Endocytosed Epidermal Growth Factor (EGF) Receptors Contributeto the EGF-Mediated Growth Arrest in A431 Cells by Inducing

a Sustained Increase in p21/CIP1Ellen Skarpen,*,1 Lene E. Johannessen,*,1 Kjetil Bjerk,* Hilde Fasteng,* Tormod K. Guren,†

Birgitte Lindeman,* G. Hege Thoresen,† Thoralf Christoffersen,† Espen Stang,*Henrik S. Huitfeldt,* and Inger Helene Madshus*,2

*Institute of Pathology, The National Hospital, The University of Oslo, 0027 Oslo; and †Department of Pharmacology,The University of Oslo, P.O. Box 1057, 0316 Oslo, Norway

We investigated the ability of endocytosed activatedepidermal growth factor receptors (EGFR) to induceexpression of the cyclin-interacting protein p21/CIP1in A431 cells. Transforming growth factor a (TGFa)and EGF both induced tyrosine phosphorylation, in-duction of p21/CIP1, and thereby inhibition of DNAsynthesis. TGFa is released from the EGFR when theTGFa–EGFR complex encounters low pH upon endo-cytosis. Consistently, we found more rapid dephos-phorylation of the EGFR and less induction of p21/CIP1 by TGFa than by EGF. This difference wasabolished upon neutralizing endosomal pH by the car-boxylic ionophore monensin or the proton ATPase in-hibitor bafilomycin A1. When surface-bound TGFa wasremoved by acid stripping and endosomal pH was neu-tralized with bafilomycin A1, TGFa stimulated EGFRtyrosine phosphorylation, induced p21/CIP1, and in-hibited DNA synthesis. This strongly suggests thatp21/CIP1 can be induced by endocytosed, activatedEGFR and that endocytosed EGFR can affect cellgrowth. © 1998 Academic Press

Key Words: growth suppression; endocytosed EGFreceptor; signal transduction; monensin; bafilomycinA1; p21/CIP1.

INTRODUCTION

Upon binding epidermal growth factor (EGF), thekinase domain of the EGF receptor (EGFR) is acti-vated, and the cytoplasmic tail of the EGFR becomestyrosine phosphorylated [1, for review see 2]. This inturn initiates several signal transduction reactions im-portant for regulation of growth and differentiation.The best characterized signaling pathway leads to ac-tivation of mitogen-activated protein kinase (MAPK)

[3, 4]. At high concentrations EGF inhibits growth inA431 cells [5–7]. Growth suppression at high concen-trations of EGF was demonstrated to correlate withincreased expression of the cyclin-dependent kinase(CDK) inhibitor p21/CIP1 [8, 9]. At growth-stimulatoryconcentrations of EGF increased levels of p21/CIP1were not observed [10]. Recently, stimulation of theEGFR was also observed to cause activation of activa-tors of transcriptions 1 and 3 (STAT1 and STAT3)[11–14], and activated STAT1 has been shown to in-duce p21/CIP1 and thereby inhibit proliferation ofA431 cells [8].

In addition to the requirement of EGFR kinase ac-tivity for initiation of signal transduction, EGFR ki-nase activity is required for initiation of endocytosis ofthe EGFR from coated pits [15] and for targeting theinternalized EGFR to lysosomes for degradation [16,17]. Endocytosed EGFR has been reported to be differ-ently phosphorylated compared to nonendocytosedEGFRs [18, for review see 19]. Furthermore, Grb-2,mSOS, and shc were shown to be enriched in the en-dosomal fraction of EGF-treated hepatocytes [20], andrecently it was demonstrated that in HeLa cells MAPKactivation was strongly inhibited when endocytosisfrom coated pits was arrested [21]. These data suggesta close interrelationship between trafficking and sig-naling of growth factor receptors and further suggestthat the subcellular localization of the EGFR is impor-tant in controlling specific signaling pathways.

We set out to investigate whether activated endocy-tosed EGFR was indeed capable of affecting gene tran-scription. For this purpose we used two EGFR-bindingligands. EGF and transforming growth factor a (TGFa)were previously shown to induce EGFR internalizationto similar degrees, but EGF stimulated EGFR degra-dation to a greater extent than did TGFa. Recovery of125I-EGF binding capacity was much faster for TGFa-treated cells, and this fast recovery did not requireprotein synthesis. Furthermore, tyrosine phosphoryla-

1 These authors contributed equally to the work.2 To whom reprint request should be addressed. Fax: 147 22 11 22

61. E-mail: i.h.madshus@labmed.uio.no.

0014-4827/98 $25.00161Copyright © 1998 by Academic Press

All rights of reproduction in any form reserved.

EXPERIMENTAL CELL RESEARCH 243, 161–172 (1998)ARTICLE NO. EX984127

tion of the EGFR in TGFa-treated cells decreased morerapidly following removal of TGFa compared to cellstreated similarly with EGF [22]. This is consistent withthe demonstration that TGFa, in contrast to EGF, dis-sociated from the EGFR at slightly acidic pH [23] andtherefore caused mainly recycling of the EGFR in con-trast to lysosomal degradation mediated by EGF.

