Mechanism of Desensitization of the Epidermal Growth Factor ...

T H E JOURNAL OF BIOLOGICAL CHEMISTRY 0 1992 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 267, No. 2, Issue of January 15, pp. 1129-1140,1992 Printed in U. S. A .

Mechanism of Desensitization of the Epidermal Growth Factor Receptor Protein-Tyrosine Kinase*

(Received for publication, July 9, 1991)

Janice L. CountawayS, Angus C. Nairng, and Roger J. Davis$T(( From the YHoward Hughes Medical Institute, $Program in Molecular Medicine, $Department of Biochemistry & Molecular Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01605 and the §Laboratory of Molecular & Cellular Neuroscience, Rockefeller University, New York, New York 10021-6399

The intrinsic protein-tyrosine kinase activity of the epidermal growth factor (EGF) receptor is required for signal transduction. Increased protein-tyrosine kinase activity is observed following the binding of EGF to the receptor. However, signaling is rapidly desensitized during EGF treatment. We report that EGF receptors isolated from desensitized cells exhibit a lower protein-tyrosine kinase activity than EGF receptors isolated from control cells. The mechanism of desensitization of kinase activity can be accounted for, in part, by the EGF-stimulated phosphorylation of the receptor at Ser'048'7, a substrate for the multifunctional calmodulin-dependent protein kinase I1 in vitro. Mu- tation of Ser'046/7 by replacement with Ala residues blocks desensitization of the EGF receptor protein- tyrosine kinase activity. Furthermore, this mutation causes a marked inhibition of the EGF-stimulated endocytosis and down-regulation of cell surface receptors. Thus, the phosphorylation site Ser'046/7 is required for EGF receptor desensitization in EGF- treated cells. This regulatory phosphorylation site is located at the carboxyl terminus of the EGF receptor within the subdomain that binds src homology 2 regions of signaling molecules.

The epidermal growth factor (EGF)' receptor is a 170-kDa transmembrane glycoprotein (reviewed by Ullrich and Schles- singer (1990)). The receptor is composed of an extracellular domain that binds EGF, a single transmembrane spanning domain, and a cytoplasmic domain with intrinsic protein- tyrosine kinase activity. Mutational analysis of the EGF receptor has demonstrated that the protein-tyrosine kinase activity is required for signal transduction (Honegger et al., 1987b; Chen et al., 1987). The binding of EGF to the extracellular domain of the receptor causes a stimulation of the cytoplasmic domain protein-tyrosine kinase activity (Ushiro

*This work was supported by Public Health Service Grants CA39240 and GM37845 from the National Institutes of Health and by a postdoctoral fellowship (to J. L. C.) from the American Cancer Society. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

)I To whom correspondence should be sent: Howard Hughes Med- ical Institute, Program in Molecular Medicine, University of Massa- chusetts Medical School, 373 Plantation St., Worcester, MA 01605.

I The abbreviations used are: EGF, epidermal growth factor; CAM kinase 11, calcium- and calmodulin-dependent protein kinase 11; CHO, Chinese hamster ovary; EGTA, [ethylenebis(oxyethylenenitrilo) Jtet- raacetic acid; HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesul- fonic acid; HPLC, high pressure liquid chromatography; Ser1"6'7, Ser1046 and Ser1047.

and Cohen, 1980). This process has been proposed to be mediated by an allosteric mechanism that involves receptor dimerization (Schlessinger, 1988). The activated receptor protein-tyrosine kinase causes phosphorylation of 1) a carboxyl- terminal subdomain of the EGF receptor (Downward et al., 1984; Margolis et al., 1989a; Walton et al., 1990)) and 2) exogenous substrate proteins. Examples of exogenous substrates that are phosphorylated by the EGF receptor protein- tyrosine kinase include GTPase-activating protein (Ellis et al., 1990; Liu and Pawson, 1991) and phospholipase C-y (Margolis et al., 198913; Meisenhelder et al., 1989; Nishibe et al., 1990; Kim et al., 1990; Wahl et al., 1990). The tyrosine phosphorylation of these substrate proteins by the EGF receptor is thought to be an important initial step in signal transduction (Ullrich and Schlessinger, 1990).

Desensitization is a process that is common to many receptors (Sibley et al., 1987). This desensitization serves an important physiological function in the control of receptor signaling. One mechanism of long term (hours) desensitization of the EGF receptor is mediated by the internalization and degradation of the receptor (Stoscheck and Carpenter, 1984). In addition, there exists a rapid (minutes) mechanism of EGF- induced EGF receptor desensitization (Davis and Czech, 1986; Cunningham et al., 1989). The mechanism of acute desensitization is not understood. Several reports have indicated that the tyrosine autophosphorylation of EGF receptors isolated from EGF-treated cells is reduced as compared with receptors isolated from control cells (Chinkers and Garbers, 1986; Lai et al., 1989; McCune et al., 1990). These observations suggest that desensitization is associated with an inhibition of the EGF receptor protein-tyrosine kinase activity. However, a direct experimental demonstration of EGF-induced desensitization of the EGF receptor kinase activity has not been reported.

Whiteley and Glaser (1986) have proposed that one potential mechanism of EGF-induced desensitization of the EGF receptor could be mediated by the phosphorylation of the EGF receptor at Thr654 by protein kinase C. This hypothesis is supported by biochemical evidence that demonstrates that the phosphorylation of the EGF receptor at Thr654 causes an inhibition of the receptor protein-tyrosine kinase activity (Friedman et al., 1984; Cochet et al., 1984; Hunter et al., 1984; Davis and Czech, 1985a; Davis et al., 1985a, 1985b; Northwood and Davis, 1989). Furthermore, site-directed mutagenesis of the EGF receptor demonstrates that the replacement of T h P 4 with Ala blocks the protein kinase C-mediated inhibition of the EGF receptor protein-tyrosine kinase activity (Davis, 1988; Countaway et al., 1990; Decker et al., 1990; Lund et al., 1990; Bowen et al., 1991). The hypothesis that there is feed- back inhibition of EGF receptor signal transduction caused by protein kinase C is therefore reasonable. However, on

1129

1130 CAM Kinase II Regulation of the EGF Receptor

closer inspection this hypothesis fails two critical tests. 1) EGF does not markedly stimulate protein kinase C in cells of mesenchymal origin (reviewed by Rozengurt (1986)). 2) Direct measurement of the stoichiometry of phosphorylation of the EGF receptor at Thr654 indicates that EGF causes only a modest and transient increase in phosphorylation at this site (Bowen et al., 1991).

Together, these considerations indicate that the phosphorylation of the EGF receptor at Thr654 may not fully account for EGF-induced EGF receptor desensitization. We have therefore considered alternative mechanisms of desensitization. Substantial evidence for an alternative mechanism of desensitization has been previously reported. For example, Friedman et al. (1989) have reported that the tumor promoter thapsigargin (which increases cytosolic Ca2+) and the calcium ionophore A23187 can inhibit EGF receptor tyrosine phosphorylation in the absence of phosphorylation of the receptor a t ThP4. A second example is provided by the observation that under some conditions phorbol ester can inhibit EGF receptor tyrosine phosphorylation by a mechanism that is independent of Thr654 phosphorylation (Livneh et aL, 1988; Lund et al., 1990). Significantly, the deletion of the carboxyl- terminal region of the EGF receptor blocks the Thr654-independent regulation of receptor tyrosine phosphorylation caused by very high concentrations of phorbol ester (Lund et al., 1990). This observation suggests that sequences within the carboxyl-terminal domain of the EGF receptor may account for the observed Thr654-independent desensitization. Previous studies have demonstrated that this carboxyl-terminal region is an important effector domain of the EGF receptor that binds to SH2 regions of signaling molecules (Margolis et al., 199Ob; Skolnik et al., 1991; Koch et al., 1991). Significantly, this carboxyl-terminal domain is also the location of the major site of EGF-stimulated serine phosphorylation of the EGF receptor, Ser'046/7 (Heisermann and Gill, 1988; Countaway et al., 1990).

The purpose of the experiments reported here was to investigate the mechanism of EGF-induced desensitization of EGF receptor signaling. The experimental approach that we employed was to measure the protein-tyrosine kinase activity of EGF receptors isolated from control and EGF-treated cells using an exogenous substrate. We report that the treatment of cells with EGF causes an inhibition of the EGF-stimulated receptor protein-tyrosine kinase activity. Furthermore, we show that the mechanism of desensitization of the EGF receptor protein-tyrosine kinase activity is mediated by the phosphorylation of the receptor at a negative regulatory site

Phosphorylation of the EGF receptor at this site in vitro by CAM kinase I1 causes an inhibition of the receptor protein- tyrosine kinase activity.

( Ser1046/7 ) located at the carboxyl terminus of the receptor.

