BIOCHEMICAL
Vol. 171, No. 2, 1990 September
AND BIOPHYSICAL
14, 1990
RESEARCH COMMUNICATIONS Pages 882-889
ANTIBODY-INDUCED ACTIVATION OF THE EPIDERMAL GROWTH FACTOR RECEPTOR TYROSINE REQUIRES THE PRESENCE OF DETERGENT
KINASE
Marcel Spaargarenly2*, Libert H.K. Defize’, Siegfried W. de Last’, and Johannes Boonstra2 ‘Hubrecht Laboratory, Netherlands Imtitute for Developmental Biology, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands 2Depan?nent of Molecular Cell Biology, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands Received
July
26,
1990
SUMMARY: Activation of the epidermaI growth factor receptor (EGF-R) tyrosine kinase was investigated in membrane preparations as well as intact A431 cells, using anti-EGF-R antibodies directed against extra- and intracellular receptor domains. In vitro assay conditions were mimicked on whole cells by a mild detergent treatment. We show that, irrespective of the recognition site on the EGF-R, antibodies induce EGF-R autophosphorylation and tyrosine kinase activity towards other endogenous and exogenous substrates, but only when detergent is present. We propose that the primary effect of detergent is to create conditions in the lipid environment of the EGF-R that allow antibodies to induce receptorreceptor interactions necessary for tyrosine kinase activation. 01990 Academic press, Inc.
The EGF-R is a transmembrane glycoprotein of 170 kD with an extracellular ligand binding domain, a single hydrophobic transmembrane stretch and an intracellular domain containing protein tyrosine kinase activity (1,2). Binding of EGF to its receptor enhances the tyrosine kinase activity, thus inducing tyrosine phosphorylation of several protein substrates including the EGF-R itself (3). Using EGF-R mutants, the protein tyrosine kinase activity was shown to be required for all receptor-mediated responses involved in mitogenic signalling (4). How extracellular EGF binding activates the intracellular EGF-R
tyrosine
kinase remains to be elucidated, but a number of recent data support an intermolecular activation mechanism, in which EGF binding causes a shift in a hypothetical equilibrium between monomeric and dimeric receptors. Dimerization of EGF-R then results in activation of the tyrosine kinase ($6). Yarden and Schlessinger have demonstrated
a reversible EGF-R
*To whom correspondence should be addressed. 0006-291X/90 $1.50 Copyright 0 1990 by Academic Press. All rights of reprudurkwz in any form
Inc. reserved.
882
oligomerization
after EGF
Vol.
171,
No.
treatment
2,
1990
BIOCHEMICAL
AND
of solubilized immunoaffinity
BIOPHYSICAL
RESEARCH
purified EGF-receptors
COMMUNICATIONS
(7,8). EGF-induced
EGF-R dimerization
could also be detected by chemical cross-linking in solubilized
EGF-R preparations (l&12). Furthermore,
(9), membrane preparations (lo), as well as in intact cells bivalent antibodies were able to induce EGF-R autophospho-
rylation and tyrosine kinase activation in in vitro phosphorylation assays, probably by inducing EGF-R dimerization (7,13,14). However, despite their bivalent nature, antibodies were not able to activate the EGF-R kinase in intact cells (12,14). Thus, apparently a discrepancy exists between the ability of the antibodies to activate the EGF-R tyrosine kinase in vitro versus in vivo (15). In this study we demonstrate
that anti-EGF-R
antibodies,
directed against
extra- and intracellular EGF-R epitopes, are able to activate the EGF-R tyrosine kinase in membrane preparations as well as in intact cells, provided a small amount of detergent is present. These data confirm and extend previous data (7,13,14), and provide additional
support for an intermolecular
mechanism of EGF-R
tyrosine
kinase activation. MATERIALS
AND METHODS
EGF (receptor grade) wa;20btained from Collaborative Research (Waltham, MA, USA), [y- P]ATP and [ PI-Orthophosphate from Amersham International (‘s Hertogenbosch, The Netherlands), Protein A-Sepharose from Pharmacia LKB Biotechnology Inc., and Plastic foil thin layer cellulose plates (5577) were from Merck. A431 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 7.5% Fetal Calf Serum and buffered with 40 mM NaHC03, under a 7.5% CO, atmosphere. Monoclonal antibody 2E9 and polyclonal antiserum 281-7 were prepared and IgG purified as previously described (13). Membrane preparations were prepared by vesiculation of A431 cells essentially as described (16). The EGF-R was immunoprecipitated using antiserum 281-7 as previously described (12). EGF-R autophosphorylation and exogenous substrate phosphorylation in membrane preparations. A431 membrane preparations (20 pg) were incubated with
ligand for 20 min at room temperature, phosphorylation reaction was started by addition of phosphorylation assay mix containing 15 PM [T-~~P]ATP (5 ,Ki), with or without 0.2% NP-40, and analyzed as described (13). After 5-15% SDS-PAGE, gel bands were cut out in order to quantitate radioactivity by liquid scintillation. 2-D phosphoamino acid analysis was performed as described (12). In order to measure exogenous substrate phosphorylation, the phosphorylation reaction was started after ligand incubation by the addition of phosphorylation assay mix, containing 15 .uM [r-32P]ATP (1 &i) and 2 mM Angiotensin I, with or without 0.2% NP-40. After 10 min on ice, the reaction was stopped and exogenous substrate phosphorylation was determined as described (13). Aspecific background labelling was corrected by substracting values of identical reactions without Angiotensin I. EGF-R autophosphotylation
and endogenous substrate phosphorylation
in cells.