In order to study the potential importance of endo-cytosed and phosphorylated EGFR for induction ofgrowth suppression in A431 cells, we studied the ef-fects of EGF and TGFa on DNA synthesis, on expres-sion of p21/CIP1, and on EGFR tyrosine phosphoryla-tion in the absence and presence of drugs known toneutralize endosomal pH. By manipulating pH wewere able to modify the tyrosine phosphorylation of theEGFR by interfering with the dissociation of ligand.We studied the accompanying changes in induction ofp21/CIP1 and in growth suppression. We further stud-ied induction of p21/CIP1 by endocytosed TGFa–EGFRcomplexes in cells in which endosomal pH had beenneutralized and ligand had been removed only fromsurface-localized receptors by acid stripping. In thefollowing, we present data indicating that endocytosed,activated EGFR can indeed induce expression of p21/CIP1 and thereby inhibit DNA synthesis following en-docytosis.

MATERIALS AND METHODS

Materials. Human recombinant EGF and TGFa were fromBachem Feinchemikalien AG (Budendorf, Switzerland). [3H]Thymi-dine (25 Ci/mmol), [g-32 P]ATP (3000 Ci/mmol), and 125I-EGF (750Ci/mmol) were from Amersham International (Buckinghamshire,UK). MEM without bicarbonate was obtained from Gibco BRL (Pais-ley, UK). All chemicals were obtained from Sigma Chemical Co. (St.Louis, MO) unless otherwise noted.

Cells. A431 cells were from the American Type Culture Collec-tion (Rockville, MD). A431 cells were grown in Costar 3000 flasks(Costar, Badhoevedorp, The Netherlands). The medium used wasDMEM (3.7 g/L sodium bicarbonate) (Flow Laboratories, Irvine,Scotland) containing 10% fetal calf serum (FCS) (Hy Clone Europe,Oud-Beiserland, The Netherlands), 2 mM L-glutamine (Gibco BRL),and 50 ng/ml gentamycin (Gibco BRL). Cells were seeded at a densityof 2.5 3 104 cells/cm2.

Measurement of DNA synthesis. Cells in 24-well microtiter plates(5 3 104 cells/well) were incubated in DMEM with 10% FCS for 48 hprior to addition of EGF or TGFa. After the indicated time points, 1mCi/ml [3H]thymidine was added, and the cells were incubated for 2additional hours. The medium was removed, and 1 ml 5% (w/v)trichloroacetic acid (TCA) was added per well for 23 10 min at roomtemperature. The precipitated DNA was then solubilized with 0.3 ml0.1 M KOH, and 1.5 ml Ultima gold scintillation fluid (PackardInstrument B. V., Groningen, The Netherlands) was added beforeliquid scintillation counting.

Western blot analysis. A431 cells in 12-well microtiter plates (1 3105 cells/well) were incubated in DMEM with 10% FCS for 48 h priorto addition of EGF or TGFa. After incubation, as indicated in legendsto the figures, the cells were washed twice with PBS (137 mM NaCl,2.7 mM KCl, 1 mM Na2HPO4, 2 mM NaH2PO4, pH 7.4) before lysisin 10 mM Tris–HCl (pH 6.8) with 4% (v/v) glycerol, 2% (w/v) sodiumdodecyl sulfate (SDS), 1 mM phenylmethylsulfonyl fluoride, 1% (v/v)

b-mercaptoethanol (M-7154), 5 mM EDTA, 50 mM NaF, 30 mMsodium pyrophosphate, 100 mM sodium orthovanadate (Na3VO4)(Stem Chemicals, Newburyport, MA), and 0.005% bromphenol blue.The samples were heated to 95°C for 10 min, centrifuged at 20,000gfor 15 s, and exposed to SDS–PAGE [24]. The proteins were thenelectrotransferred to nitrocellulose membranes (Micron Separations,Inc., Westborough, MA) [25]. Membranes were washed four times inTris-buffered saline (TBS) (10 mM Tris, pH 7.6, 137 mM NaCl) with0.1% (v/v) Tween 20 and preincubated for 60 min in 5% (w/v) fat-freedry milk in TBS/Tween 20. The membranes were incubated over-night at 4°C with antibodies to p21/CIP1 (1:3000, sc-397; Santa CruzBiotechnology, Inc., Santa Cruz, CA), to phosphotyrosine (1:5000,05-321; Upstate Biotechnology, Inc., Lake Placid, NY), and to phos-phorylated erk (1:1000, 9101S; New England Biolabs, Inc., Beverly,MA). All antibodies were diluted in 1% (w/v) fat-free dry milk inTBS/Tween 20. The membranes were then washed four times for 15min in TBS/Tween 20 and preincubated for 60 min in 5% (w/v)fat-free dry milk in TBS/Tween 20 prior to incubation with horse-radish peroxidase-conjugated anti-rabbit IgG (1:5000, A-6154;Sigma) or anti-mouse IgG (1:4000, 715-035-151; Jackson ImmunoResearch Laboratories, West Grove, PA) diluted in 1% (w/v) fat-freedry milk in TBS/Tween 20 for 2 h. Again, the filters were washed fourtimes for 15 min in TBS/Tween 20 before the immunobinding wasdetected by the enhanced chemiluminescence method (Amersham)following the manufacturer’s instructions.