EXPERIMENTAL PROCEDURES

Materials-CAM kinase I1 was purified from rat forebrain as described (McGuiness et al., 1985). 1 unit of CAM kinase I1 activity was defined as the amount of enzyme that phosphorylates 1 pmol/ min of the substrate peptide Syntide 2 (1 mg/ml) at 22 "C. Rabbit muscle enolase and phorbol myristate acetate were from Sigma. Calmodulin was isolated from rabbit brain as described (Grand et al., 1979). The synthetic peptides PLARTLSVAGLPGKK (Syntide 2) and KRTLRR were purchased from GIBCO-BRL and Peninsula Laboratories, Inc., respectively. The synthetic peptides RRLIEDAE- YAARG and RRFLQRYSSDPTGAL were obtained from the Peptide Synthesis Core Facility, University of Massachusetts Medical School. Murine EGF was purified as described (Savage and Cohen, 1972; Matrisian et al., 1982) and iodinated to a specific activity of 70-90 Ci/g (Pessin et al., 1983). [Y-~'P]ATP was prepared from [32P]phosphate (Du Pont-New England Nuclear) using a Gamma-Prep A kit (Promega Biotech), according to the manufacturer's directions. NalZ5I

and [35S]methionine were from Amersham International PLC. "'11- Goat anti-mouse Ig antibody and lZ5I-protein A were purchased from Du Pont-New England Nuclear. The monoclonal antibody PY20 was from ICN Biomedicals, Inc. The rabbit anti-human EGF receptor antibody has been described previously (Davis and Czech, 1985a). Membranes were prepared from tissue culture cells as described (Davis and Czech, 1985b).

Construction and Purification of Bacterially Expressed Fusion Pro- teins-The plasmid pGEX-TK6 has been described by Koland et al. (1990). This plasmid directs the synthesis of a protein in bacteria that consists of EGF receptor cytoplasmic domain sequences fused to glutathione-S-transferase. Site-directed mutagenesis was used to re- place SeP4'j and Ser"" with alanine residues (Countaway et al., 1990). The resulting plasmid was designated pGEX-[A1"6/7]TK6. The fusion proteins (TK6 and [A1046/']TK6) were expressed and purified as described by Koland et al. (1990), except that Triton X-100 was not used during glutathione-agarose affinity chromatography.

Tissue Culture-CHO cells expressing wild-type and mutated human EGF receptors have been described previously (Countaway et al., 1990). The cells were maintained in Ham's F12 medium supplemented with 5% fetal bovine serum (GIBCO-BRL). A431 human epidermoid carcinoma cells were cultured in Dulbecco's modified Eagle's medium supplemented with 5% calf serum (GIBCO-BRL).

Expression of a Protein-Tyrosine Kinase Defective EGF Receptor in CHO Cells-Oligonucleotide-directed mutagenesis of Lys'" to Arg was carried out using a 17-mer oligonucleotide (5' gct atc agg gaa tta ag 3'), according to Zoller and Smith (1984) using methods described previously (Davis and Meisner, 1987). The mutation was confirmed by sequencing, using ["SIdATP, dideoxy NTPs, and Sequenase (San- ger et al., 1977). The mutated EGF receptor cDNA was then cloned as a 4-kilobase XbaI-Hind111 fragment into the plasmid pX (Coun- taway et al., 1989). This expression vector contains the murine dihydrofolate reductase gene as a selectable marker and allows the expression of the EGF receptor cDNA using the SV40 early promoter and polyadenylation signals (Countaway et al., 1989). This plasmid was designated pXER(Arg7"). CHO cells expressing [Arg7"]EGF receptors were obtained by transfection with the plasmid pXER(Arg'") using the calcium phosphate technique. After 3 days, the cells were passaged and selected using minimal essential medium- (Y supplemented with 5% dialyzed fetal bovine serum and 1 p M amethopterin. Stable colonies were isolated using cloning rings and screened for the expression of EGF receptors by measuring the cell surface binding of Iz51-EGF at 4 "C. Control experiments demonstrated that the [Arg'"]EGF receptor did not express protein-tyrosine kinase activity.

Phosphorylation of Synthetic Peptides by CAM Kinase II-Syn- thetic peptides (1 mg/ml) were incubated with 1 microunit of CAM kinase I1 in 25 mM HEPES, 10 mM MgC12, 0.4 mM EGTA, and 0.6 p~ calmodulin in the presence and absence of 1 mM CaC12 (final volume, 50 pl). The phosphorylation reactions were initiated by the addition of 20 p~ [Y-~'P]ATP (50 pCi/nmol), and the assays were terminated after 10 min at 22 "C by the addition of 5 pl of 250 mM EDTA, 10 mg/ml bovine serum albumin. The samples were placed on ice, and 20 pl of 50% (w/v) trichloroacetic acid was added. After incubation for 60 min at 4 "C, the samples were centrifuged for 10 min in a microcentrifuge. Aliquots of the supernatant were spotted on phosphocellulose paper (Whatman P-81). The phosphocellulose paper was washed three times with 1 M acetic acid, 4 mM sodium pyrophosphate, and the radioactivity associated with the filter paper was measured using a liquid scintillation counter. Nonspecific incorporation of radioactivity was determined in control incubations without synthetic peptide.

Immune Complex Protein-Tyrosine Kinase Assays-CHO cells expressing human EGF receptors were seeded in 35-mm wells and grown to a density of 2 X lo5 cells/well. The cells were washed with 120 mM NaC1,6 mM KC1,1.2 mM CaC12,l mM MgC12,25 mM HEPES (pH 7.4) and incubated for 30 min in Ham's F-12 medium. The cells were treated without or with EGF at 37 "C and subsequently lysed in 50 mM HEPES (pH 7.4), 50 mM NaCl, 1% Triton x-100, 10 mM EGTA, 50 mM NaF, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, and 10 pg/ml leupeptin. In some cases, 0.1 mM sodium orthovanadate was included in the lysis buffer. Cell extracts were centrifuged at 100,000 X g for 30 min, and EGF receptors were isolated by immunoprecipitation of the supernatant with the monoclonal antibody m108.1 prebound to protein-A Sepharose as described (Honegger et al., 1987a). These immunoprecipitates were employed to investigate EGF receptor autophosphorylation and the tyrosine phosphorylation of an exogenous substrate.

CAM Kinase 11 Regulation of the EGF Receptor 1131

In vitro autophosphorylation of the EGF receptors was measured using an immune complex kinase assay. The immunoprecipitates bound to protein A-Sepharose were resuspended in 30 pl of HNTG buffer (50 mM HEPES, pH 7.4, 50 mM NaCl, 0.1% Triton X-100, 10% glycerol) supplemented with 100 nM EGF, 5 mM MgC12, 20 mM MnC12, and 10 pg/ml leupeptin. The phosphorylation reaction (4 "C) was initiated by the addition of 10 pl of 50 p~ ATP, 50 mM HEPES (pH 7.4). After 2 min, the reaction was terminated by the addition of 1 ml of HNTG buffer containing 10 mM EDTA. The immunoprecipitates were washed and analyzed by polyacrylamide gel electrophoresis. Tyrosine phosphorylation of the EGF receptor was investigated by a Western blot procedure using a monoclonal anti-phosphotyrosine antibody (PY20) and lZ5I-goat anti-mouse Ig antibody, as described (Countaway et al., 1990).

The protein-tyrosine kinase activity of the EGF receptor toward exogenous substrates was measured in an immune complex kinase assay using rabbit muscle enolase as a substrate (Cooper et al., 1984). The washed immunoprecipitates bound to protein A-Sepharose were resuspended in 30 p1 of HNTG buffer supplemented with 5 mM MgC12, 20 mM MnC12, 6 p M enolase, and 10 pg/ml leupeptin. The phosphorylation reaction was initiated by the addition of 10 p1 of 100 p~ [Y-~'P]ATP (50 pCi/nmol). After 10 min at 22 "C, the reaction was terminated by the addition of 100 pl of Laemmli sample buffer. The samples were centrifuged in a microcentrifuge (5 min), and 100 pl of the supernatant was removed and heated at 90 "C for 2 min. Phosphorylated proteins were resolved by polyacrylamide gel electrophoresis and visualized by autoradiography. The incorporation of radioactivity into enolase was quantitated by liquid scintillation counting.

CAM Kinase ZI Regulation of the EGF Receptor Protein-Tyrosine Kinase Activity-The effect of CAM kinase I1 on the protein-tyrosine kinase activity of the EGF receptor was investigated using membranes isolated from A431 epidermoid carcinoma cells. Membranes (2 pg of protein) were incubated with 100 nM EGF in 25 mM HEPES (pH 7.4), 10 mM MgCl,, 0.4 mM EGTA (Buffer A) for 15 min at 22 "C in a final volume of 10 pl. Following the preincubation with EGF, the reaction volume was brought to 40 pl by the addition of Buffer A supplemented with 4 microunits of CAM kinase I1 and RR-SRC peptide (1 mg/ml final concentration). The effect of the addition of 1 mM CaC12 and 6 p~ calmodulin to the incubations was investigated. The phosphorylation reaction was initiated by the addition of 10 p1 of 100 p M [y-32P]ATP (50 pCi/nmol). The reaction was stopped after 10 min at 22 "C by the addition of 5 pl of 250 mM EDTA containing 10 mg/ml bovine serum albumin. Protein was precipitated with trichloroacetic acid at 4 "C, and peptide phosphorylation was quantitated by spotting on phosphocellulose paper as described above. Nonspecific incorporation of radioactivity was determined in control incubations without the synthetic peptide RR-SRC.