Assays were performed in A431 cells, essentially as described (17). Briefly, cells, grown to subconfluency in 12-well tissue culture clusters (Costar), were incubated with antibody for 20 min at room temperature in DMEM/Hepes (10 mM). After 883
Vol.
171,
No.
2,
1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
this incubation cells were washed two times with cold PBS and once with cold permeabilization buffer containing 20 mM Hepes (pH 7.4), 145 mM NaCl, 5.4 mM KU, 1 mM CaC12, 2 mM MgCl,, 2 mM MnCl,, 20 PM Na3V0,, 10 mM PNPP, 1 mM PMSF. Phosphotylation was started b addition of permeabilization buffer containing 0.005% Saponin and 15 PM [y- x P]ATP (5 &i) and where indicated EGF, for 10 min on ice. The reaction was stopped by washing twice with cold Ca2+ and Mg2+-free PBS containing 200 PM Na,VO, followed by the addition of sample buffer. Substrate phosphorylation was measured after 5-15% SDS-PAGE by scanning of the autoradiograph on a Joyce-Loebl Chromoscan 3 apparatus. RESULTS Earlier studies have shown that anti-EGF-R antibodies can enhance the EGF-R autophosphorylation in isolated plasma membranes or purified receptor preparations, but not in intact cells (7,12-15).
We have investigated
this phenomenon
by
comparing the effects of EGF with those of monoclonal antibody 2E9, directed against the extracellular EGF-R domain and competing for EGF binding to low affinity receptors, and polyclonal antiserum 281-7, directed against the cytoplasmic EGF-R domain (a.a. 984-996). Since in vitro phosphorylation assays are usually carried out in the presence of detergent, it was investigated whether this influences antibody-induced EGF-R activation, and we mimicked these conditions on intact cells by adding low concentrations
of saponin.
As shown in Fig. 1, EGF induces EGF-R phosphorylation in isolated membrane preparations regardless of the presence of detergent. In contrast, 2E9 and 281-7 are able to enhance EGF-R phosphorylation under these conditions only if a detergent like NP-40 is present in the assay mixture. In isolated membrane preparation
only
low affinity EGF-R are detectable (18) and thus 2E9 antagonized EGF-induced EGF-R phosphorylation. Interestingly, simultaneous addition of antiserum 281-7 and EGF resulted in an approximately additive effect on EGF-R phosphorylation. In all cases EGF-R phosphorylation
occurred specifically on tyrosine residues, as shown
by 2-D phosphoamino
acid analysis for EGF and 2E9 (Fig. 1B). These results
demonstrate that the antibodies requires the EGF and anti-EGF-R addition they show that
enhancement of EGF-R phosphorylation by anti-EGF-R presence of detergent. The molecular mechanisms by which antibodies exert their effects are thus not identical. In the activation of the EGF-R by antibodies does not depend
on the receptor epitope to which these antibodies are directed. It has recently become clear that enhanced EGF-R phosphorylation
is not
necessarily indicative for receptor autophosphorylation, but can also occur through cross-phosphoryl~ltion of non-active receptors by active kinase receptors (12.19-22). To discriminate between antibody-induced kinase activation and facilitated intermolecular cross-phosphorylation we analysed the capacity of EGF and antibodies 884
Vol.
171,
No.
