Alteration of Epidermal Growth Factor Receptor Activity by Mutation ...

THE JOURNAL OF BIOLOGICAL CHEMISTRV Q 1988 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 263, No. 8, Issue of March 15, pp. 3610-3617,1988 Printed in U. S. A.

Alteration of Epidermal Growth Factor Receptor Activity by Mutation of Its Primary Carboxyl-terminal Site of Tyrosine Self-phosphorylation*

(Received for publication, June 9,1987)

Paul J. BerticsS, William S. Cheng, Laura Hublerq, Cheri S . Lazarll, Michael G. Rosenfeld**$$, and Gordon N. Gill11 From the Department of Physiological Chemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706 and the IlDepartmnt of Medicine, Division of Endocrinology and Metabolism, the $Department of Chemistry, and the **Howard Hughes Medical Institute, University of California-San Diego, La J o h , California 92093

The epidermal growth factor (EGF) receptor, which exhibits intrinsic protein tyrosine kinase activity, undergoes a rapid, intramolecular self-phosphorylation reaction following EGF activation. The primary sites of tyrosine self-phosphorylation in vivo are lo- cated in the extreme carboxyl-terminal region of the molecule, principally Tyr-1173. To test the biological and biochemical consequences of this EGF receptor self-phosphorylation, we made the mutation TyrdPhe- 1173. Membranes containing the mutated receptor exhibited an ED60 for EGF activation of tyrosine kinase activity equivalent to control receptor at both high and low substrate levels, but exhibited reduced basal and EGF-stimulated tyrosine kinase activity at low, nonsaturating substrate levels. The TyrdPhe- 11 73 mutant possessed high affinity EGF binding and could still self-phosphorylate other tyrosine sites in an intramolecular fashion with a low K, for ATP (200 nM), suggesting that this alteration did not grossly change receptor structure. When EGF-dependent growth of Chinese hamster ovary cells expressing comparable levels of control or mutant EGF receptor was measured, the ability of the mutant receptor to mediate cell growth in response to EGF was reduced by approximately 60%, yet both receptors exhibited a similar affinity and EDao for EGF. These results support the concept that this self-phosphorylation site can act as a competitive/alternate substrate for the EGF receptor, and that this region of the molecule is important in modulating its maximal biological activity.

Numerous studies on cell growth control and transforma-

* These studies were supported by research grants from the Life and Health Insurance Medical Research Fund, Grant 5-605 from the March of Dimes, the Shaw Scholars program of the Milwaukee Foundation, and the Medical and Graduate Schools of the University of Wisconsin-Madison (to P. J. B.) and by awards from the American Cancer Society, the National Institutes of Health, and the Council for Tobacco Research-USA, Inc. (to G. N. G. and M. G. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisemnt” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom correspondence should be addressed Dept. of Physio- logical Chemistry, 1300 University Ave., University of Wisconsin- Madison, Madison, WI 53706.

(I Predoctoral student supported in part by a grant from the Lucille P. Markey Charitable Trust, Miami, FL, and by United States Public Health Service Training Grant T32 GM07215.

$$ A scholar of the Howard Hughes Medical Institute.

tion have shown that many retroviral transforming proteins are themselves protein tyrosine kinases or are ligands for receptors with intrinsic protein tyrosine kinase activity (1-3). The epidermal growth factor (EGF)’ receptor, which is one of the more extensively characterized enzyme/receptor systems involved in growth control, is a 170-kDa transmembrane glycoprotein possessing EGF-stimulable protein tyrosine kinase activity (3-5). EGF binding to the extracellular receptor domain activates its tyrosine kinase activity and leads to increased intracellular substrate phosphorylation and self- phosphorylation (4, 6). The cytoplasmic protein tyrosine kinase domain of the EGF receptor is homologous to erb B, the transforming protein of the avian erythroblastosis virus (7- 13), and is structurally similar to other cellular tyrosine kinases identified as the proto-oncogene products of certain retroviruses (1-3, 9).

The EGF receptor exists as a phosphoprotein in vivo, containing 2-3 mol of phosphate/mol of receptor protein (14). These phosphates are primarily distributed on serine and threonine residues (6, 14). EGF activation results in receptor self-phosphorylation on tyrosines found in the extreme carboxyl-terminal region of the molecule (15). The major site is tyrosine 1173, but receptor phosphorylation on tyrosines 1068 and 1148 also occurs (15, 16). Because self-phosphorylation can regulate several protein tyrosine kinases (17-21), as well as the CAMP-dependent protein kinase (22), calmodulin kinase I1 (23-25), phosphorylase kinase (26, 27), and protein kinase C (28-31), it may be that this type of “auto”-regulation serves as an important control mechanism for protein kinases in general.