Analysis of EGF Receptor Tryptic 32P-Labeled Phosphopeptides- A431 membranes (10 pg of protein) were incubated without and with 100 nM EGF at 22 'C in 50 p1 of Buffer A. After 15 min, the samples were diluted with an additional 50 pl of Buffer A supplemented without and with 2 mM CaC12, 12 pM calmodulin, and 10 microunits of CAM kinase 11. The phosphorylation reaction was initiated by the addition of 80 pl of 100 p~ [T-~'P]ATP (90 pCi/nmol) in Buffer A and was terminated after 10 min by the addition of 1 ml of 25 mM HEPES (pH 7.4), 500 mM NaCl, 50 mM NaF, 10 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1 mM phenylmethylsulfonyl fluoride, 0.1 mM sodium orthovanadate, and 10 pg/ml leupeptin. EGF receptors were isolated by immunoprecipitation and polyacrylamide gel electrophoresis as described (Davis and Czech, 1987). The EGF receptors were digested with 1 pg of tosylphenylalanyl chloromethyl ketone-treated trypsin in 100 mM N - ethylmorpholine (pH 8.0). After 5 h, a second addition of trypsin was made, and the incubation was allowed to proceed for a further 19 h. Phosphopeptide mapping of the trypsin-digested EGF receptor was performed by reverse-phase HPLC using a Vydac CIS column (0.46 X 25 cm) equilibrated with 0.1% trifluoroacetic acid (Davis and Czech, 1987). Peptides were eluted with a linear gradient of acetonitrile (0.5%/min) in 0.1% trifluoroacetic acid at a flow rate of 1.5 ml/min. Fractions were collected at 1-min intervals, and the 32P-labeled phosphopeptides were detected by measuring the Cerenkov radiation associated with each fraction. The tryptic 32P-labeled phosphopeptide corresponding to Ser1046'/7 was isolated from [3ZP]phosphate-labeled A431 cells by immunoprecipitation of the EGF receptors, polyacrylamide gel electrophoresis, trypsin digestion, and reverse-phase HPLC, as described by Countaway et al. (1990).

Phosphoamino Acid Analysis-Phosphoamino acid analysis was

performed by partial acid hydrolysis (1 h at 110 "C in 6 M HCl) and thin layer electrophoresis by the method of Hunter and Sefton (1980) as described (Davis et al., 1985a).

Measurement of EGF Receptor Degradation-CHO cells expressing wild-type EGF receptors were seeded in 35-mm dishes and grown to a density of 2 X lo6 cells/well. The medium was aspirated and replaced with 1 ml of methionine-free minimal essential medium (Flow Lab- oratories) supplemented with 1% fetal calf serum and 50 p~ ["SI methionine (50 pCi/ml). The cells were incubated for 18 h. The medium was aspirated and replaced with Ham's F-12 medium. The cells were then treated without and with EGF for defined times. Subsequently, the cells were lysed in 50 mM HEPES (pH 7.4), 50 mM NaCl, 1% Triton X-100, 10 mM EGTA, 50 mM NaF, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 10 pg/ml leupeptin. Cell extracts were centrifuged at 100,000 X g for 30 min, and EGF receptors were isolated by immunoprecipitation using the monoclonal antibody m108.1 prebound to protein-A Sepharose as described (Honegger et al., 1987a). The immunoprecipitates were solubilized with sodium dodecyl sulfate4 and analyzed by polyacrylamide gel electrophoresis. The radioactivity associated with the EGF receptors was visualized and quantitated using a Phosphorimager (Molecular Dynamics).

Analysis of EGF Receptor Oligomerization by Covalent Cross-linking"A431 membranes (5 pg) were incubated in 25 mM HEPES (pH 7.5), 10 mM MgC12, 0.5 mM EGTA. The effect of CAM kinase I1 was investigated in incubations containing 10 microunits of CAM kinase 11, 6 p~ calmodulin, and 1 mM CaC12. The phosphorylation reaction was initiated by the addition of 1 mM ATP. After 10 min at 22 "C, the phosphorylation reaction was terminated by the addition of 70 pl of 25 mM HEPES (pH 7.5), 50 mM NaF, 5 mM EGTA. Subsequently, lZ5I-EGF (1 nM) was added to each incubation. Nonspecific binding of lZ5I-EGF was determined in incubations containing 500 nM EGF. After 30 min, the samples were cooled to 10 "C, and covalent cross- linking was initiated with 0.2 mM disuccinimidyl suberate. The cross- linking reaction was terminated after 15 min by the addition of 1 ml of lysis buffer (100 mM Tris (pH 8.0), 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 50 mM NaF, 0.5 M NaCl, 10 pg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 5 mM EGTA). The samples were incubated for 5 min and then centrifuged at 100,000 X g for 30 min. The supernatant was removed and incubated with 20 p1 of protein A-Sepharose prebound to rabbit anti-EGF receptor antibody. The immune complexes were collected after 60 min by centrifugation and were washed three times with lysis buffer. The immunoprecipitates were solubilized with sodium dodecyl sulfate and analyzed by linear gradient (3-9%) polyacrylamide gel electrophoresis and autoradiography (Northwood and Davis, 1988, 1989).

Measurement of the Cell Surface Expression of the EGF Receptor- CHO cells expressing EGF receptors were seeded in 16-mm dishes and grown to a density of 5 X lo' cells/well. The cells were washed with serum-free medium and incubated for 30 min at 37 "C in 120 mM NaCl, 6 mM KCl, 1.2 mM MgC12, 1 mM CaC12, 25 mM HEPES (pH 7.4), and 30 p~ bovine serum albumin. The cells were incubated with 1 nM EGF for defined times. The medium was removed and rapidly replaced with medium (4 "C) supplemented with 10 pg/ml rabbit anti-EGF receptor antibody (Davis and Czech, 1985a). After 2 h at 4 "C, the monolayers were washed and then incubated with '"I- protein A (2 X lo6 cpm/ml) for an additional 2 h at 4 "C. The monolayers were washed three times with cold medium and solubilized with 900 p1 of 1 M NaOH. Radioactivity associated with the cells was quantitated with a Beckman y counter. Nonspecific binding of the Iz6I-protein A was estimated in incubations without the rabbit anti-EGF receptor antibody. In control experiments using the paren- tal CHO cells that do not express EGF receptors, no specific binding of '251-protein A was detected.

Measurement of the Rate of Internalization of the EGF Receptor- The apparent first-order endocytotic rate constant for EGF receptor internalization was measured using the In/Sur method of Wiley and Cunningham (1982). The method involves the measurement of the rate of intracellular accumulation of ligand under conditions where the number of occupied cell surface receptors is constant and no release of accumulated ligand occurs. CHO cells were seeded in 16- mm wells and grown to a density of 5 X lo4 cells/well. The cells were washed with 120 mM NaC1, 6 mM KC1, 1.2 mM CaC12, 1 mM MgC12, 25 mM HEPES (pH 7.4), 30 p~ bovine serum albumin and subsequently incubated in the same medium for 30 min at 37 "C. The cells were then incubated with 1 nM lZ5I-EGF for 2,4,6,8, 10, and 12 min at 37 "C and then rapidly cooled to 4 "C. The cells were washed without and with 50 mM NaCl, 150 mM glycine, pH 3.0, for 3 min at 4 "C. Control experiments demonstrated that the acid-washing pro-

1132 CAM Kinase 11 Regulation of the EGF Receptor tocol caused the dissociation of greater than 95% of the specific cell surface-hound ""I-EGF. 1nt.racellular EGF was estimated by deter- mination of cell-associated ""I-EGF following acid washing (Haigler et al., 1980). Cell surface ""I-EGF was estimated by suhtraction of the estimated intracellular radioactivity from the measured total cell associated ""I-EGF. Nonspecific binding and accumulation of ""I- EGF was determined in experiments using a 500-fold excess of EGF. The first-order endocytotic rate constant was calculated from these data as described by Wiley and Cunningham (1982).

Analysis of "'I-EGF HindinE to C ~ l l Surfacr EGF Rrceptors- Binding assays were performed on cells seeded in 16 mm-wells and grown to a density of 5 X 10' cells/well. The cells were washed with serum-free medium and incubated for 30 min a t 37 "C in 120 mM

7.4), and 30 p~ bovine serum albumin. The cells were incubated with 10 nM phorhol myristate acetate or 100 nM EGF for defined times. The medium was removed and rapidly replaced with medium a t 4 "C. The cells were washed with 50 mM NaCI, 150 mM glycine (pH 3.0) for 3 min a t 4 "C to remove EGF hound to cell surface receptors (Haigler et a/., 1980). The cells were then washed twice a t 4 "C with serum-free medium and subsequently incubated with different concentrations of ""I-EGF for 180 min a t 4 "C. The monolayers were washed three times with cold medium and solubilized with 900 p1 of 1 M NaOH. Radioactivity associated with the cells was quantitated with a Reckman y counter. Nonspecific binding was estimated in incubations with a 200-fold excess of unlabeled ligand.