2,
1990
BIOCHEMICAL
-
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
NP-40
A 2E9 m
281-7
C
-EGF
C
2E9
281-7
+EGF
EGF
2E9
B Fig. 1. Effect of detergent on antibody-induced EGF-R autophosphorylation in membranes. A431 membranes were incubated with buffer (C) or the indicated antibodies (50 pg/ml), with (crossed bars) or without (black bars) EGF (1 pg/ml), for 20 min at room temperature before carrying out the phosphorylation reaction, in the absence or presence of 0.2% NP-40, for 10 min on ice. After 515% SDS-PAGE and autoradiography, EGF-R phosphorylation was quantitated by Cerenkov radiation of the excised 170 kD gel-bands. Inset, autoradiograph of the region containing the excised gel bands (A). Protein, eluted from the gel-bands, was subjected to 2-D phosphoamino acid analysis, P-TYR is phosphotyrosine (B).
to induce EGF-R kinase mediated phosphorylation
of Angiotensin
I, an exogenous
tyrosine-containing
peptide, in the presence and absence of detergent (Fig. 2). EGF
stimulates EGF-R
kinase activity in this assay, while addition
of detergent
results
in a higher basal activity, probably due to increased substrate accessibility. and a similar 5 fold stimulation
by EGF. Anti-EGF-R
tyrosine kinase activity, but only when detergent 885
antibodies indeed enhance EGF-R is present. as shown for 2E9.
Vol.
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No.
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2, 1990
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
0
+ NP-40
- NP-40
Fip. 2. Effect of detergent on antibody-induced exogenous substrate phosphorylation in membranes. A431 membranes were incubated in the absence (black bars) or
presence of 50 pg/ml 2E9 (hatched bars) or 1 pg/rnl EGF (crossed bars) for 20 min at room temperature. Phosphorylation mixture containing [T-~~P]ATPand 2 mM Angiotensin I was added in the absence or presence of 0.2% NP-40. After 10 mm on ice the reaction was stopped by TCA precipitation. The phosphorylated peptide was collected on Whatman P81 and radioactivity was determined (see Materials and Methods).
Several earlier reports have demonstrated that in intact cells anti-EGF-R antibodies are not able to activate the EGF-R (12-15). To investigate whether addition
of detergent
would enable antibody to stimulate EGF-R
autophospho-
206
116 97
46
29
Fig. 3. Antibody-induced phosphorylation
EGF-R autophosphorylation and endogenous substrate in permeabilized cells. A431 cells were, as indicated, preincubated
for 20 min with 2E9 (50 pg/mI) at room temperature. Permeabilization buffer containing 0.005% saponin and [T-~~P]ATP,and where indicated EGF (1 ,ug/ml), was added for 10 min on ice as described in Materials and Methods. The phosphorylation was stopped by scraping the cells in sample buffer and phosphoproteins were analysed by 515% SDS-PAGE and subsequent autoradiography (170 kD band is EGF-R).
886
Vol.
171, No. 2, 1990
BIOCHEMICAL
AND BIOPHYSICAL
rylation in whole cells, we preincubated
RESEARCH COMMUNICATIONS
A431 cells with ligand, followed by the
addition of permeabilizing buffer containing 0.005% saponin and [Y-~~P]ATP. Fig. 3 shows that both EGF and 2E9 stimulate EGF-R phosphorylation in this assay. As deduced from 2-D phosphoamino
acid analysis after immtmoprecipitation,
EGF-R
phosphorylation occurred specifically on tyrosine residues, and was stimulated by anti-EGF-R antibodies 528, 225, and 2Dll as well (not shown). None of these antibodies activated the EGF-R in intact cells (12-14). Similar results were obtained using 32P-orthophosphate labeled intact cells, in which, upon incubation and subsequently a 0.5% TX-100 containing buffer, enhanced EGF-R rylation was detected (not shown). Furthermore,
with 2E9 phospho-
both EGF and 2E9 induced the
phosphorylation of several endogenous substrates among which a 35 kD protein (Fig. 3). lJpon omission of Ca2’ from the permeabilization buffer this 35 kD band was no longer observed, suggesting that this protein is the Ca2+-dependent substrate Calpactin II, also known as Lipocortin
EGF-R
I (17,23). Other major substrates
were 82, 62, and 22 kD proteins which have been described before (17,24). DISCUSSION In this study we compared EGF and anti-EGF-R
antibodies, directed against extra-
or intracellular EGF-R domains, for their ability to stimulate EGF-R kinase in membrane preparations as well as intact A431 cells. In vitro assay conditions were mimicked on whole cells by a mild detergent treatment. Our data demonstrate antibody-induced