Self-phosphorylation appears to have a biological role in modulating EGF receptor activity (32, 33). Studies on EGF receptor self-phosphorylation using purified protein have shown that the reaction is a rapid, intramolecular process (34, 35) which is maximal at 30-37 “C and exhibits a rC, for ATP (200 nM) that is much less than that calculated for exogenous substrate phosphorylation (32-36). Our earlier work indicated that these self-phosphorylation sites act as alternate substrates for the EGF receptor (32, 33) and that the inhibition of self-phosphorylation by exogenous substrates observed by us and others (32, 36-38) is a result of competition for the enzyme active site. These studies showed that the self-phosphorylation sites can competitively inhibit the EGF receptor

The abbreviations used are: EGF, epidermal growth factor; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid; CHO, Chinese hamster ovary; DME, Dulbecco’s modified Eagle’s minimal essential medium; PBS, phosphate-buffered saline; HEPES, 442- hydroxyethy1)-1-piperazineethanesulfonic acid; HPLC, high pressure liquid chromatography.

3610

EGF Receptor Self-phosphorylation Mutant 361 1

but have no effect on the maximal catalytic competence ( Vma) of the enzyme. This effect appears different from that pro- posed for insulin receptor self-phosphorylation (17, 18, 39), which appears to alter the Vmax of the enzyme and render it ligand-insensitive.

Wi th the EGF receptor, ligand binding is the major regu- lator of enzyme activity. However, self-phosphorylation may act to suppress its basal tyrosine kinase activity so as to decrease undesired substrate phosphorylation in the unstim- ulated state (32). The kinetic mechanism for exogenous substrate phosphorylation is an ordered Bi Bi sequential pattern wherein protein/peptide substrate binding precedes that of ATP (36). Product release is also ordered with phosphopep- tide dissociation preceding that of ADP. This kinetic mechanism is of interest because other protein kinases, both serine/ threonine (40) and tyrosine (41) kinases, have generally been found to have an ordered mechanism wherein ATP, not the protein substrate, binds to the enzyme first. These unusual kinetics may be an important feature of the primary structure of the EGF receptor, and this reaction may be critical in the suppression of EGF receptor tyrosine kinase activity by competition with the self-phosphorylation site. The receptor would be subject to competitive binding of the self-phosphorylation site regardless of the ATP level, thereby allowing for a continual and potent blockade of enzyme activity until enzyme activation occurs via EGF binding followed by self- phosphorylation as a rapid secondary response. According to this model, the phosphorylation rate of a given substrate would depend, in part, on its concentration and affinity for the enzyme relative to that of the self-phosphorylation site. This hypothesis for the effect of self-phosphorylation o n EGF receptor activity appears to be in opposition to that of Down- ward et al. (16) who concluded that self-phosphorylation had no effect on enzyme activity.

To clarify the role of self-phosphorylation in EGF receptor regulation, a mutant EGF receptor was prepared containing a phenylalanine instead of a tyrosine residue at amino acid 1173, which is the major in uiuo site of self-phosphorylation. It was postulated that the substitution of a structurally similar, but non-phosphate-accepting amino acid would allow this region to still function as a competitive inhibitor versus exogenous substrates.

In this paper, we show that the mutant receptor exhibits impaired kinase activity at low substrate levels but essentially normal kinase activity at saturating substrate levels, indicating the mutation did not nonspecifically inactivate the enzyme. The mutant receptor exhibits high affinity EGF binding, a normal ED, for EGF activation, and can undergo phosphorylation on the remaining self-phosphorylation sites in an intramolecular manner with a low K, for ATP (200 nM). When the EGF-dependent growth of CHO cells containing similar levels of control or mutant EGF receptor was measured, it was observed that the ability of the mutant receptor to mediate cell growth in response to EGF was reduced by approximately 50%, although both receptors had a similar affinity (KD = 0.5 nM) and ED,, (0.02 nM) for EGF. These results support the hypothesis that the carboxyl-terminal region of the molecule acts as a competitive/alternate substrate for the receptor and is important in modulating the receptor's maximal biological activity.