NRCI, 6 mM KCI, 1.2 mM MgCI?, 1 mM CaCI?, 25 mM HEPES (pH

RESULTS

In initial experiments, the effects of EGF on the tyrosine phosphorylation of the EGF receptor were investigated. CHO cells expressing wild-type human EGF receptors were employed for these studies. After incubation without and with 100 nM EGF for 5 or 30 min a t 37 "C, the CHO cells were lysed in a detergent buffer containing phosphatase inhibitors, and the EGF receptors were isolated by immunoprecipitation. EGF receptor tyrosine phosphorylation was examined using a Western blot procedure employing the anti-phosphotyrosine antibody PY20. Addition of EGF caused a marked increase in the level of EGF receptor tyrosine phosphorylation after 5 min of EGF treatment (Fig. 1, lanes 1 and 2). However, the marked extent of EGF-stimulated tyrosine phosphorylation of the receptor was not sustained, and a significantly lower level of phosphorylation was found after 30 min of EGF treatment (Fig. 1, lane 3) . This decrease in EGF receptor tyrosine phosphorylation observed after prolonged treatment of cells with EGF could be caused by two different mechanisms: 1) a reduction in EGF receptor number or 2) a decrease in the stoichiometry of EGF receptor tyrosine phosphorylation.

We tested the hypothesis that EGF receptors are degraded during the incubation of cells with EGF and that this decrease in receptor number accounts for the observed decrease in EGF receptor tyrosine phosphorylation. CHO cells expressing wild- type EGF receptors were pulse-labeled with [:''S]methionine and then chased in the presence and absence of EGF for different times. The EGF receptors were then isolated by immunoprecipitation and polyacrylamide gel electrophoresis. I t was observed that EGF treatment of the cells for 1-3 h caused a marked decrease in the level of EGF receptor expression (Fig. 2). However, no significant effect of EGF to decrease EGF receptor expression was observed after 5-15 min of EGF treatment (Fig. 2). These data demonstrate that EGF receptor degradation can account for long term (hours) actions of EGF to decrease EGF-stimulated tyrosine phosphorylation. How- ever, the acute decrease in tyrosine phosphorylation (Fig. l) is mediated by a mechanism that is independent of receptor degradation (Fig. 2).

I f the decreased EGF receptor tyrosine phosphorylation (Fig. 1) is not caused by a decrease in receptor number (Fig. Z ) , it must therefore be the result of a lower stoichiometry of

Orthovanadate

"- 0 5 30 0 5 30 0 5 30 ECP Treatment

-r " . - (minutes)

' b. - ECP Receptor

"- - - + In Vitro Phosphorylation

FIG. 1. Desensitization of EGF receptor tyrosine phosphorylation in situ. CHO cells expressing wild-type E(;F receptors were incubated without and with 100 nM EGF for 5 or 30 min. The EGF receptors were then isolated hy immunoprecipitation. The ty - rosine phosphatase inhihitor orthovanadate was used to maintain the tyrosine phosphorylation state of the EGF receptor during the immunoprecipitation procedure. Immunoprecipitates prepared in the absence of orthovanadate were incubated with 10 p~ ATP and 100 nM EGF to study the in uifro autophosphorylation of the EGF receptor. Tyrosine phosphorylation of the EGF receptor was examined hy Western blot analysis using the anti-phosphotvrosine nnti- body PY20 and ""I-goat anti-mouse Ig. An autoradiograph (48-h exposure) of the dried blot is presented. Similar results were obtained in three separate experiments.

120,

.ll " 0 so 00 80 110 1w 180

TIME. mlnntes

FIG. 2. Pulse-chase analysis of EGF receptor degradation. CHO cells expressing wild-type EGF receptors were Iabelcd t)y incubation with [ "Slmethionine for 18 h. The medium was then replaced and the cells were treated without (0) and with 1 nM E(;F ( A ) or 1 0 0 nM EGF (0) for defined times. Thfa EGF receptors were isolated hy immunoprecipitation and polyacrylamide gel electrophoresis. The gel was dried, and the receptors were detected and quantitated using a Phosphorimager (Molecular Dynamics). The radioactivity associated with the EGF receptors (mean & S.D., n = 3 ) is presented.

phosphorylation. The change in phosphorylation stoichiometry could be caused by 1) a decrease in the rate of tyrosine phosphorylation or 2) an increase in the rate of receptor dephosphorylation. Alterations in enzyme activity or subcellular localization may account for the decreased EGF receptor tyrosine phosphorylation ohserved. One potential mechanism is that EGF causes desensitization of the EGF receptor protein-tyrosine kinase actvity. Further experiments were de- signed to test this hypothesis.

EGF Causes Desensitization of the ECF Receptor Protein-Tyrosine Kinase Activity

In preliminary experiments, we examined the effect of the treatment of cells with EGF on the in vitro autophosphorylation of the EGF receptor. Fig. 1 shows that ECF receptors isolated by immunoprecipitation in the absence of the tyrosine phosphatase inhibitor orthovanadate exhibit a very low level of tyrosine phosphorylation. Addition of ATP to these immunoprecipitates initiates an autophosphorylation reaction.


Marked EGF-stimulated tyrosine autophosphorylation of receptors isolated from control cells was observed (Fig. 1, lanes 6 and 7). However, the in vitro EGF-stimulated tyrosine autophosphorylation of receptors isolated from cells treated with 100 nM EGF for 5 or 30 min was significantly decreased as compared with receptors isolated from cells treated without EGF (Fig. 1, lanes 7-9). The decreased in vitro autophosphorylation on tyrosine suggests that the addition of EGF to cells causes a desensitization of the EGF receptor protein-tyrosine kinase activity.

Autophosphorylation (Fig. 1) is not a rigorous method for the examination of protein kinase activity. Therefore, to confirm that EGF causes desensitization of the EGF receptor protein-tyrosine kinase activity, we investigated the tyrosine phosphorylation of an exogenous substrate (enolase) by the receptor. Fig. 3 shows that treatment of cells with 1 nM EGF caused an inhibition of the exogenous substrate phosphorylation by the EGF receptor in vitro. A significant decrease in phosphorylation was observed after 2 min of EGF treatment and was sustained for at least 30 min (Fig. 3). As half-maximal stimulation of proliferation was observed after treatment of these cells with 2 nM EGF (data not shown), it is likely that the sustained desensitization of EGF receptor protein-tyrosine kinase activity caused by 1 nM EGF is physiologically significant.

A, 1nM EGF 120,

I

0 1 s 1 0 1 0

TIME. mlnnter

B. lOOnM EGF 110,

o 1 s ~ o a o s o

TIME. minuter FIG. 3. Time course of desensitization of the EGF receptor

protein-tyrosine kinase activity. CHO cells expressing wild-type [Thr654,Thr669,Ser'046,Ser1M7]EGF receptors (filled bars) or mutated [Ala654,Ala669,Ala1046,Ala1~7]EGF receptors (open bars) were incubated with 1 nM EGF (panel A ) or 100 nM EGF (panel B ) for defined times a t 37 'C. The EGF receptors were isolated by immunoprecipitation, and the protein-tyrosine kinase activity was measured using enolase and [-y-32P]ATP as substrates. The phosphorylation reaction was terminated after 10 min at 22 "C. The enolase was isolated by polyacrylamide gel electrophoresis, and the incorporation of radioactivity was measured by liquid scintillation counting. The data presented are the means k S.D. of three independent determinations.

In order to characterize the desensitization of the EGF receptor protein-tyrosine kinase activity, we measured the kinetic parameters for phosphorylation of the exogenous substrate enolase. CHO cells expressing wild-type EGF receptors were treated without (control) and with 100 nM EGF for 5 min at 37 "C. The EGF receptors were then isolated by immunoprecipitation, and the receptor protein-tyrosine kinase activity was measured at 22 "C. The K,.,, for enolase and ATP for the EGF receptor isolated from control cells were 14 & 3 and 1.6 f 0.4 p ~ , respectively (mean f S.D., n = 3). After treatment of the cells with EGF, the Kmapp for enolase (11 f 2 pM) and ATP (2.1 +. 0.3 p ~ ) were not significantly different from the values measured for receptors isolated from control cells (mean f S.D., n = 3). In contrast, the Vmaxapp of receptors isolated from EGF-treated cells was found to be 45 f 5% (mean f S.D., n = 3) of the V,,,,, determined for receptors isolated from control cells. These data demonstrate that the observed inhibition of EGF receptor protein-tyrosine kinase activity is caused by a decrease in Vmax rather than a change in K , for substrates.

Further experiments were performed to investigate the effect of EGF concentration on the extent of desensitization. It was observed that the addition of high concentrations of EGF (100 nM) to cells did not cause a more profound desensitization of protein-tyrosine kinase activity than that observed after 1 nM EGF (Fig. 3). Furthermore, the desensitization caused by 100 nM EGF was transient, with maximal effects occurring at 5 min (Fig. 3). A second phase of apparent desensitization that was reproducibly observed after 30 min of treatment with 100 nM EGF may be the result of EGF receptor degradation. The transient desensitization caused by 100 nM EGF is in marked contrast to the sustained desensitization caused by 1 nM EGF (Fig. 3).