EGF-R autophosphorylation
that
and EGF-R tyrosine kinase activity
towards other endogenous as well as exogenous substrates is absolutely dependent on the presence of detergent. This holds true for isolated membranes as well as intact cells, and thus explains the apparent discrepancy emerged from earlier studies (7,12-15), in which antibody-induced
EGF-R autophosphorylation
was found only
under in vitro conditions, i.e. in the presence of detergent. In addition these data show that, under the appropriate conditions, antibodies activate EGF-R, irrespective of their recognition site on the receptor, suggesting that this ability relies solely on their bivalent character. EGF will activate EGF-R under all conditions which allow EGF binding, as expected. Interestingly, the simultaneous addition of saturating concentrations of EGF and non-competing antibodies to membrane preparations resulted in enhanced, approximately additive, EGF-R autophosphorylation when detergent formation
was present, as if the antibodies facilitate/stabilize EGF-induced of EGF-R oligomers. Together, these results render further support for
an intermolecular activation mechanism of the EGF-R tyrosine kinase. Interestingly, antibodies have been reported to stimulate the insulin receptor tyrosine kinase in detergent solubilized receptor preparations (25), in contrast to 887
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intact cells (26). This indicates that the detergent dependency of antibody induced EGF-R activation as reported in this study may be a general phenomenon for growth factor receptor tyrosine kinases. The mechanism by which detergent enables antibody-mediated activation of the EGF-R possibly relates to EGF-R/lipid interactions.
tyrosine kinase is not known, but The existence of such a detergent
sensitive factor involved in EGF-R activation has been suggested previously (27). In this study, the authors proposed that membrane lipids associated to the EGF-R may regulate EGF-R activation and thus be influenced by a detergent treatment. Evidence for an influence of lipids on the EGF-R comes from the observation that the ganglioside GM3 inhibited, sphingosine stimulated EGF-R
while de-N-acetyl-GM3 and the lysosphingolipid tyrosine kinase activity (28). In view of these
observations and the existing evidence in favour of an intermolecular mechanism
for the EGF-R
treatments,
as used in our experiments,
environment interactions
of
the
EGF-R
we propose that the primary that
allow
effect of detergent
is to create conditions antibody-induced
activation in the lipid
receptor-receptor
necessary for tyrosine lcinase activation.
Acknowledement: This research was supported by the Centre for Developmental Biology, Utrecht, The Netherlands.
REFERENCES 1. Ullrich, A., Coussens, L., Hayflick, J.S., Dull, T.J., Gray, A., Tam, A.W., Lee, J., Yarden, Y., Libermann, T.A., Schlessinger, J., Downward, J., Mayes, E.L.V., Whittle, N., Waterfield, M.D., and Seeburg, P.H. (1984) Nature 309, 418-425. 2. Carpenter, G. (1987) Amm. Rev. Biochem. 56, 881-914. 3. Hunter, T., and Cooper, J.A. (1985) Annu. Rev. Biochem. 54, 897-930. 4. Schlessinger, J. (1988) Biochemistry 27, 3119-3123. 5. Schlessinger, J. (1986) J. Cell Biol. 103, 2067-2072. 6. Schlessinger, J. (1988) Trends Biochem. Sci. 13, 443-447. 7. Yarden, Y., and Schlessinger, J. (1987) Biochemistry 26, 1434-1442. 8. Yarden, Y., and Schlessinger, J. (1987) Biochemistry 26, 1443-1451. 9. Boni-Schnetzler, M., and Pilch, P.F. (1987) Proc. Natl. Acad. Sci. USA 84, 78327836. 10. Northwood, I.C., and Davis, R.J. (1988) J. Biol. Chem. 263, 7450-7453. 11. Fanger, B-O., Austin, K.S., Earp, H.S., and Cidlowski, J.A. (1986) Biochemistry 25, 6414-6420. 12. Defize, L.H.K., Boonstra, J., Meisenhelder, J., Kruijer, W., Tertoolen, L.G.J., Tilly, B.C., Hunter, T., van Bergen en Henegouwen, P.M.P., Moolenaar, W.H., and de Laat, S.W. (1989) J. Cell Biol. 109, 2495-2507. 13. Defize, L.H.K., Moolenaar, W.H., van der Saag, P.T., and de Laat, S.W. (1986) EMBO J. 5, 1187-1192. 14. Gill, G.N., Kawamoto, T., Cachet, C., Le, A., Sato, J.D., Masui, H., McLeod, C., and Mendelsohn, J. (1984) J. Biol. Chem. 259, 7755-7760. 15. Defize, L.H.K., Mummery, CL., Moolenaar, W.H., and de J-sat, SW. (1987) Cell Diff. 20, 87-102. 888
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No.