125I-EGF–cell interaction experiments. A431 cells were plated in24-well microtiter plates as described above. The cells were incu-bated with or without 0.1 mM bafilomycin A1 for 10 min at 37°C.Then 1028 M 125I-EGF was added to the cells on ice in MEM withoutHCO3

2 and with or without bafilomycin A1. After 10 min at 4°C thecells were washed three times with PBS to remove unbound ligand.The cells were then chased in MEM without HCO3

2, with or withoutbafilomycin A1, and with 0.1% bovine serum albumin (BSA) at 37°Cfor the indicated time periods. The medium was collected, and the125I-EGF was precipitated using 5% trichloroacetic acid and 1%phosphotungstic acid (Sigma) (TCA–PTA) as described [26]. Both theTCA-precipitable and the TCA-soluble radioactivity was counted in agamma counter. Cells were washed three times with PBS, treatedwith 0.2 M sodium acetate buffer (pH 4.5 or 7.4) and 0.5 M NaCl(SAB, pH 4.5/SAB, pH 7.4) on ice for 10 min, and washed once withthe same buffer. Then 125I-EGF was precipitated from the cells withTCA–PTA. Finally, the precipitate was dissolved with 1 M NaOH,and the radioactivity was counted. Counts per minute (cpm) fromcells treated with SAB, pH 4.5, represent internalized 125I-EGF,while cpm from cells treated with SAB, pH 7.5, represent bothinternalized and surface-localized 125I-EGF.

Immunocytochemistry. A431 cells grown in 5-cm-diameter petridishes (5 3 105 cells/dish) were incubated for 24 h in DMEM with10% FCS before ligand was added. After incubation the cells werewashed twice in PBS, fixed in 96% (v/v) ethanol for 10 min, and thenwashed twice in PBS for 10 min before incubation with a combina-tion of sheep antibody to the EGFR (1:800, 13287-016; Gibco BRL,Gaithersburg, MD) and mouse antibody to activated (ligand-bound)EGFR (1:160, E12120; Transduction Laboratories, Lexington, KY)diluted in PBS containing 1% (w/v) BSA. After incubation overnightthe cells were washed twice with PBS for 10 min and exposed to amixture of fluorescein isothiocyanate (FITC)-conjugated donkey an-ti-sheep IgG and Texas Red-conjugated donkey anti-mouse IgG (713-095-147 and 715-075-15, respectively, from Jackson Immuno Re-search Laboratories, both diluted 1:80). The cells were washed twicein PBS and mounted with buffered polyvinyl alcohol, pH 8.5. Stainedcells were examined with a Nikon Labophot microscope (Nikon,Tokyo, Japan) containing an epifluorescence attachment andequipped with a Bio-Rad MRC 600 confocal laser scan unit with akrypton/argon laser, a K1 double-dichroic-excitation filter block, anda K2 dichroic-emission filter block (Bio-Rad Microscience Division,Hertfordshire, UK). This combination allowed simultaneous detec-

162 SKARPEN ET AL.

tion of FITC and Texas Red fluorescence. A polaroid freeze-frameunit (Polaroid, Cambridge, MA) was attached for photographic doc-umentation of acquired images.

MAP kinase assay. A431 cells in 5-cm-diameter petri dishes (5 3105 cells/dish) were grown in DMEM with 10% FCS for 48 h. Thecells were then exposed to 1 or 10 nM EGF or TGFa for 5, 10, and 30min and for 24 h. The cells were assayed for MAP kinase activitywith myelin basic protein (MBP) as substrate in the presence ofprotein kinase A inhibitor essentially as described [27, 28].

RESULTS

Effect of EGF and TGFa on EGFR tyrosine phosphor-ylation. Different trafficking of endocytosed EGFRfollowing binding of EGF and TGFa in NIH-3T3 cellshas been reported [22]. This can possibly be explainedby the demonstration that TGFa is released from theEGFR at slightly acidic pH, while this is not the casefor EGF [23]. We wanted to make sure that the re-ported difference in pH-dependent dissociation of EGFand TGFa applied to A431 cells and to investigatewhether dissociation of ligand in fact caused dephos-phorylation of the EGFR. Tyrosine phosphorylation ofthe EGFR by EGF or TGFa was induced by addition ofligand to cells incubated at 4°C to avoid endocytosis ofthe EGFR. The cells were subsequently incubated onice with medium adjusted to neutral and low pH asindicated. The tyrosine phosphorylation was analyzedby Western blotting, using an antibody reacting specif-ically to phosphotyrosine (Fig. 1), recognizing a 170-kDa band corresponding to the EGFR. To ensure thatthis tyrosine-phosphorylated protein was EGFR, EGFRwas immunoprecipitated with an antibody to EGFR, and

Western blots were labeled with antibodies to phospho-tyrosine and to EGFR (data not shown). Furthermore, anantibody to an activated form of the EGFR identified thesame protein (data not shown). As shown in Fig. 1, treat-ment of cells with pH 7.0 buffer did not differentiallyaffect the level of tyrosine phosphorylation caused bybinding of EGF or TGFa (see Fig. 1, lanes 1 and 2). At pH6.0, the tyrosine phosphorylation caused by binding ofTGFa was strongly reduced, while the same buffer re-duced the tyrosine phosphorylation caused by EGF to amuch smaller extent (Fig. 1, compare lanes 4 and 5).Upon treatment with pH 5.0, the tyrosine phosphoryla-tion induced by both EGF and TGFa (Fig. 1, comparelanes 7 and 8) was comparable to the level of phosphor-ylation in cells incubated without added ligand (Fig. 1,lanes 3, 6, and 9). This confirms the previously reportedpH dependence of dissociation of EGF and TGFa from theEGFR [23].

Since EGF and TGFa dissociate from the EGFR atdifferent pH, we induced tyrosine phosphorylation ofthe EGFR on ice with EGF/TGFa ligand and chasedthe cells at 37°C for increasing time periods. EGF orTGFa at 1028 M was added to cells for 10 min. The cellswere then washed three times with ice-cold DMEM toremove unbound ligand and subsequently incubated at37°C for the times indicated in Fig. 2A. As illustrated,the EGFR was dephosphorylated more rapidly whenthe tyrosine phosphorylation had been induced byTGFa compared to induction by EGF (Fig. 2A). Thesame time course of dephosphorylation was observedwhen the cells were incubated with 1029 M EGF andTGFa (data not shown). The data are consistent withthe notion that TGFa dissociates from the EGFR fol-lowing endocytosis due to the acidic endosomal pH.

