., Nuclear protein tyrosine kinases. TIBS, I9 (1994) 373-376. Yang, E.B.. Mack. P. and Cheng, L.Y.. Studies of epidermal growth factor receptor in plasma membrane from rat liver and hepatoma tissues. Biochem. Mol. Biol. Int.. 33 (1994) 22 I-228.