2,
1990
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RESEARCH
COMMUNICATIONS
16. Cohen, S., Ushiro, H., Stoschek, C., and Chinkers, M. (1982) J. Biol. Chem. 257, 1523- 1531. 17. Guigni, T.D., James, L.C., and Haigler, H.T. (1985) J. Biol. Chem. 260, 1508115090. 18. Berkers, J., van Bergen en Henegouwen, P.M.P., Verkleij, A.J., and Boonstra, J. (1990:) Biochim. Biophys. Acta 1052, 453-460. 19. Honegger, A.M., Kris, R.M., Ullrich, A., and Schlessinger, J. (1989) Proc. Natl. Acad. Sci. USA 86, 925-929. 20. Honegger, A.M., Schmidt, A., Ullrich, A., and Schlessinger, J. (1990) Mol. Cell. Biol., in press. 21. King, C.R., Borello, I., Bellot, F., Comoglio, P., and Schlessinger, J. (1988) EMBO J. 7, 1647-1651. 22. Stern, D.F., and Kamps, M. (1988) EMBO J. 7, 995-1001. 23. Fava, R.A., and Cohen, S. (1984) J. Biol. Chem. 259, 2636-2645. 24. Hunter, T., and Cooper, J.A. (1981) Cell 24, 741-752. 25. O’Brien, R.M., Soos, M.A., and Siddle, K. (1987) EMBO J. 6, 4003-4010. 26. Soos, M.A., O’Brien, R.M., Brindle, N.P.J., Stigter, J.M., Okamoto, AK., Whittaker, J., and Siddle, K. (1989) Proc. Natl. Acad. Sci. USA 86, 5217-5221. 27. Rubin, R-A., and Earp, H.S. (1983) J. Biol. Chem. 258, 5177-5182. 28. Hannun, Y.A., and Bell, R.M. (1989) Science 243, 500-507.
889
Vol. 171, No. 2, 1990 September
AND BIOPHYSICAL
14, 1990
RESEARCH COMMUNICATIONS Pages 882-889
ANTIBODY-INDUCED ACTIVATION OF THE EPIDERMAL GROWTH FACTOR RECEPTOR TYROSINE REQUIRES THE PRESENCE OF DETERGENT
KINASE
Marcel Spaargarenly2*, Libert H.K. Defize’, Siegfried W. de Last’, and Johannes Boonstra2 ‘Hubrecht Laboratory, Netherlands Imtitute for Developmental Biology, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands 2Depan?nent of Molecular Cell Biology, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands Received
July
26,
1990
SUMMARY: Activation of the epidermaI growth factor receptor (EGF-R) tyrosine kinase was investigated in membrane preparations as well as intact A431 cells, using anti-EGF-R antibodies directed against extra- and intracellular receptor domains. In vitro assay conditions were mimicked on whole cells by a mild detergent treatment. We show that, irrespective of the recognition site on the EGF-R, antibodies induce EGF-R autophosphorylation and tyrosine kinase activity towards other endogenous and exogenous substrates, but only when detergent is present. We propose that the primary effect of detergent is to create conditions in the lipid environment of the EGF-R that allow antibodies to induce receptorreceptor interactions necessary for tyrosine kinase activation. 01990 Academic press, Inc.
The EGF-R is a transmembrane glycoprotein of 170 kD with an extracellular ligand binding domain, a single hydrophobic transmembrane stretch and an intracellular domain containing protein tyrosine kinase activity (1,2). Binding of EGF to its receptor enhances the tyrosine kinase activity, thus inducing tyrosine phosphorylation of several protein substrates including the EGF-R itself (3). Using EGF-R mutants, the protein tyrosine kinase activity was shown to be required for all receptor-mediated responses involved in mitogenic signalling (4). How extracellular EGF binding activates the intracellular EGF-R
tyrosine
kinase remains to be elucidated, but a number of recent data support an intermolecular activation mechanism, in which EGF binding causes a shift in a hypothetical equilibrium between monomeric and dimeric receptors. Dimerization of EGF-R then results in activation of the tyrosine kinase ($6). Yarden and Schlessinger have demonstrated
a reversible EGF-R
*To whom correspondence should be addressed. 0006-291X/90 $1.50 Copyright 0 1990 by Academic Press. All rights of reprudurkwz in any form
Inc. reserved.