Expression of the cyclin-dependent kinase inhibitorp21/CIP1 upon incubation of A431 cells with EGFand TGFa. High concentrations of EGF, inhibitingDNA synthesis in A431 cells, induce expression ofthe CDK inhibitor p21/CIP1 [8 –10]. In order to in-vestigate whether the difference in EGFR phospho-tyrosine content caused differences in downstreamsignaling, we studied the EGFR-mediated inductionin p21/CIP1. A431 cells in microtiter plates wereexposed to 1028 M EGF or TGFa for 10 min at 4°C.Then the cells were washed three times with ice-coldDMEM and further incubated at 37°C for the indi-cated time periods (Fig. 2B). Expression of p21/CIP1was studied by Western blotting. p21/CIP1 wasstrongly induced after incubation at 37°C for 10 honly when EGF had been added to the cells (Fig. 2B).TGFa did not significantly induce expression of p21/CIP1 when the ligand was bound to the EGFR on iceand the cells were subsequently incubated at 37°C(Fig. 2B). When 1029 M EGF was added, the induc-tion of p21/CIP1 was the same as with 1028 M EGF

FIG. 1. Effect of pH on EGF- or TGFa-induced EGFR tyrosinephosphorylation. A431 cells were grown in DMEM with 10% FCS for48 h as described under Materials and Methods. The medium wasreplaced by MEM without bicarbonate. The cells were chilled on ice for5 min. 1028 M EGF, TGFa, or no ligand was added, and the cells wereincubated on ice for an additional 10 min. Then the medium wasremoved, and MEM supplemented with 10 mM MES and adjusted topH 7.0, 6.0, and 5.0 was added with or without 1028 M EGF or TGFa asindicated on ice for 20 min. Then the cells were washed, lysed, andsubjected to SDS–PAGE and Western blotting with an antibody tophosphotyrosine as described under Materials and Methods.

163EGF RECEPTOR SIGNALING AFTER ENDOCYTOSIS

(data not shown). The expression of p21/CIP1 there-fore correlated with the sustained level of EGFRphosphotyrosine content seen upon incubation withEGF, but not with TGFa (compare Figs. 2A and 2B).

Effect of the carboxylic ionophore monensin on EGF/TGFa-mediated EGFR phosphorylation, expression ofp21/CIP1, and reduction in DNA synthesis. The ob-served difference in EGFR phosphotyrosine contentupon addition of EGF or TGFa to cells can likely beexplained by the differential effect of low pH on disso-ciation of ligand from the EGFR. To study this moreclosely, we made use of drugs known to neutralizeendosomal pH, the assumption being that the differ-ences in EGFR phosphorylation and in downstreamsignaling would be abolished upon neutralization ofpH. We studied how the effects of EGF and TGFa onEGFR phosphorylation, p21/CIP1 expression, and DNAsynthesis were influenced by treatment with monensin(10 mM), a cation proton ionophore (see [29] for a re-view). A431 cells were incubated with or without 10mM monensin in the presence of 1029 M EGF or TGFafor the indicated time periods. In the absence of mo-nensin, the EGFR phosphorylation induced by TGFadecreased more rapidly compared to phosphorylationinduced by EGF (compare Figs. 3A and 3B). However,in the presence of monensin, TGFa gave the samesustained tyrosine phosphorylation of the EGFR as didEGF (compare Figs. 3A and 3B). The same sustainedreceptor tyrosine phosphorylation was observed alsoin the presence of 0.1 mM bafilomycin A1 (data notshown). Bafilomycin A1 increases endosomal pH byinhibiting V-ATPases, like the proton ATPase respon-sible for acidification of endosomes and lysosomes [30].The data show that when endosomal pH was neutral-ized, the EGFR phosphotyrosine content induced byTGFa was as sustained as after induction with EGF,while the EGFR phosphotyrosine content induced by

FIG. 2. Time-dependent EGFR tyrosine phosphorylation andp21/CIP1 induction by EGF or TGFa. A431 cells were incubated for48 h in DMEM with 10% FCS, as described under Materials andMethods. Then the cells were chilled on ice and 1028 M EGF or TGFawas added. After 10 min on ice the cells were washed three timeswith ice-cold DMEM to remove unbound ligand, and the cells weresubsequently incubated with DMEM at 37°C for the indicated timeperiods. The cells were washed and lysed as described under Mate-rials and Methods, and the lysed cells were subjected to SDS–PAGEand electrotransferred to nitrocellulose membranes. The membraneswere incubated with an antibody specifically recognizing phosphoty-rosine (A) or with an antibody to p21/CIP1 (B), as described underMaterials and Methods.

FIG. 3. Effect of monensin on EGFR tyrosine phosphorylation and on induction of p21/CIP1 by TGFa and EGF. A431 cells were incubated inDMEM with 10% FCS for 48 h, as described under Materials and Methods. The cells were incubated with or without 10 mM monensin for 10 minbefore addition of 1029 M TGFa (A) or 1029 M EGF (B) and then were incubated for the indicated time periods. Then the cells were washed, lysed,and subjected to SDS–PAGE, as described under Materials and Methods. The proteins were electrotransferred to nitrocellulose membranes, andthe membranes were incubated with antibodies to phosphotyrosine and p21/CIP1 as described in the legend to Fig. 2.