EXPERIMENTAL PROCEDURES

Construction of the Y1173 and F"" EGF Receptor cDNA Transcrip- tion Units-The construction of the chimeric plasmid pXER was carried out as described previously (42). The EGF receptor transcription unit utilized the SV40 early promoter/enhancer and the SV40 late splicefpolyadenylation site. A mutant dihydrofolate reductase

gene provided a dominant selectable marker (43). F1173 EGF receptor construction was accomplished by in vitro mutagenesis using a synthetic oligonucleotide complementary to the region indicated by a bar in Fig. 1 to generate the tyrosine to phenylalanine substitution at amino acid 1173. This synthetic oligonucleotide was used for site- directed mutagenesis using the Yn7' EGF receptor cDNA XbaI- HindIII fragment introduced into M13 as a template (44). Mutant clones were selected by hybridization to the 32P-labeled oligonucleotide. Positive plaques were further purified and checked for the appearance of a unique EcoRI site generated by the introduced mutation, and constructions were then verified by DNA sequencing. The F117' EGF receptor XbaI-HindIII fragment was replaced into the pXER vector.

Selection of Mutants-Vectors containing the Y1173 and F1173 EGF receptor were introduced into mouse L (B82) and CHO cell lines using a calcium phosphate coprecipitation technique (45) and B82 transfectants were selected for and grown using 400 nM methotrexate in DME medium with 10% dialyzed fetal calf serum. Gene amplifi- cation was accomplished using stepwise increases in methotrexate to a final concentration of 10 FM. CHO transfectants were selected using cotransfection with pSV2-neo and growth in 100 pg/ml G418. Mouse B82L and CHO cell recipients lack EGF receptor mRNA and protein (46).

Preparation of Cell Membranes and Purification of the EGF Recep- tor-Membranes were prepared from ten 15-cm diameter plates of near confluent cells. The cells were washed three times with ice-cold Caz+-free phosphate-buffered saline containing 2.5 mM EDTA and EGTA, and then lysed in a 20 mM HEPES, pH 7.4, buffer containing 1.5 mM MgC12, 2.5 mM EDTA and EGTA, 1 mM phenylmethylsulfonyl fluoride, 6 mM 2-mercaptoethanol, 4 mM benzamidine, 4 mM iodoa- cetic acid, 0.5 units/ml aprotinin, and 20 pg/ml leupeptin. All procedures were done at 0-4 "C. The cells were subjected to Dounce homogenization followed by sonication. The homogenates were first centrifuged at 1500 X g for 10 min, and the supernatant was then centrifuged at 18,000 X g for 30 min. The final membrane pellet was resuspended in a 20 mM HEPES, pH 7.4, buffer containing 6 mM 2- mercaptoethanol, 10 pg/ml leupeptin, and 1 mM EGTA. Aliquots were stored at -70 'C.

Purified and enzymatically active control or mutant EGF receptor/ kinase was prepared from B82L cell lines as described previously (34, 47). Briefly, the EGF receptor was purified from Triton X-100- solubilized cells by immunoaffinity chromatography using an anti- EGF receptor monoclonal antibody (528) which recognizes the receptor's EGF binding region (48). The receptor was eluted from the immobilized antibody using EGF and is active as a protein tyrosine kinase (34). This receptor preparation is highly purified (homogene- ous) as judged by sodium dodecyl sulfate-polyacrylamide gel electro- phoresis and amino-terminal sequence analysis (34).

Assay of EGF Receptor Tyrosine Kinase Activity-The tyrosine kinase activity of the purified EGF receptor was assayed in reaction mixtures containing 20 mM HEPES, pH 7.4, 2 mM MnClz, 5 mM MgCl,, 50 p~ Na3V04, 0.5 mg/ml bovine serum albumin, 0.1-10.0 PM [y3*P]ATP (specific activity = 0.07-1.5 X lo6 cpm/pmol), 0-600 nM EGF (as indicated), the appropriate purified enzyme or cell membranes, and without or with exogenous peptide substrates (32, 34).

bated with the indicated concentrations of EGF for 45 min at 4 "C When membranes were used as the enzyme source, they were incu-

and 10-20 min at 22 'C in a buffer containing 20 mM HEPES, pH 7.4, before initiating assays of protein tyrosine kinase activity (5,34). In each experiment, control incubations contained all assay components except enzyme. The peptide substrates used in these studies were angiotensin I1 and the src-peptide (a synthetic peptide corresponding to Y41s in pp60-u-src) (32, 34). Enzyme reactions were carried out a t 4 or 30 "C for various time intervals and were termi- nated via the addition of trichloroacetic acid. Peptide phosphorylation was measured using a previously described phosphocellulose binding procedure (32), and self-phosphorylation was determined using a filter binding assay (32,34).