882
oligomerization
after EGF
Vol.
171,
No.
treatment
2,
1990
BIOCHEMICAL
AND
of solubilized immunoaffinity
BIOPHYSICAL
RESEARCH
purified EGF-receptors
COMMUNICATIONS
(7,8). EGF-induced
EGF-R dimerization
could also be detected by chemical cross-linking in solubilized
EGF-R preparations (l&12). Furthermore,
(9), membrane preparations (lo), as well as in intact cells bivalent antibodies were able to induce EGF-R autophospho-
rylation and tyrosine kinase activation in in vitro phosphorylation assays, probably by inducing EGF-R dimerization (7,13,14). However, despite their bivalent nature, antibodies were not able to activate the EGF-R kinase in intact cells (12,14). Thus, apparently a discrepancy exists between the ability of the antibodies to activate the EGF-R tyrosine kinase in vitro versus in vivo (15). In this study we demonstrate
that anti-EGF-R
antibodies,
directed against
extra- and intracellular EGF-R epitopes, are able to activate the EGF-R tyrosine kinase in membrane preparations as well as in intact cells, provided a small amount of detergent is present. These data confirm and extend previous data (7,13,14), and provide additional
support for an intermolecular
mechanism of EGF-R
tyrosine
kinase activation. MATERIALS
AND METHODS
EGF (receptor grade) wa;20btained from Collaborative Research (Waltham, MA, USA), [y- P]ATP and [ PI-Orthophosphate from Amersham International (‘s Hertogenbosch, The Netherlands), Protein A-Sepharose from Pharmacia LKB Biotechnology Inc., and Plastic foil thin layer cellulose plates (5577) were from Merck. A431 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 7.5% Fetal Calf Serum and buffered with 40 mM NaHC03, under a 7.5% CO, atmosphere. Monoclonal antibody 2E9 and polyclonal antiserum 281-7 were prepared and IgG purified as previously described (13). Membrane preparations were prepared by vesiculation of A431 cells essentially as described (16). The EGF-R was immunoprecipitated using antiserum 281-7 as previously described (12). EGF-R autophosphorylation and exogenous substrate phosphorylation in membrane preparations. A431 membrane preparations (20 pg) were incubated with
ligand for 20 min at room temperature, phosphorylation reaction was started by addition of phosphorylation assay mix containing 15 PM [T-~~P]ATP (5 ,Ki), with or without 0.2% NP-40, and analyzed as described (13). After 5-15% SDS-PAGE, gel bands were cut out in order to quantitate radioactivity by liquid scintillation. 2-D phosphoamino acid analysis was performed as described (12). In order to measure exogenous substrate phosphorylation, the phosphorylation reaction was started after ligand incubation by the addition of phosphorylation assay mix, containing 15 .uM [r-32P]ATP (1 &i) and 2 mM Angiotensin I, with or without 0.2% NP-40. After 10 min on ice, the reaction was stopped and exogenous substrate phosphorylation was determined as described (13). Aspecific background labelling was corrected by substracting values of identical reactions without Angiotensin I. EGF-R autophosphotylation
and endogenous substrate phosphorylation
in cells.
Assays were performed in A431 cells, essentially as described (17). Briefly, cells, grown to subconfluency in 12-well tissue culture clusters (Costar), were incubated with antibody for 20 min at room temperature in DMEM/Hepes (10 mM). After 883
Vol.
171,
No.
2,
1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
this incubation cells were washed two times with cold PBS and once with cold permeabilization buffer containing 20 mM Hepes (pH 7.4), 145 mM NaCl, 5.4 mM KU, 1 mM CaC12, 2 mM MgCl,, 2 mM MnCl,, 20 PM Na3V0,, 10 mM PNPP, 1 mM PMSF. Phosphotylation was started b addition of permeabilization buffer containing 0.005% Saponin and 15 PM [y- x P]ATP (5 &i) and where indicated EGF, for 10 min on ice. The reaction was stopped by washing twice with cold Ca2+ and Mg2+-free PBS containing 200 PM Na,VO, followed by the addition of sample buffer. Substrate phosphorylation was measured after 5-15% SDS-PAGE by scanning of the autoradiograph on a Joyce-Loebl Chromoscan 3 apparatus. RESULTS Earlier studies have shown that anti-EGF-R antibodies can enhance the EGF-R autophosphorylation in isolated plasma membranes or purified receptor preparations, but not in intact cells (7,12-15).