164 SKARPEN ET AL.

EGF was equally sustained with or without increasedendosomal pH.

We further studied the effect of monensin on theexpression of p21/CIP1. While TGFa in the presence ofmonensin increased the level of p21/CIP1 and caused asustained increase in p21/CIP1 compared to in theabsence of monensin (Fig. 3A), there was no additionalincrease in the level of p21/CIP1 when cells were incu-bated in the presence of monensin and stimulated withEGF (Fig. 3B).

Since it has previously been demonstrated that thelevel of p21/CIP1 correlated with suppression of DNAsynthesis [8–10], we studied the effects of EGF andTGFa on DNA synthesis in the absence or presence ofmonensin as a function of incubation time. As can beseen from Fig. 4A, 1029 M EGF decreased DNA syn-thesis in the absence of monensin more rapidly andmore efficiently than TGFa, while both ligands inhib-ited DNA synthesis with the same time course andefficiency in the presence of monensin (Fig. 4A). Thedifference in suppression of DNA synthesis correlated

with the different level of p21/CIP1, as demonstratedin Fig. 4B. In the absence of monensin the level ofp21/CIP1 was consistently increased more by EGFthan by TGFa. In the presence of monensin p21/CIP1was increased similarly by EGF and TGFa (data notshown). This illustrates that the increase in phospho-tyrosine content caused by neutralization of endosomalpH had functional consequences both with regard tothe efficiency of the p21/CIP1 gene induction and withregard to the suppression of DNA synthesis upon ad-dition of TGFa. Control experiments showed that ad-dition of monensin did not affect the initial tyrosinephosphorylation of the EGFR induced by either EGF orTGFa on ice (data not shown).

Effect of acid stripping and treatment with bafilomy-cin A1 on TGFa-mediated reduction in EGFR phos-phorylation, expression of p21/CIP1, and DNA synthe-sis. In order to more directly assay the effect of endo-cytosed and phosphorylated EGFR on expression ofp21/CIP1 and DNA synthesis, we acid-stripped TGFa-

FIG. 4. (A) Time-dependent suppression of DNA synthesis by EGF or TGFa in the absence or presence of monensin. A431 cells weregrown in DMEM with 10% FCS for 48 h as described under Materials and Methods. Then the cells were incubated in the absence or presenceof 10 mM monensin for 10 min before 1029 M EGF or TGFa was added. The cells were incubated with or without monensin in the presenceof ligand for 2, 5, 10, 15, and 24 h. 1 mCi/ml [3H]thymidine was present during the last 2 h of the incubation. The TCA-precipitableradioactivity was measured as described under Materials and Methods. The data represent the means of four independent experiments witherror bars. (B) Time-dependent induction of p21/CIP1 by EGF or TGFa. 1029 M EGF or TGFa was added to cells, and the cells were incubatedfor the indicated time periods. Then the cells were lysed and exposed to Western blotting with an antibody to p21/CIP1 as described in thelegend to Fig. 2.

165EGF RECEPTOR SIGNALING AFTER ENDOCYTOSIS

treated cells in the presence or absence of bafilomycinA1. Bafilomycin A1 inhibits the proton ATPase respon-sible for acidification of endosomal compartments [30],and in the presence of bafilomycin A1 the pH of endo-somes and lysosomes will therefore be neutral even ifcells are incubated in medium with low pH. This treat-ment should therefore remove TGFa only from surface-localized EGFR while intracellular TGFa–EGFR com-plexes should not be dissociated (see Fig. 5). This is incontrast to monensin, which dissipates pH gradientsdue to exchange with cations. The endosomal pH willpresumably be acidic during incubation of cells withlow pH and monensin.

In order to study the effect of ligand-bound endocy-tosed EGFR on the induction of p21/CIP1, the cellswere preincubated with or without 0.1 mM bafilomycinA1 for 10 min before addition of 1028 M TGFa andfurther incubation at 37°C for 60 min. The cells werethen incubated with MEM, pH 6.0, with or withoutbafilomycin A1 at 37°C for 10 min in order to removesurface-bound ligand. In one experiment the cells werelysed and subjected to SDS–PAGE and Western blot-ting with an antibody to phosphotyrosine, while inanother experiment the cells were further incubatedwith DMEM at 37°C in the presence or absence ofbafilomycin A1. In the later experiment the cells werelysed after 6 h and subjected to SDS–PAGE and West-ern blotting with an antibody to p21/CIP1. As shown inFig. 6, in acid-stripped cells TGFa-stimulated EGFRwas significantly phosphorylated in the presence ofbafilomycin A1 (lanes 4 and 5), while in the absence ofbafilomycin A1, the EGFR was only slightly phosphor-ylated (lanes 2 and 3) compared to cells treated withlow pH only (lane 1) and to cells treated with low pHand bafilomycin A1 in the absence of ligand (lane 6).

After 6 h of incubation, p21/CIP1 was furthermoresignificantly more induced in the presence of bafilomy-cin A1 (lanes 4 and 5) compared to in the absence ofbafilomycin A1 (lanes 2 and 3). The effect of bafilomy-cin A1 on TGFa-mediated suppression of DNA synthe-sis was studied 24 h after acid stripping. As shown inFig. 7, DNA synthesis was strongly inhibited by 1028

M TGFa in acid-treated cells only in the presence ofbafilomycin A1. When bafilomycin A1 was replaced bymonensin during acid stripping, DNA synthesis wasnot inhibited.