Desensitization of the EGF Receptor Protein-Tyrosine Kinase Is Absent in Mutated EGF Receptors Lacking the

Phosphorylation Site Ser1046/7 The EGF receptor is phosphorylated at multiple sites

(Hunter and Cooper, 1981). To investigate whether EGF receptor phosphorylation is relevant to the mechanism of desensitization, we constructed a mutated EGF receptor in which all of the major sites of serine/threonine phosphorylation (Thr654, Thr6'j9, Serlo4'j, and Ser'047) were replaced with alanine residues. The phosphorylation-defective EGF receptor was expressed in CHO cells (Countaway et al., 1990). Treatment of these cells with EGF caused no significant desensitization of the receptor protein-tyrosine kinase activity (Fig. 3). This result suggests that serine/threonine phosphorylation of the EGF receptor may be required for desensitization.

To identify which EGF receptor phosphorylation site is mechanistically relevant to desensitization, we examined the properties of further mutant EGF receptors. Substitution of the major sites of threonine phosphorylation (Thr654 and Thr'j6') with Ala residues caused no significant change in the extent of desensitization observed as compared with the wild- type EGF receptor (Fig. 4). In contrast, the substitution of the major site of serine phosphorylation (Ser1046'7) with Ala blocked the desensitization of the EGF receptor protein- tyrosine kinase activity (Fig. 4). This result is consistent with the hypothesis that the EGF-stimulated phosphorylation of the EGF receptor at Ser1046/7 (Heisermann and Gill, 1988; Countaway et al., 1990) accounts for the observed desensitization of the EGF receptor protein-tyrosine kinase activity.

In previous studies, the phosphorylation of the EGF receptor at Ser'046/7 has been studied in detail. Heisermann and


689 888 1046/7

FIG. 4. Effect of point mutation at the major sites of EGF receptor serinelthreonine phosphorylation on the desensitization of the EGF receptor protein-tyrosine kinase activity. CHO cells expressing [Thr654,Thr669,Ser'046,Ser1047]EGF receptors (wild-type ( W T ) , [Ala654,Ala669]EGF receptors, [Ala'046,Ala'047]EGF receptors, and [Ala654,Ala669,Ala'046,Ala'047]EGF receptors were grown in 35-mm wells. The cells were incubated without (open burs) or with (closed bars) 100 nM EGF for 5 min at 37 "C. The EGF receptors were isolated by immunoprecipitation, and the protein-tyrosine kinase activity was measured using enolase and [y3'P]ATP as substrates. The phosphorylation reaction was terminated after 10 min at 22 "C. The enolase was isolated by polyacrylamide gel electrophoresis, and the incorporation of radioactivity was measured by liquid scintillation counting. The data presented are the means * S.D. of three independent determinations.

Gill (1988) have reported that Ser1046/7 is the principal site of EGF-stimulated serine phosphorylation of the EGF receptor. The major site of phosphorylation was located at Ser'047, but some phosphorylation at both Ser'04'j and SerIM7 was found (Heisermann and Gill, 1988; Countaway et aL, 1990). Muta- tional analysis indicates that the replacement of Ser'04'j with Ala caused no significant change in the stoichiometry of EGF receptor phosphorylation at the major site, Ser1047 (Countaway et al., 1990). In contrast, the replacement of Ser1047 with Ala caused a marked increase in phosphorylation of the EGF receptor at Ser'O4'j (Countaway et al., 1990). Serine phosphorylation was blocked only when both SerIo4'j and Ser'047 were simultaneously replaced with Ala (Countaway et al., 1990).

EGF Receptor Ser1O4'jl7 Is a Substrate for Phosphorylation by CAM Kinase IZ in Vitro

The mechanism of EGF-stimulated phosphorylation of the EGF receptor at Ser1M6/7 is not understood (Heisermann and Gill, 1988; Countaway et al., 1990). Inspection of the primary sequence of the EGF receptor surrounding Ser'047 (RYSS) indicates that it conforms to the consensus sequence for phosphorylation by CAM kinase I1 (Pearson et al., 1985). Significantly, Ser1047 is the only Ser residue located within the EGF receptor cytoplasmic domain that is consistent with this consensus sequence. We therefore investigated whether Ser1046/7 is a substrate for CAM kinase 11. In initial studies, we prepared a synthetic peptide (RRFLQRYSSDPTGAL) corresponding to the sequence of the EGF receptor (residues 1041-1053) and examined whether this peptide is a CAM kinase I1 substrate. Table I shows that this synthetic peptide was phosphorylated by CAM kinase 11. The rate of phosphorylation observed was 81% of that measured using the CAM kinase I1 substrate peptide Syntide 2 (Table I). Phos- phoamino acid analysis of the Ser'M6/7 peptide demonstrated the presence of [32P]phosphoserine but not [32P]phosphothre- onine or [32P]phosphotyrosine (data not shown).

In further studies, we investigated the phosphorylation of a bacterially expressed glutathione-S-transferase fusion protein (TK6) that contains sequences derived from the EGF receptor cytoplasmic domain (Koland et aL, 1990). It was observed that glutathione-S-transferase was not a substrate

TABLE I Phosphorylation of synthetic peptide substrates by CAM kinase II

CAM kinase I1 in 25 mM HEPES, 10 mM MgC12, 0.4 mM EGTA, and Synthetic peptides (1 mg/ml) were incubated with 1 microunit of

0.6 p~ calmodulin in the presence and absence of 1 mM CaC12 (final volume, 50 pl). The phosphorylation reaction was initiated by the addition of 20 PM [y-32P]ATP (50 pCi/nmol) and was terminated after 10 min at 22 "C. The incorporation of radioactivity into the synthetic peptides was measured using the phosphocellulose paper assay. The results obtained are expressed as the mean ( n = 3) rate of peptide phosphorylation.

Rate of Peptide substrate Sequence phosphorylation

-Ca2+ +Ca2+

fmollmin Syntide 2 PLARTLSVAGLPGKK 0 1030 EGF receptor (Ser'0466/') RRFLQRYSSDPTGAL 0 830 EGF receptor (Thr654) KRTLRR 0 0 RR-SRC RRLIEDAEYAARG 0 0

for CAM kinase 11. However, the fusion protein TK6 was phosphorylated by CAM kinase 11. [32P]Phosphoserine was the only phosphoamino acid detected (data not shown). To test the hypothesis that the site of phosphorylation of this fusion protein was Ser'M6/7, we replaced these residues with Ala. It was observed that the mutated protein was an ex- tremely poor substrate for phosphorylation by CAM kinase I1 (data not shown). This result is consistent with the hypothesis that Ser1046/7 is a substrate for phosphorylation by CAM kinase 11.

We next examined whether the full-length EGF receptor is a substrate for phosphorylation by CAM kinase 11. The EGF receptor was found to be phosphorylated after the addition of [y3'P]ATP in the presence and absence of activated CAM kinase I1 (Fig. 5A) . Phosphoamino acid analysis demonstrated tyrosine phosphorylation of the EGF receptor (Fig. 5B) . Ac- tivation of CAM kinase I1 caused a marked increase in EGF receptor serine phosphorylation, indicating that the EGF receptor is a substrate for CAM kinase I1 (Fig. 5B) . In order to localize the major site(s) of EGF receptor phosphorylation by CAM kinase 11, we digested the receptor with trypsin and separated the 32P-labeled phosphopeptides obtained by reverse-phase HPLC (Fig. 5C) . Phosphoamino acid analysis of the peptides eluted from the column demonstrated the presence of [32P]phosphotyrosine. However, a phosphopeptide containing [32P]phosphoserine was observed to be eluted from the column at 33% acetonitrile (Fig. 5C). This phosphopeptide corresponds to the major site of serine phosphorylation of the EGF receptor by CAM kinase 11. To identify this major site of serine phosphorylation, we prepared tryptic peptides from EGF receptors that were isolated from [32P]pho~phate-labeled A431 epidermoid carcinoma cells (Countaway et al., 1990). The phosphopeptide corresponding to Ser'046/7 phosphorylated in uiuo co-eluted from the reverse-phase column with the EGF receptor tryptic peptide phosphorylated by CAM kinase I1 in uitro (Fig. 5C). Together, these data strongly support the hypothesis that the EGF receptor is phosphorylated a t Ser'046/7 by CAM kinase 11.'

' In further experiments, we have investigated CAM kinase I1 action using purified EGF receptors isolated from detergent-solubilized A431 membranes by 1) affinity chromatography (Davis and Czech, 1984) and 2) immunoprecipitation with the monoclonal antibody m108.1. It was observed that the purified EGF receptor was a poor substrate for CAM kinase I1 and that only a very low stoichiometry of phosphorylation of the receptor at Ser1046/7 could be achieved. This result contrasts with the marked phosphorylation of the EGF receptor a t Ser'"6/7 observed in membrane preparations (Fig. 5). In control experiments, we investigated the phosphorylation


A. EGF Receptor Phosphorplation

- + - + calcium - - + + EGF - 0 0 .II. c 170 kDa

- + - + " 8 a m - m m - * m

AcetonlMlc 1%)

FIG. 5. Phosphorylation of the EGF receptor by CAM kinase 11. Membranes were prepared from A431 cells that express wild-type EGF receptors. The membranes (10 pg of protein) were incubated with 100 nM EGF, 6 p~ calmodulin, and 10 microunits of CAM kinase I1 in the presence and absence of Ca'+. The phosphorylation reaction was initiated by the addition of [y3*P]ATP and was terminated after 10 min of incubation a t 22 "C. The EGF receptors were isolated by immunoprecipitation and polyacrylamide gel electrophoresis. Panel A, an autoradiograph of the dried polyacrylamide gel. Panel B, phosphoamino acid analysis of the EGF receptors was performed by partial acid hydrolysis and thin layer electrophoresis. Panel C, tryptic ["P]phosphopeptide mapping of the EGF receptor phosphorylated by CAM kinase I1 in the presence of Ca'+ and calmodulin. The elution of the purfied EGF receptor tryptic ["PI phosphopeptide corresponding to the phosphorylation site Ser"6/7 is also presented. T o improve the clarity of presentation of the data, 400 cpm were added to each data point obtained for the phosphopeptide map of the EGF receptor phosphorylated by CAM kinase 11. The results of phosphoamino acid analysis of the phosphopeptides eluted from the HPLC column are shown (S, phosphoserine; Y, phosphotyrosine).