We have investigated
this phenomenon
by
comparing the effects of EGF with those of monoclonal antibody 2E9, directed against the extracellular EGF-R domain and competing for EGF binding to low affinity receptors, and polyclonal antiserum 281-7, directed against the cytoplasmic EGF-R domain (a.a. 984-996). Since in vitro phosphorylation assays are usually carried out in the presence of detergent, it was investigated whether this influences antibody-induced EGF-R activation, and we mimicked these conditions on intact cells by adding low concentrations
of saponin.
As shown in Fig. 1, EGF induces EGF-R phosphorylation in isolated membrane preparations regardless of the presence of detergent. In contrast, 2E9 and 281-7 are able to enhance EGF-R phosphorylation under these conditions only if a detergent like NP-40 is present in the assay mixture. In isolated membrane preparation
only
low affinity EGF-R are detectable (18) and thus 2E9 antagonized EGF-induced EGF-R phosphorylation. Interestingly, simultaneous addition of antiserum 281-7 and EGF resulted in an approximately additive effect on EGF-R phosphorylation. In all cases EGF-R phosphorylation
occurred specifically on tyrosine residues, as shown
by 2-D phosphoamino
acid analysis for EGF and 2E9 (Fig. 1B). These results
demonstrate that the antibodies requires the EGF and anti-EGF-R addition they show that
enhancement of EGF-R phosphorylation by anti-EGF-R presence of detergent. The molecular mechanisms by which antibodies exert their effects are thus not identical. In the activation of the EGF-R by antibodies does not depend
on the receptor epitope to which these antibodies are directed. It has recently become clear that enhanced EGF-R phosphorylation
is not
necessarily indicative for receptor autophosphorylation, but can also occur through cross-phosphoryl~ltion of non-active receptors by active kinase receptors (12.19-22). To discriminate between antibody-induced kinase activation and facilitated intermolecular cross-phosphorylation we analysed the capacity of EGF and antibodies 884
Vol.
171,
No.
2,
1990
BIOCHEMICAL
-
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
NP-40
A 2E9 m
281-7
C
-EGF
C
2E9
281-7
+EGF
EGF
2E9
B Fig. 1. Effect of detergent on antibody-induced EGF-R autophosphorylation in membranes. A431 membranes were incubated with buffer (C) or the indicated antibodies (50 pg/ml), with (crossed bars) or without (black bars) EGF (1 pg/ml), for 20 min at room temperature before carrying out the phosphorylation reaction, in the absence or presence of 0.2% NP-40, for 10 min on ice. After 515% SDS-PAGE and autoradiography, EGF-R phosphorylation was quantitated by Cerenkov radiation of the excised 170 kD gel-bands. Inset, autoradiograph of the region containing the excised gel bands (A). Protein, eluted from the gel-bands, was subjected to 2-D phosphoamino acid analysis, P-TYR is phosphotyrosine (B).
to induce EGF-R kinase mediated phosphorylation
of Angiotensin
I, an exogenous
tyrosine-containing
peptide, in the presence and absence of detergent (Fig. 2). EGF
stimulates EGF-R
kinase activity in this assay, while addition
of detergent
results
in a higher basal activity, probably due to increased substrate accessibility. and a similar 5 fold stimulation
by EGF. Anti-EGF-R
tyrosine kinase activity, but only when detergent 885
antibodies indeed enhance EGF-R is present. as shown for 2E9.
Vol.
171,
No.
BIOCHEMICAL
2, 1990
AND
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RESEARCH
COMMUNICATIONS
0
+ NP-40
- NP-40
Fip. 2. Effect of detergent on antibody-induced exogenous substrate phosphorylation in membranes. A431 membranes were incubated in the absence (black bars) or
presence of 50 pg/ml 2E9 (hatched bars) or 1 pg/rnl EGF (crossed bars) for 20 min at room temperature. Phosphorylation mixture containing [T-~~P]ATPand 2 mM Angiotensin I was added in the absence or presence of 0.2% NP-40. After 10 mm on ice the reaction was stopped by TCA precipitation. The phosphorylated peptide was collected on Whatman P81 and radioactivity was determined (see Materials and Methods).