Endocytosis and sorting of EGF–EGFR in the ab-sence and presence of drugs neutralizing endosomalpH. As it has previously been demonstrated thatEGF–EGFR complexes can indeed recycle to the cellsurface upon internalization in A431 cells [31], weinvestigated whether complexes of EGF and EGFRwere recycled under our experimental conditions.This was important in order to exclude the possibil-ity that the more potent effect of EGF compared toTGFa could be explained by recycling of EGF–EGFRcomplexes. 125I-EGF was bound to cells on ice for 10min. The cells were washed and subsequently chasedat 37°C for the times indicated in Fig. 8. Potentialrecycling of the EGF was assayed by measuringTCA-precipitable radioactivity released to the me-dium. Additionally, surface-localized EGF was re-leased from the EGFR by low-pH treatment, as pre-viously described [31]. As shown in Fig. 8, 125I-EGFbound to cells on ice was rapidly internalized both inthe absence (Fig. 8A) and in the presence (Fig. 8B) ofbafilomycin A1, and there was no detectable recy-cling of 125I-EGF in either case. In the absence ofbafilomycin A1, TCA-soluble material was released

FIG. 5. Schematic drawing illustrating the effect of low pH on EGFR–TGFa interaction in the absence and presence of bafilomycin A1.Endosomal pH becomes acidic due to proton ATPases in the limiting membrane. This acidification is efficiently counteracted by bafilomycin A1 bothat pH 6.0 and pH 7.4 in the surrounding medium. TGFa has been reported to dissociate from the EGFR at slight reduction in pH [23].

166 SKARPEN ET AL.

to the medium after a 30-min chase, and after 3 happroximately 70% of the initially bound 125I-EGFhad been degraded (Fig. 8A), consistent with thetransport of EGF–EGFR complexes to lysosomes. Incontrast, in the presence of bafilomycin A1, no deg-radation of 125I-EGF could be detected (Fig. 8B).

In order to investigate whether EGF–EGFR com-plexes would recycle when endosomal pH was neutral-ized, we added 1028 M EGF to cells preincubated withmonensin. After 60 min the cells were fixed and incu-bated with antibodies recognizing all EGFR or onlyligand-bound EGFR (activated EGFR). Two-color im-munofluorescence staining (Fig. 9) showed that in thepresence of monensin, activated EGFR localized mainlyto vesicular-like structures. This indicates that EGF–EGFR complexes under these conditions are upconcen-trated in endosomes and do not significantly recycle.This is consistent with previous reports demonstratingthat monensin in fact inhibits recycling of EGF–EGFRcomplexes [23, 32, 33].

Activation of MAPK by EGF and TGFa. Effects ofdifferent concentrations of EGF on DNA synthesis inA431 cells have previously been explained by differ-ent activation of MAPK [34, 35]. We therefore inves-

tigated whether EGF and TGFa activated MAPKdifferently at 1029 M at which the DNA synthesiswas suppressed to a different extent (see Fig. 4A).A431 cells in microtiter plates were incubated withand without 1029 M EGF or TGFa for increasingtime periods and exposed to Western blotting. MAPKactivation was indirectly detected with an antibodyreacting specifically to the phosphorylated forms oferk 1 and erk 2. It should be noted that erk 2 ispresent in a much higher concentration than erk 1 inA431 cells, and phosphorylated erk 1 is hardly visi-ble in Fig. 10A. There was a transient increase inMAPK activity, peaking at 5 min following additionof ligand (Figs. 10A and 10B). However, there wereno ligand-specific differences at the indicated timepoints in reactivity to phosphorylated erk1/erk2when 1029 M EGF or TGFa had been added to thecells (Fig. 10A). We also measured the activity ofMAPK using myelin basic protein as substrate. Asshown in Fig. 10B, there was a time-dependent acti-vation of MAPK, with maximal activity after 5 min ofincubation with either ligand at 1029 and 1028 M.However, there was no ligand-specific difference inactivation of MAPK.

FIG. 6. Effect of TGFa on EGFR tyrosine phosphorylation and induction of p21/CIP1 upon acid stripping in the presence or absence ofbafilomycin A1. A431 cells were grown in DMEM with 10% FCS for 48 h as described under Materials and Methods. The cells were incubatedwith or without 0.1 mM bafilomycin A1 for 10 min at 37°C prior to addition of 1028 M TGFa. After 60 min incubation with or without ligand,the medium was removed and replaced by MEM adjusted to pH 6.0 with or without 0.1 mM bafilomycin A1. The cells were acid-stripped for10 min at 37°C with or without bafilomycin A1. In one case the cells were washed, lysed, and subjected to SDS–PAGE and Western blottingwith an antibody to phosphotyrosine as described in the legend to Fig. 2. In another case the cells were incubated in DMEM with or withoutbafilomycin A1 for 6 h following acid stripping and were subsequently washed, lysed, and subjected to SDS–PAGE and Western blotting usingan antibody to p21/CIP1 as described (legend to Fig. 2).