CAM Kinase Z I Inhibits the EGF Receptor Protein-Tyrosine Kinase Activity

Addition of ATP to the wild-type EGF receptor initiates an autophosphorylation reaction that results in the phosphorylation of the receptor on tyrosine residues (Ushiro and Cohen, 1980). This autophosphorylation reaction may occur by both inter- and intramolecular mechanisms (Honegger et al., 1989, 1990a, 1990b). The in uitro autophosphorylation of the EGF receptor on tyrosine residues is shown in Fig. 5B. Activation of CAM kinase I1 in the presence of Ca'+ and calmodulin caused a marked decrease in EGF receptor tyrosine phosphorylation (Fig. 5B) . The decreased tyrosine phosphorylation was associated with increased serine phosphorylation of

of the bacterially expressed EGF receptor carboxyl-terminal domain (TK6). It was observed that the addition of detergent (Triton X-100 or Nonidet P-40) caused a marked decrease in the CAM kinase I1 phosphorylation of the bacterially expressed protein. It is therefore likely that the modest phosphorylation of the purified EGF receptor observed is a result of the detergent used to solubilize the receptor.

- + - + calcium " + + "II

FIG. 6. CAM kinase I1 regulation of the EGF receptor protein-tyrosine kinase activity. A431 epidermoid carcinoma cell membranes (2 pg of protein) were incubated with 100 nM EGF. The EGF receptor protein-tyrosine kinase activity was determined using [-y-"P]ATP and the synthetic peptide RR-SRC as an exogenous substrate. The effect of the addition of Ca2+ and calmodulin/CAM kinase I1 on the measured protein-tyrosine kinase activity was measured. The results presented are the means S.D. of the determinations made in three separate experiments. The data are normalized to the rate of phosphorylation observed for A431 membranes in the absence of calcium, calmodulin, and CAM kinase I1 (52 fmol/min/ pg; 100%).

the EGF receptor (Fig. 5 B ) . This result suggests that the phosphorylation of the EGF receptor a t Ser1046/7 by CAM kinase I1 caused a decrease in the EGF receptor protein- tyrosine kinase activity. To test this hypothesis, we measured the EGF receptor protein-tyrosine kinase activity using an exogenous peptide substrate (RR-SRC) that is not phosphorylated by CAM kinase I1 (Table I). Fig. 6 shows that the activation of CAM kinase I1 caused a decrease in the rate of tyrosine phosphorylation of the exogenous substrate by the EGF receptor. We conclude that the activation of CAM kinase I1 causes an inhibition of the EGF receptor protein-tyrosine kinase activity.

Regulation of the Oligomeric State of the EGF Receptor by CAM Kinase ZZ

It has been proposed that the mechanism by which EGF stimulates the EGF receptor protein-tyrosine kinase activity is mediated by the EGF-stimulated dimerization of the receptor (Schlessinger, 1988). An inhibition of receptor dimerization could therefore account for the inhibition of EGF receptor protein-tyrosine kinase activity caused by CAM kinase 11. We therefore tested the hypothesis that CAM kinase I1 reg- ulates the EGF-stimulated dimerization of the EGF receptor. The experimental approach that we used was to employ the covalent cross-linking reagent disuccinimidyl suberimidate and lZ5I-EGF as a ligand. Control experiments demonstrated that there was no significant effect of CAM kinase I1 on "'1- EGF binding to the EGF receptor (data not shown). Cross- linking analysis did not demonstrate any significant effect of CAM kinase I1 on EGF receptor aggregation (data not shown).

Role of Ser'046'7 in the Down-regulation and Transmodulation of Cell Surface EGF Receptors

It has previously been proposed that EGF-induced desensitization of the EGF receptor can be accounted for by 1) internalization and sequestration of the receptor from the cell surface (down-regulation), 2) a decrease in the high affinity component of the EGF-binding sites expressed at the cell surface (transmodulation), and 3) an inhibition of the receptor protein-tyrosine kinase activity. There is a role for the phosphorylation of the EGF receptor a t Ser1046/7 in the regulation of the EGF receptor protein-tyrosine kinase activity (Figs. 4- 6). However, it is not known whether the phosphorylation of the EGF receptor at Ser104fi/7 is mechanistically relevant to the down-regulation and transmodulation of the EGF receptor

1136 CAM Kinase 11 Regulation of the EGF Receptor

that is caused by EGF. We therefore investigated the effect of point mutation of the EGF receptor at Ser1046/7 on these processes.

Transmodulation-Wiley et al. (1989) have reported that EGF causes a rapid loss of the high affinity EGF-binding sites expressed at the cell surface (transmodulation). This transmodulation of the EGF receptor is similar to that observed in cells treated with phorbol ester or platelet-derived growth factor (Countaway et al., 1989). The EGF-stimulated transmodulation of the EGF receptor may be significant for the process of desensitization. We therefore investigated the lZ5I- EGF-binding isotherm in cells treated with 100 nM EGF for 60 min at 37 "C. Table 11 shows that the expression of high affinity EGF-binding sites was markedly reduced after EGF treatment of cells expressing the wild-type EGF receptor. A similar loss of high affinity EGF-binding sites was observed in cells treated with phorbol ester (Table 11). The EGF- stimulated transmodulation of the EGF receptor was also observed in experiments using cells expressing [Ala'046/7]EGF receptors (Table 11). This is consistent with observations made in previous studies that phorbol ester and platelet- derived growth factor cause transmodulation of the [A1a'046/7] EGF receptor (Countaway et al., 1990). Together, these data indicate that the Ser'046/7 phosphorylation site is not required for EGF receptor transmodulation.

Endocytosis-The first-order rate constant for the receptor- mediated endocytosis of EGF was measured by incubating cells with 1 nM Iz5I-EGF at 37 "C (Fig. 7). The measured endocytotic rate constant in experiments using cells expressing the wild-type EGF receptor was 0.23 -+ 0.02 min" (mean k S.D., n = 3). In contrast, the rate of internalization of the [Ala'046/7]EGF receptor was determined to be 0.06 f 0.01 min". Thus, the [Ala'046/7]EGF receptor was internalization- defective as compared with the wild-type EGF receptor. It has previously been reported that protein-tyrosine kinase- inactive EGF receptors are also internalization-defective (Glenney et al., 1988). We therefore constructed a kinase- defective EGF receptor by replacing the lysine located in the active site (residue 721) with arginine. It was observed that the kinase-inactive [Arg7"]EGF receptor internalized at a

TABLE I1 Analysis of the '"I-EGF binding isotherm

CHO cells expressing wild type or mutated EGF receptors were treated without or with 10 nM phorbol myristate acetate (PMA) for 15 min at 37 "C. The cells were then treated without and with 100 nM EGF for an additional 60 min. The specific binding of 20 PM, 50 pM, 100 pM, 200 pM, 500 pM, 1 nM, 5 nM, and 10 nM '251-EGF to cell surface receptors was measured at 4 "C, as described under "Experi- mental Procedures." Similar data were obtained in three separate experiments. The "'1-EGF-binding isotherm was analyzed using the computer program LIGAND (Munson and Rodbard, 1980) to fit the data to a one-site model and to a two-site model for the binding of "'1-EGF to cell surface receptors. The fit of the experimental data (mean f S.E.) to the one-site model is presented unless the fit obtained for the two-site model was significantly better ( F test, p > 0.9).

Site 1 Site 2

Kd Sites/cell K d Sites/cell nM X l K 3 nM X 10-3

Wild-type EGF receptor Control 2.4 f 0.8 136 f 11 0.08 f 0.05 4.1 f 2.0 EGF 3.7 f 0.7 65 f 12 PMA 4.4 1- 1.1 121 f 17

Control 3.2 f 1.1 145 f 15 0.2 f 0.08 8.2 f 4.1 EGF 4.1 f 1.6 110 f 19 PMA 2.2 t- 0.6 134 f 14

[Ala1046/']EGF receptor

0 . ~ 1

WT A-1046/7 R-721

FIG. 7. Effect of mutation at Ser'046/7 on the rate of internalization of the EGF receptor. CHO cells expressing wild-type ( W T ) or mutated EGF receptors were used to measure the rate of internalization of 1 nM Iz5I-EGF. The first-order endocytotic rate constant was calculated using the In/Sur method of Wiley and Cunningham (1982) as described under "Experimental Procedures." The data presented are the means of three independent experiments f S.D.