Several earlier reports have demonstrated that in intact cells anti-EGF-R antibodies are not able to activate the EGF-R (12-15). To investigate whether addition
of detergent
would enable antibody to stimulate EGF-R
autophospho-
206
116 97
46
29
Fig. 3. Antibody-induced phosphorylation
EGF-R autophosphorylation and endogenous substrate in permeabilized cells. A431 cells were, as indicated, preincubated
for 20 min with 2E9 (50 pg/mI) at room temperature. Permeabilization buffer containing 0.005% saponin and [T-~~P]ATP,and where indicated EGF (1 ,ug/ml), was added for 10 min on ice as described in Materials and Methods. The phosphorylation was stopped by scraping the cells in sample buffer and phosphoproteins were analysed by 515% SDS-PAGE and subsequent autoradiography (170 kD band is EGF-R).
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rylation in whole cells, we preincubated
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A431 cells with ligand, followed by the
addition of permeabilizing buffer containing 0.005% saponin and [Y-~~P]ATP. Fig. 3 shows that both EGF and 2E9 stimulate EGF-R phosphorylation in this assay. As deduced from 2-D phosphoamino
acid analysis after immtmoprecipitation,
EGF-R
phosphorylation occurred specifically on tyrosine residues, and was stimulated by anti-EGF-R antibodies 528, 225, and 2Dll as well (not shown). None of these antibodies activated the EGF-R in intact cells (12-14). Similar results were obtained using 32P-orthophosphate labeled intact cells, in which, upon incubation and subsequently a 0.5% TX-100 containing buffer, enhanced EGF-R rylation was detected (not shown). Furthermore,
with 2E9 phospho-
both EGF and 2E9 induced the
phosphorylation of several endogenous substrates among which a 35 kD protein (Fig. 3). lJpon omission of Ca2’ from the permeabilization buffer this 35 kD band was no longer observed, suggesting that this protein is the Ca2+-dependent substrate Calpactin II, also known as Lipocortin
EGF-R
I (17,23). Other major substrates
were 82, 62, and 22 kD proteins which have been described before (17,24). DISCUSSION In this study we compared EGF and anti-EGF-R
antibodies, directed against extra-
or intracellular EGF-R domains, for their ability to stimulate EGF-R kinase in membrane preparations as well as intact A431 cells. In vitro assay conditions were mimicked on whole cells by a mild detergent treatment. Our data demonstrate antibody-induced
EGF-R autophosphorylation
that
and EGF-R tyrosine kinase activity
towards other endogenous as well as exogenous substrates is absolutely dependent on the presence of detergent. This holds true for isolated membranes as well as intact cells, and thus explains the apparent discrepancy emerged from earlier studies (7,12-15), in which antibody-induced
EGF-R autophosphorylation
was found only
under in vitro conditions, i.e. in the presence of detergent. In addition these data show that, under the appropriate conditions, antibodies activate EGF-R, irrespective of their recognition site on the receptor, suggesting that this ability relies solely on their bivalent character. EGF will activate EGF-R under all conditions which allow EGF binding, as expected. Interestingly, the simultaneous addition of saturating concentrations of EGF and non-competing antibodies to membrane preparations resulted in enhanced, approximately additive, EGF-R autophosphorylation when detergent formation
was present, as if the antibodies facilitate/stabilize EGF-induced of EGF-R oligomers. Together, these results render further support for
an intermolecular activation mechanism of the EGF-R tyrosine kinase. Interestingly, antibodies have been reported to stimulate the insulin receptor tyrosine kinase in detergent solubilized receptor preparations (25), in contrast to 887
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intact cells (26). This indicates that the detergent dependency of antibody induced EGF-R activation as reported in this study may be a general phenomenon for growth factor receptor tyrosine kinases. The mechanism by which detergent enables antibody-mediated activation of the EGF-R possibly relates to EGF-R/lipid interactions.
tyrosine kinase is not known, but The existence of such a detergent
sensitive factor involved in EGF-R activation has been suggested previously (27). In this study, the authors proposed that membrane lipids associated to the EGF-R may regulate EGF-R activation and thus be influenced by a detergent treatment. Evidence for an influence of lipids on the EGF-R comes from the observation that the ganglioside GM3 inhibited, sphingosine stimulated EGF-R
while de-N-acetyl-GM3 and the lysosphingolipid tyrosine kinase activity (28). In view of these
observations and the existing evidence in favour of an intermolecular mechanism
for the EGF-R
treatments,
as used in our experiments,
environment interactions
of
the
EGF-R
we propose that the primary that
allow
effect of detergent
is to create conditions antibody-induced
activation in the lipid
receptor-receptor
necessary for tyrosine lcinase activation.
Acknowledement: This research was supported by the Centre for Developmental Biology, Utrecht, The Netherlands.
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