167EGF RECEPTOR SIGNALING AFTER ENDOCYTOSIS

DISCUSSION

In this study we have addressed the question ofwhether endocytosed EGFR is capable of completing afunctional signaling cascade all the way to altered geneexpression. We have taken advantage of the findingsthat the two ligands EGF and TGFa have differentisoelectric points [23] and that TGFa presumably dis-sociates from the EGFR in early endosomes, while EGFis receptor associated all the way to lysosomes. Theprevious observation that EGFR becomes dephospho-rylated upon loosing its bound ligand [18] is an impor-tant prerequisite for our studies. We found that bothEGF and TGFa inhibited DNA synthesis in A431 cells.However, EGF inhibited DNA synthesis more effi-ciently than did TGFa and caused a more sustainedphosphorylation of the EGFR and a higher expressionof the CDK inhibitor p21. This is consistent with alonger lasting signal, again explained by the possibilitythat EGF affects signaling also after internalization.We found that the difference in effect between EGF and

TGFa was abolished by treatment of cells with the car-boxylic ionophore monensin. This is consistent with thenotion that monensin inhibits dissociation of TGFa fromthe endocytosed EGFR by neutralizing pH. When EGFwas added, the tyrosine phosphorylation of the EGFRwas not changed significantly, depending on whethermonensin was present or not. Acid-stripping of ligandin TGFa-treated cells with or without bafilomycin A1showed that in the presence of bafilomycin A1, the endo-cytosed EGFR was not dephosphorylated, demonstratingthat TGFa did not dissociate from the receptor at neutralpH. Ligand-bound endocytosed EGFR was capable of ef-ficiently inducing p21/CIP1 and thereby reducing DNAsynthesis. By this work we have demonstrated that ty-rosine-phosphorylated EGFR can induce expression ofp21/CIP1 from endocytic compartments.

EGF–EGFR complexes have previously been demon-strated to persist for a long time period in the para-Golgiregion in A431 cells [26], and tyrosine-phosphorylatedintracellular EGFRs have been described [18]. After im-

FIG. 7. Effect of TGFa on DNA synthesis upon acid stripping in the presence or absence of bafilomycin A1. A431 cells were grown inDMEM with 10% FCS for 48 h as described under Materials and Methods. The cells were incubated with or without 0.1 mM bafilomycin A1for 10 min at 37°C prior to addition of 1028 M TGFa. After 60 min the medium was removed and the cells were incubated with MEM adjustedto pH 6.0 with or without 0.1 mM bafilomycin A1 at 37°C for 10 min. The low-pH medium was removed and DMEM with or without 0.1 mMbafilomycin A1 was added. After 20 h 1 mCi/ml [3H]thymidine was added, and the cells were incubated for 4 h more. The TCA-precipitableradioactivity was measured as described under Materials and Methods. The left bar represents cpm in cells treated with TGFa only aspercentage of cpm in nontreated cells, whereas the right bar represents cpm in cells treated with TGFa and bafilomycin as percentage of cpmin cells treated with bafilomycin A1 only.

168 SKARPEN ET AL.

munoprecipitation of the tyrosine-phosphorylated EGFRfrom endosomal fractions, a 55-kDa protein was copre-cipitated and was termed pyp55 [36]. This protein wassubsequently identified as the adaptor protein shc [37].Furthermore, a complex of phosphorylated EGFR, shc,the adaptor protein Grb2, and associated mSOS wasfound enriched in the endosomal fraction of EGF-treatedhepatocytes [20]. It has, however, not been previouslydemonstrated that endocytosed EGFR can in fact induceexpression of a defined gene product. We have demon-strated induction of p21/CIP1 from endocytosed EGFR,and this together with the demonstration that endocy-

tosed EGFR is capable of suppressing DNA synthesis isimportant in proving the endosome is a functional locusfor signal transduction.

Our data further emphasize the correlation betweenEGFR tyrosine phosphorylation and inhibition of DNAsynthesis in A431 cells. The data suggest that theendocytosed EGFR actively transduces signals impor-tant for growth suppression by way of increased p21levels. The mechanisms whereby EGF suppressesgrowth in A431 cells have been studied extensively,and several possible explanations have been sug-gested. Chajry et al. [34, 35] reported a relationship

FIG. 8. Internalization and degradation of 125I-EGF. A431 cells were incubated without (A) or with (B) 0.1 mM bafilomycin A1 asdescribed under Materials and Methods. 1028 M 125I-EGF was added to the cells on ice. After binding on ice for 10 min the cells were washedand chased at 37°C as described under Materials and Methods. The cells were treated with low-pH buffer to release EGFR-boundsurface-localized 125I-EGF. At the indicated time points the cells were analyzed for surface-bound (E) and intracellular (F) 125I-EGF, and themedium was analyzed for released 125I-EGF (�) and degraded 125I-EGF (‚) as described under Materials and Methods. Each point representsthe mean of two parallels of one typical experiment. The data are expressed as percentage of cpm 125I-EGF initially bound.

FIG. 9. Localization of ligand-bound EGFR in the presence of monensin. A431 cells were grown in DMEM with 10% FCS for 24 h asdescribed under Materials and Methods. The cells were incubated with 10 mM monensin for 10 min at 37°C prior to addition of 1028 M EGF.Then the cells were incubated for 1 h at 37°C. The medium was removed and the cells were fixed in ethanol and stained by two-colorimmunofluorescence with antibodies to EGFR and ligand-bound EGFR as described under Materials and Methods. The antibody to EGFR(A) stained both in the plasma membrane and intracellularly, whereas antibody to ligand-bound EGFR (B) gave only a strong punctateintracellular staining, consistent with endosomal localization of the activated EGFR. (C) The confocal laser scan images of EGFR (green color)and ligand-bound EGFR (red color) were overlayed. Colocalization is seen as yellow staining. The bars in A and B indicate 5 mm.