1101

Time, minutes

FIG. 8. Effect of mutation at Ser'046/7 on the cell surface expression of the EGF receptor. CHO cells expressing wild-type EGF receptors, [Ala1046/7]EGF receptors, and [Arg'21]EGF receptors were incubated for defined times with 1 nM EGF. The cells were then washed, and the cell surface expression of EGF receptors was examined by measuring the binding of a rabbit anti-EGF receptor antibody to the cell monolayers at 4 "C. The data presented are the means of five independent experiments f S.D. The rate of down-regulation of the wild-type EGF receptor was significantly more rapid ( t test, p > 0.05) than the mutated EGF receptors. However, no statistically significant difference between the rate of down-regulation of the two mutated EGF receptors was observed.

rate similar to that of the phosphorylation-defective [A1a'04G/7] EGF receptor. These data indicate that the substitution of Ser'04G/7 with Ala causes a marked decrease in the rate of EGF receptor endocytosis.

Down-regulation-The internalization-defective phenotype of the [Ala'04G/7]EGF receptor suggests that this receptor may also exhibit defects in down-regulation. We therefore investigated the time course of EGF receptor down-regulation in cells incubated with 1 nM EGF. Fig. 8 shows that the cell surface expression of the wild-type EGF receptor is rapidly decreased during incubation of cells with EGF. In contrast to the rapid down-regulation of the wild-type EGF receptor, the internalization-defective [Ala'046/7]EGF receptor was observed to be down-regulated very slowly when cells were incubated with EGF (Fig. 8). A similar slow rate of down- regulation was observed for the protein-tyrosine kinase defective [Arg7"]EGF receptor (Fig. 8).

DISCUSSION

Integration of Signaling Pathways Signal transduction by the EGF receptor is acutely regu-

lated by the status of activated second messenger pathways in the cell (Fig. 9). This regulatory network provides a mech-


EGF Receptor

EGF

Feed-back Stgnalllng Pathway

I I 1 1

' 1047

FIG. 9. Schematic representation of the regulation of EGF receptor protein-tyrosine kinase activity by serine/threonine phosphorylation. The sites of EGF receptor phosphorylation by CAM kinase I1 (Ser'"fi'7) and protein kinase C ( T h P ) are shown. Phosphorylation of the EGF receptor a t these sites is associated with a decreased EGF receptor protein-tyrosine kinase activity. Phos- phorylation at T h P accounts for the inhibition of EGF receptor protein-tyrosine kinase activity caused by phorbol ester (Davis, 1988). In contrast, phosphorylation at Sert"46'7 accounts for the desensitization of the EGF receptor protein-tyrosine kinase activity caused by EGF.

anism for integrating the process of signal transduction in response to complex mitogens (e.g. serum) during the control of cellular proliferation. The molecular details of how this integration of signaling occurs is incompletely understood. However, the control of EGF receptor function can be accounted for, in part, by changes in the state of phosphorylation of the EGF receptor (Fig. 9).

The regulation of EGF receptor function by protein kinase C has been studied in detail. Protein kinase C activation caused by diacylglycerol results in the phosphorylation of the EGF receptor at Thr654 (Hunter et al., 1984; Davis and Czech, 1985a). This phosphorylation site is located close to the cytoplasmic surface of the plasma membrane between the ligand-binding domain and the protein-tyrosine kinase domain of the EGF receptor (Fig. 9). It has been demonstrated that the phosphorylation of the EGF receptor a t ThrfiS4 causes an inhibition of the receptor protein-tyrosine kinase activity (Cochet et al., 1984; Friedman et al., 1984; Downward et al., 1985; Davis et al., 1985a, 1985b; Davis, 1988; Countaway et al., 1990; Decker et al., 1990; Lund et al., 1990).

A second pathway of regulation of the EGF receptor is represented by the phosphorylation of the receptor at serln4s/; . Increased phosphorylation at this site is associated with an inhibition of the receptor protein-tyrosine kinase activity (Fig. 6). This phosphorylation site is located within the carboxyl-terminal subdomain of the EGF receptor (Fig. 9). I n uitro experiments demonstrate that this site is a substrate for phosphorylation by CAM kinase I1 (Fig. 5). EGF is known to increase cytosolic Ca2+ (Sawyer and Cohen, 1981; Morris et al., 1984; Moolenaar et al., 1984; Magni et al., 1991) and has been shown to stimulate CAM kinase I1 activity in fibroblasts (Ohta et al., 1988; Miyamoto et al., 1990). The stimulation of CAM kinase I1 by other peptide growth factors has also been demonstrated (MacNicol et al., 1990). I t is therefore possible that CAM kinase I1 accounts for the phosphorylation of the EGF receptor a t Ser1046/i in situ. The observation that the calmodulin antagonist trifluoperazine

blocks EGF-induced desensitization of EGF receptor function is consistent with this hypothesis (Cunningham et al., 1989; Kuppuswamy and Pike, 1989). Furthermore, a CAM kinase I1 pathway of EGF receptor regulation can account for the effects of increased cytosolic Ca'+ (observed after the treatment of cells with the ionophore A23187 or the tumor promoter thapsigargin) to cause an inhibition of the EGF receptor protein-tyrosine kinase activity by a mechanism that is independent of protein kinase C and phosphorylation a t T h P 4 (Friedman et al., 1989). Together, these data indicate that CAM kinase I1 (Colbran et al., 1989; Nairn, 1990) is a candi- date enzyme that could account for the phosphorylation of

in situ, but a role for other protein kinases cannot be excluded. Examples of protein kinases that may phosphoryl- ate the EGF receptor a t Ser104fi/i in situ include c-raf-1 (Li et al., 1991) and the S6 kinases (Erikson, 1991).

Distinct protein kinase signaling pathways control the EGF receptor protein-tyrosine kinase activity by receptor phosphorylation (Fig. 9). I t is likely that EGF receptor desensitization is mediated by the combined state of activation of these signal transduction pathways in different tissues. This integration of different signals is analogous to observations that have been made for the desensitization of the @-adrenergic receptor that can be accounted for by the phosphorylation of the receptor by the CAMP-dependent protein kinase and p- adrenergic receptor kinase (Hausdorff et al., 1990).

In many cells of mesenchymal origin, EGF causes no de- tectable changes in phosphatidylinositol turnover (Besterman et al., 1986; Tsuda et al., 1986) and only modest increases in protein kinase C activity (Blackshear et al., 1985). However, EGF significantly increases cytosolic Ca2+ in these cells (Moolenaar et dl., 1984; Morris et al., 1984). The major mechanism of acute homologous desensitization of EGF receptor function in these cells may therefore be accounted for by the EGF-stimulated phosphorylation of the EGF receptor at the CAM kinase I1 site, Ser1n46/7 (Fig. 4). The lack of a role for ThrfiS4 phosphorylation in this process is indicated by the observation that the replacement of ThrfiS4 with Ala does not cause a significant decrease in the extent of EGF-stimulated desensitization of the EGF receptor (Fig. 4). However, for some tumor cells of epithelial origin, it is likely that EGF- stimulated desensitization is caused by the phosphorylation of the EGF receptor a t both Thrfis4 and Ser1046/i (Fig. 9). This is because EGF causes marked increases in the rate of phosphatidylinositol turnover, the level of diacylglycerol, and protein kinase C activity in A431 epidermoid carcinoma cells, MDA468 breast cancer cells, and liver epithelial cells (Sawyer and Cohen, 1981; Macara, 1986; Whiteley and Glaser, 1986; Moolenaar et al., 1986; Pike and Eakes, 1987; Wahl et al., 1987; Wahl and Carpenter, 1988; Earp et al., 1988; Cun- ningham et al., 1989).

Ser1046/5

Mechanism of Inhibition of the EGF Receptor Protein- Tyrosine Kinase Actiuity by SerinelThreonine

Phosphorylation It has been proposed that the mechanism by which EGF

increases the protein-tyrosine kinase activity of the EGF receptor is mediated by the EGF-stimulated dimerization of the receptor (Schlessinger, 1988). The inhibition of protein- tyrosine kinase activity caused by serinelthreonine phosphorylation of the EGF receptor could therefore be accounted for by the regulation of EGF receptor aggregation. We have previously reported that the phosphorylation of the EGF receptor at T h P 4 by protein kinase C does not alter EGF receptor dimerization (Northwood and Davis, 1989). In this study, we have investigated whether the phosphorylation of


the EGF receptor at Ser1046/7 by CAM kinase I1 inhibits EGF receptor dimerization. No marked effect of CAM kinase I1 on EGF receptor dimerization was observed in experiments using covalent cross-linking analysis. Thus, it is unlikely that regulation of receptor dimerization accounts for CAM kinase I1 inhibition of the EGF receptor protein-tyrosine kinase activity (Fig. 6). If regulation of EGF receptor aggregation does not account for the inhibition of protein-tyrosine kinase activity, it is therefore necessary that an alternative mechanism be proposed that can serve as a working hypothesis. Two general classes of mechanisms can be proposed.

1) The phosphorylation of T h P 4 and Se~'" j /~ may alter the conformation of the EGF receptor. Such a conformational change could directly inhibit the receptor protein-tyrosine kinase activity (Fig. 9). Alternatively, the putative phosphorylation-induced conformation could change the structure of the active dimeric form of the EGF receptor in a manner that is not detected by cross-linking analysis.