EGF Receptor Binding Assays-Binding assays using '9-EGF were performed as described (49) with the following modifications. Cul- tured cells were washed with phosphate-buffered saline (PBS) and incubated for 4 h at 4 'C with PBS containing 0.1% bovine serum albumin, 0.05 nM lZ5I-EGF, and increasing amounts of unlabeled EGF. Blank values that were measured in the presence of 1 PM unlabeled EGF were subtracted from the total binding. Duplicate plates were used to quantitate cell number. Data were analyzed for binding affinities (KD values) and the number of binding sites by computerized nonlinear regression methods (46). As shown in the

3612 EGF Receptor Self-phosphorylation Mutant text and as described by many other groups, Scatchard analysis of EGF binding to intact cells yields curvilinear plots (46, 50). As discussed by Carpenter (50), the meaning of such curvilinear Scat- chard plots is uncertain. In the text, the apparent KO and E,, values are indicated for the high and low affinity components of the curves, as well as the values calculated using all of the data points and assuming a single binding class.

Phosphpeptide Mopping-B82L membranes prepared from cells expressing Y11'3 and F"" EGF receptors (360 and 170 pg of membrane protein, respectively) were incubated for 20 min at 22 "C in a 20 mM HEPES, pH 7.4, buffer with 1 p~ EGF and 100 pg/ml leupeptin. The membranes were then added to reaction mixtures that had a final level of 200 (IM NasVO,, 2 mM MnC12, and 0.5 pM [Y-~'P]ATP (5 X 10' cpm), and incubated for 15 min at 4 "C to self-phosphorylate the enzyme extensively (34, 51). The reactions were stopped and the membranes solubilized by adding 0.5 ml of 20 mM HEPES, pH 7.4, 0.5% Triton X-100, 0.1% sodium dodecyl sulfate, 10% glycerol, 150 mM NaC1, 2 mM EGTA, 200 p~ NhVO,, 10 pg/ml leupeptin, and 5 pM unlabeled ATP. The mixtures were centrifuged at 15,000 X g for 15 min, and the supernatants were incubated with 528 IgG-Sepharose for 2 h at 4 'C (47). After washing, the EGF receptors were eluted from the immunoaffinity matrix with 100 mM acetic acid, lyophilized, resuspended in 50 mM NaHCO,, pH 8.3, and extensively digested with L-1-tosylamido-2-phenylethyl choromethyl ketone-treated trypsin. Tryptic digests were analyzed on a Cls reverse phase HPLC column (4.6 X 25 cm, 10 pm) using a 3-30% acetonitrile, 0.1% trifluoroacetic acid gradient at a flow rate of 1 ml/min.

Qwntitution of EGF Receptor Protein-Membranes from B82L cells without or with Y1173 or F"', EGF receptors were solubilized using Laemmli sample buffer and separated on a 7.5% sodium dodecyl sulfate-polyacrylamide gel (100-120 pg/lane). Proteins were trans- ferred to nitrocellulose (52) and identified using a 1:400 dilution of a polyclonal anti-hEGF receptor serum (53) with detection by the avidin-biotin-Staph A procedure (Vectastain ABC Kit, Vector Lab- oratories, Buiington, CA).

The mass of EGF receptor protein in membranes was also determined by radioimmunoassay using the rabbit polyclonal antibody generated against purified hEGF receptor prepared from A431 cells (53). The antisera, which are directed against protein epitopes in the extracellular ligand binding domain, were diluted 1:400 and a standard curve generated using the purified 'T-EGF receptor extracellular domain secreted by A431 cells. Each membrane preparation was solubilized using 1% Triton X-100 and 10% glycerol, and three dilutions performed in triplicate were used for each determination of EGF receptor mass? Membranes from untransfected B82L cells gave no displacement confirming antibody specificity.

Muterials-[~-~~P)ATP (3000 Ci/mmol) was obtained from Amer- sham Corp., and angiotensin I1 and the tyrosine kinase substrate (src-peptide) corresponding to the site of self-phosphorylation in pp60-src were purchased from Sigma. EGF was purified from adult mouse submaxillary glands (54); purity was verified by HPLC and amino acid analysis.