169EGF RECEPTOR SIGNALING AFTER ENDOCYTOSIS

between the stimulatory/inhibitory effect of EGF andthe activity of the serine/threonine mitogen-activatedprotein kinase. A moderate, but persistent, activationof MAPK at growth-stimulatory concentrations wasreported, while an early peak of MAPK activation,rapidly falling below the basal level, was observed at

growth-inhibitory concentrations of EGF. It was fur-ther reported that the EGF-dependent inhibition ofMAPK was caused by protein tyrosine phosphatase 2A[35]. It has previously been speculated that the dura-tion of the MAPK signal is important for determiningwhether cells proliferate or differentiate (see [38] forreview). As EGF and TGFa in our experiments gave asimilar initial activation and time course of erk 1 anderk 2, while the two ligands affected DNA synthesisdifferently, our results do not support a role of MAPKregulation in the growth inhibition in A431 cells.

Grb2 and mSOS were reported to complex with en-docytosed EGFR in EGF-treated hepatocytes [20], andit was recently demonstrated that activation of MAPKwas suppressed when endocytosis was inhibited in aHeLa cell line stably transfected with a dominant neg-ative interfering dynamin mutant [21]. Together, theavailable information supports the notion that intra-cellular trafficking of growth factor receptors is impor-tant in controlling mitogenic signaling. However, wedid not observe any difference between EGF and TGFawith respect to activation of MAPK, suggesting thatMAPK in our system is activated mainly from the cellsurface. There is a very high number of EGFR in A431cells [39, 40], and the MAPK pathway could thereforebe saturated at ligand concentrations not being satu-rated with respect to binding and tyrosine phosphory-lation of the EGFR. Our finding that 1028 and 1029 MEGF activated MAPK to the same extent supports thisnotion. Furthermore, MAPK was activated very rap-idly, with a maximal response within 10 min of addedligand (our own data and [34]). In contrast, phosphor-ylated EGFRs are endocytosed relatively slowly inA431 cells at high receptor occupancy [31, 41]. Afterbinding of EGF to EGFRs, the occupied receptors werefound to cluster to each other, and these clusters local-ized over coated pits on the cell surface before endocy-tosis [42–45]. In cells with few EGFR, like fibroblasts,the endocytosis has been reported to be faster, andthere was no evidence that a receptor–receptor cluster-ing was required for EGF-induced internalization [46,47]. The different kinetics of endocytosis could there-fore explain why activation of MAPK was apparentlyaffected by endocytosis in some cells [21] and not inothers. Clearly, cell-type-specific differences are ex-pected to be found, since stoichiometric relations be-tween receptors and proteins involved in signaling andin intracellular trafficking vary between cells.

Our finding that TGFa gave an increased expressionof the cyclin-dependent kinase inhibitor p21/CIP1 inthe presence, but not in the absence, of bafilomycin A1following acid stripping of ligand from surface-local-ized receptors indicates that the EGF receptor recruitssecond messengers after endocytosis and actively sig-nals from endosomes as long as the receptor is ligandbound. Since p21/CIP1 was suggested to be induced by

FIG. 10. Activation of MAPK by EGF and TGFa as a function oftime. (A) A431 cells were cultured in DMEM with 10% FCS for 48 h(as described under Materials and Methods) prior to incubation with1029 M EGF (lanes 1, 3, 5, 7, 9), 1029 M TGFa (lanes 2, 4, 6, 8, 10),or no ligand (lane 11). At the indicated times the cells were washed,lysed, and subjected to 10% SDS–PAGE. The proteins were electro-transferred to nitrocellulose membranes, and phosphorylated erk1(44 kDa) and erk2 (42 kDa) were identified using an antibody tophosphorylated erk, as described under Materials and Methods. (B)A431 cells were incubated for 48 h in DMEM with 10% FCS asdescribed under Materials and Methods. Then the cells were incu-bated with 1028 and 1029 M EGF or TGFa for 5 min, 30 min, and24 h or incubated without added ligand for 24 h. Then the cells werewashed and lysed as described under Materials and Methods. TheMAPK was purified by phenyl Sepharose-affinity chromatographyand eluted with 60% ethylene glycol. Then the MAPK activity wasmeasured using [g-32P]ATP and MBP as substrate. RadiolabeledMBP was spotted onto Whatman paper, and the radioactivity wasmeasured by liquid scintillation counting as described under Mate-rials and Methods.

170 SKARPEN ET AL.

STAT1 in A431 cells [8], it will be of great interest toinvestigate in more detail how STAT1 is activated inA431 cells. Regulation of p21/CIP1 turns out to becomplicated, and we are currently studying whetherthe main regulation of p21/CIP1 upon addition of EGFis exerted at the transcriptional, posttranscriptional,or posttranslational level. Stabilization of p21-encod-ing mRNA has been reported to occur in a p53-inde-pendent way after addition of tumor necrosis factor aand after addition of interleukin-1 [48, 49], and stabi-lization of the p21 protein was described to occur inpreadipocyte differentiation [50].

In conclusion, we have confirmed that growth factorreceptors are tyrosine phosphorylated upon endocyto-sis when bound to ligand. Additionally, we have dem-onstrated that endocytosed receptors can indeed altergene transcription. This indicates that growth factorreceptors are functional in intracellular vesicles andthat growth can be affected by conditions alteringmembrane trafficking. Our data support the notionthat the EGFR can actively transduce signals bothfrom the cell surface and following endocytosis. It re-mains to be seen whether the specificity of the signal-ing differs depending on the origin of the signal.

This work was supported by the Norwegian Research Council forScience and the Humanities, the Norwegian Cancer Society, theNovo Nordic Foundation, Bruuns Legacy, Blix Legacy, Torsted’sLegacy, and Anders Jahres Foundation. We are grateful to BjørgFjell and Arnhild Tollefsrud for expert technical assistance.

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Received January 28, 1998

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