2) The serine/threonine phosphorylation of the EGF receptor may alter the interaction of the receptor with other biomolecules within the receptor signaling complex (Ullrich and Schlessinger, 1990). According to this model, the inhibition of protein-tyrosine kinase activity caused by serine/ threonine phosphorylation is an indirect action.

Role of the Carboxyl-terminal Domain of the EGF Receptor The negative regulatory phosphorylation site Ser'046/7 is

located within the carboxyl-terminal domain of the EGF receptor (Fig. 9). This location is consistent with the results of previous studies that have demonstrated that a negative regulatory site is present within this domain of the EGF receptor. These studies have shown that the removal of the carboxyl-terminal domain by truncation causes a marked increase in the EGF receptor protein-tyrosine kinase activity (Walton et al., 1990) and that the transfer of this domain to the erbB-2 gene product causes a suppression of protein- tyrosine kinase activity (Di Fiore et al., 1990). I t has been suggested that the inhibition of protein-tyrosine kinase activity caused by the carboxyl-terminal domain of the EGF receptor may be the result of competition between exogenous substrate phosphorylation and autophosphorylation (Bertics and Gill, 1985; Bertics et al., 1988; Hsu et al., 1991; Helin and Beguinot, 1991; Sorkin et al., 1991), but supporting evidence has not been obtained by others (Downward et al., 1985; Honegger et al., 1988). The data reported here indicate that the negative regulation of protein-tyrosine kinase activity caused by the carboxyl-terminal domain of the EGF receptor can be accounted for, in part, by the regulatory phosphorylation site Ser1046/7 (Fig. 9).

Previous studies have indicated two independent functions of the carboxyl-terminal domain of the EGF receptor.

Binding of Substrates-Activation of the EGF receptor causes the physical association of the receptor with several cytoplasmic substrates for tyrosine phosphorylation, including phospholipase C-7 and GTPase-activating protein (reviewed by Ullrich and Schlessinger, 1990). The binding of these substrates can be accounted for by the presence of conserved src homology domains (SH2 and SH3) in these proteins (Anderson et al., 1990; Koch et al., 1991). The binding site on the EGF receptor has been identified as the tyrosine phosphorylated carboxyl-terminal domain of the receptor (Margolis et al., 1990a, 199Ob; Skolnik et al., 1991; Koch et al., 1991). This carboxyl-terminal region therefore represents an important effector domain for EGF receptor function that is required for the interaction with this class of signaling molecules. Consequently, it may be significant that the neg-

ative regulatory phosphorylation site Ser'046/7 is contained within this carboxyl-terminal domain of the EGF receptor (Fig. 9). One possibility is that the phosphorylation of the EGF receptor at Ser'046/7 may alter the interaction of the receptor with substrates. Such a change in substrate interaction may result in an apparent decrease in protein-tyrosine kinase activity of the EGF receptor measured with model substrates. This hypothesis can be directly tested by compar- ing the interaction of substrates with the wild-type and [Ala'046/7]EGF receptor.

Receptor Internalization-Deletion mutations within the carboxyl-terminal domain of the EGF receptor have been shown to cause an inhibition of EGF receptor endocytosis (Chen et al., 1989). The importance of this subdomain of the EGF receptor for endocytosis is consistent with the observation that the replacement of Ser'"6/7 with Ala causes a marked inhibition of EGF receptor internalization (Fig. 7). It is possible that the mutation of Ser'046/7 alters the functional conformation of a putative internalization signal located within the carboxyl-terminal domain of the EGF receptor (Chen et al., 1989). Alternatively, it is possible that the phosphorylation of the EGF receptor at Ser'046/7 is required for the rapid internalization of the EGF receptor. At present, insufficient information is available to distinguish between these hy- potheses.

One result of the internalization-defective phenotype of the [Ala'046/7]EGF receptor is that the rate of down-regulation of this receptor from the cell surface is significantly slower than wild-type EGF receptor (Fig. 8). The defect in down-regulation may be significant for the process of desensitization. This is because the wild-type EGF receptor is rapidly internalized, and the protein-tyrosine kinase activity of the receptor is inhibited in EGF-treated cells. In contrast, the [Ala'046/7]EGF receptor is not down-regulated during short term EGF treatment (Fig. s), and no inhibition of the receptor protein- tyrosine kinase activity is observed (Fig. 4). It is therefore possible that internalization of the EGF receptor (and the consequent change in subcellular compartmentation) is required for the desensitization of the receptor protein-tyrosine kinase activity. Previous studies have demonstrated that internalization-defective EGF receptors exhibit a potentiation of mitogenic signaling and may therefore be defective in desensitization (Wells et al., 1990).3 Further work will be required to address whether internalization is required for the acute inhibition of the EGF receptor protein-tyrosine kinase activity that is observed in EGF-treated cells.

Sarcomagenic Potential of the v-erbB Oncogene Correlates with the Deletion of the Negative Regulatory

Phosphorylation Site Ser'046/7

Avian leukosis viruses can cause erythroleukemia in chick- ens after a long latent period by inserting within the c-erbB gene (reviewed by Maihle and Kung, 1988). This insertion results in the expression of a truncated EGF receptor that lacks the extracellular ligand-binding domain. The insertion- ally activated c-erbB is an oncogene that is exclusively leu- kemogenic (Pelley et al., 1988). In contrast, acute transform- ing viruses that carry the erbB oncogene (such as the R and

The internalization-defective EGF receptor used to demonstrate a potentiation of mitogenic signal transduction was constructed by truncation of the carboxyl terminus of the receptor at residue 973 (Wells et aL, 1990). This truncated receptor lacks the Ser'046'7 phosphorylation site. It is therefore possible that the absence of this negative regulatory phosphorylation site may contribute to the potentiation of signal transduction observed for this mutant receptor.


H strains of avian erythroblastosis virus) induce both fibro- sarcomas and erythroleukemias (Maihle and Kung, 1988). The expanded oncogenic potential of u-erbB can be attributed to sequence alterations (point mutations and deletions) within the cytoplasmic domain (Maihle and Kung, 1988). It has been demonstrated that carboxyl-terminal truncations do not contribute to the sarcomagenic potential of erbB (Pelley et al., 1989). However, two structural alterations have been found to closely correlate with the ability of erbB to transform fibroblasts. First, Shu et al. (1990) have reported that a point mutation within the ATP-binding site of the protein-tyrosine kinase domain of erbB H accounts for fibroblast transformation. Other point mutations may contribute to the sarcomagenic potential of erbB R (Shu et al., 1990). Second, it has been shown that deletions within the carboxyl-terminal domain close to the P3 tyrosine autophosphorylation site correlate with the expanded oncogenic potential of erbB (Gamett et al., 1986; Pelley et al., 1989). It has been shown that the deletion of tyrosine autophosphorylation sites is not relevant to fibroblast transformation (Pelley et al., 1989). However, comparison of the deletions present within erbB in a series of avian viruses that exhibit an expanded disease tropism indicates that the deletion of the negative regulatory phosphorylation site equivalent to Ser'046/7 in the human EGF receptor is a common event (Tracy et al., 1985; Gamett et al., 1986; Tracy, 1988; Raines et al., 1988; Pelley et al., 1988; Pelley et al., 1989).

Oncogenic conversion by the loss of negative regulatory elements is a frequent mechanism of formation of viral on- cogenes (Lewin, 1991; Cantley et al., 1991; Hunter, 1991). One example is provided by the observation that the deletion of a negative regulatory phosphorylation site (Tyr'") in pp60"" contributes to fibroblast transformation by u-src (Cooper et al., 1986). It is therefore likely that the deletion of the negative regulatory phosphorylation site Ser'046/7 in erbB may contribute to the sarcomagenic potential of this oncogene. Indirect evidence that supports this hypothesis has recently been obtained from studies of chimeric erbB genes in which the cytoplasmic and transmembrane regions of u-erbB were fused to the extracellular ligand-binding domain of the human EGF receptor (Massoglia et al., 1990). The chimeric proteins were found to cause EGF-dependent transformation of NIH 3T3 cells. However, a carboxyl-terminal deletion of residues including the phosphorylation site Ser'046/7 was associated with the EGF-independent transformation of NIH 3T3 cells (Mas- soglia et al., 1990). These data indicate that there is a close association between the increased sarcomagenic potential of erbB and the carboxyl-terminal deletion of residues, including Ser'046/7. An important goal for further studies will be to perform a rigorous test of this hypothesis. This can be achieved by constructing recombinant avian leukosis viruses containing point mutations at the phosphorylation site within the erbB gene and examination of the tissue specificity of tumor induction. These experiments are currently in progress in this laboratory.

Acknowledgments-We thank Dr. T. Hunter and J. Meisenhelder for providing the monoclonal antibody m108.1, Dr. R. Cerione for the plasmid pGEX-TK6, and Nkia Giron6s for constructing the plasmid pGEX-[A'"6'7]TK6 and isolating the bacterially expressed fusion proteins. The excellent technical assistance of Krista Stanley and Debra Latour is greatly appreciated. Margaret Shepard is thanked for providing expert administrative assistance.

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