RESULTS

Generation of Cell Lines Expressing Mutant Human EGF Receptors-To characterize the influence of self-phosphorylation on EGF receptor tyrosine kinase activity and its biological effects, we prepared two transcription units containing the entire coding region of the human EGF receptor with either a tyrosine or a phenylalanine at amino acid position 1173, which is the major in vivo self-phosphorylation site (15, 16). The construction of the eukaryotic expression vectors encoding these control (Y1173) or mutant (F1173) EGF receptors is shown in Fig. 1. In these experiments, both constructs contained an alanine at amino acid position 654, which is the site of protein kinase C phosphorylation (46, 55), to preclude any possible effects of differential phosphorylation at this site between the two receptor forms.

The Y1173 and F1173 EGF receptor genes were transfected into Chinese hamster ovary and mouse B82L cells which do not contain endogenous EGF receptor mRNA or protein (46).

C. D. Carpenter, J. M. Sowadski, C. S. Lazer, C. Cochet, G. N. Gill, and M. C. Rosenfeld (1987) submitted for publication.

Permanent transfected cell lines were analyzed by lZ5I-EGF binding and by a monoclonal antibody (528) that exclusively recognizes the amino-terminal portion of the human EGF receptor (48). EGF receptor mass, quantitated by immunoblotting and radioimmunoassay, indicated that both receptors were synthesized and processed into fully mature 170-kDa forms (Fig. 1). Confirmation of the loss of the major self- phosphorylation site is detailed in Fig. 1. The principal in vivo and in vitro self-phosphorylation site is designated P1 (15) and is absent from the mutant EGF receptor as shown in Fig. 1. These analyses of the mutant receptor also indicate that, although the major site of self-phosphorylation has been removed, this enzyme can still phosphorylate two other prin cipal sites, i.e. tyrosines 1068 and 1148 (15) which are indicated as P2 and P3 in Fig. 1, as well as other unidentified sites (15, 51).

Fig. 2 shows that both the Y1173 and F1173 EGF receptors exhibit curvilinear Scatchard plots characteristic of Y-EGF binding to intact cells (46, 50). The values for the high and low affinity components of these curves are given in the inset to Fig. 2. Because there is some question as to the interpre- tation of nonlinear Scatchard plots, the data were also analyzed as a single component curve. Using B82L cells, the data yielded an apparent KD = 0.3 nM and a B,,, = 53,300 receptors/cell ( r 2 = 0.74) for the Y1173 EGF receptor and a KD = 0.6 nM and a B,, = 113,000 receptors/ceII for the F1I7' EGF receptor. These findings indicate that the F1173 substitution does not appreciably alter the binding characteristics of the EGF receptor expressed on the surface of these cells.

Kinetic Analysis of F1173 EGF Receptor Protein Tyrosine Kinase Activity--If the mechanism of action of self-phosphorylation is consistent with a competitive model, one would predict that the receptor would exhibit impaired kinase activity a t low substrate levels but essentially normal kinase activity at saturating substrate levels, thereby indicating that the mutation did not generate a nonspecific enzyme inactivation. If self-phosphorylation activates the receptor regardless of whether ligand is bound, as is the case with the insulin receptor (17, 18, 39), then the activity of the mutant enzyme should be reduced at both high and low substrate concentrations.

Fig. 3 illustrates that B82L cell membranes containing the mutant F1173 EGF receptor exhibit reduced EGF-stimulated tyrosine kinase activity at low angiotensin I1 levels when compared to membranes containing the normal Y1173 receptor, but both receptors possess similar maximal velocities indicating that self-phosphorylation does not appreciably alter the maximal catalytic competence of the enzyme. In these experiments, the Y1173 receptor exhibited a V,,, = 196 & 8 pmol/ min/pmol EGF binding and an apparent K,,, = 2.63 4 0.20 mM for angiotensin 11, whereas the Y1173 receptor exhibited a Vmax = 253 f 5 pmol/min/pmol EGF binding and an apparent K,,, = 0.93 f 0.04 mM for angiotensin 11, which is similar to the K, of 0.8 mM that has been previously reported for the wild type A431 receptor (34). A similar increase in K,,, using F1173 compared to Y1173 EGF receptors was observed with a second peptide substrate which corresponds to Y416 in pp60- src (Table I). Immunoaffinity-purified F1'73 and Y1173 EGF receptors also possessed comparable maximal velocities (data not shown).

Since these reactions were run using 10 p M ATP at 30 "C for 2 min, which allows for rapid and extensive self-phosphorylation (32-34), both enzyme preparations should be fully self-phosphorylated early in the assay time course. However, to confirm that the calculated K,,, values were for fully phosphorylated enzyme, both forms were first incubated with 15

EGF Receptor Self-phosphorylation Mutant 3613

AAT OCA GAA Asn Ala

‘Iu %? C% GTC Val

170 --c -1 kDa Null

y r t 7 3

Flt73

(pmollmg membrane EGF Receptor

protein)

0.0

7.0

9.8

Phe Acetonitrile (94)

FIG. 1. Preparation and characterization of a mutant human EGF receptor containing phenylalanine at position 1173, the major in vivo site of tyrosine self-phosphorylation. Left, construction of a eukaryotic expression vector encoding the mutant human EGF receptor. The F”73 EGF receptor mutation was made, as described under “Experimental Procedures,” on the second nucleotide of codon 1173 (A to T) using a synthetic oligonucleotide complementary to the region indicated by the bar generating the point mutation ( T y r to Phe). Center, quantitation of Y“” and F”” EGF receptors in membranes prepared from B82L cells. Protein was detected by immunoblotting and radioimmunoassay as described under “Experimental Procedures.” Right, tryptic phospho- peptide map of EGF receptors isolated from cell lines expressing the Y1173 or F”73 receptor. The receptors were extensively self-phosphorylated, isolated by immunoaffinity adsorption, and digested with trypsin as described under “Experimental Procedures.” The digest was analyzed by HPLC using a Cl* reverse phase column and a 3- 30% acetonitrile gradient a t a flow rate of 1 ml/min. The major sites of tyrosine self-phosphorylation are indicated (PI-P3); PI corresponds to tyrosine 1173 (15).

0 (PM)

FIG. 2. Binding of ‘“I-EGF to Y1ln and F”73 EGF receptor containing cells. Binding assays were carried out a t 4 “C for 4 h as described under “Experimental Procedures,” and the data were analyzed by the method of Scatchard (46). Panel A, B82L cells expressing Y’l” EGF receptors; Panel B, B82L cells expressing F”73 EGF receptors. Receptor number was calculated assuming one EGF binding site per molecule (34). Inset shows calculations assuming high and low affinity binding components.

PM ATP at 30 “C for 1 min followed by a measurement of their K,,, for the src-peptide. As seen in Table I, this treatment did not substantially alter the estimates of the K,,, for both the Y1173 and F1’73 receptor forms. The fact that mutant receptors are essentially as active as control receptors at saturating substrate levels, where effective competition occurs, but are much less active at lower nonsaturating substrate levels and have a larger apparent K,,, for peptide substrates,

suggests that the self-phosphorylation site can act as a tight binding competitive inhibitor that cannot be removed by phosphorylation of Y1173 in mutant receptors.

Because the exogenous peptide substrates are in great ex- cess to enzyme, this analysis provides valid apparent K , and VmaX values for the two forms of the EGF receptor. Kinetic analysis of tight binding competitive inhibitors present at concentrations equal to or less than enzyme yield converging lines on double-reciprocal plots indicating comparable Vmm values in the absence or presence of the inhibitor but different apparent K,,, values (56, 57). In the present case, the concentration of the carboxyl terminus equals the concentration of the receptor, but the mutant F1173 EGF receptor may have a higher effective inhibitor concentration since a reversible site has been removed. Comparison of the two forms of the EGF receptor may thus be analogous to varying the inhibitor concentration and, as shown in Fig. 3 and Table I, yields results that could be expected for a tight binding competitive inhibitor (56,57).

To analyze whether the Tyr+Phe-1173 mutation influ- ences its EGF-dependent activation, we measured the ability of mutant and control receptors to phosphorylate high (1.5 mM) and low (0.2 mM) levels of angiotensin 11 following incubation with increasing amounts of EGF. Fig. 4 shows that the Y1’73 and the F’173 receptors possess a comparable EDw for EGF with respect to activation of tyrosine kinase activity both at high and low substrate levels. At high substrate concentrations, both the basal ([EGF] = 0) and fully EGF- stimulated protein tyrosine kinase activities are similar for the control and mutant receptor. However, at low substrate levels, the mutant receptor has a greatly reduced basal and EGF-stimulated protein tyrosine kinase activity. These ob- servations support the idea that the self-phosphorylation sites, which can interact with the enzyme active site inde-

3614 EGF Receptor Self-phosphorylation Mutant

- 200

p 180

0 C

d I60

13 W

2 4 0 a

2 0 - >

m c I ~ a a C

LL 13 w

B

0.04

0 0.02

(Y n

W - 2 0 2 4 6 8 IO ~~ < l/[Angiotmrin II](mM)"

FIG. 3. Kinetic analysis of control Y1173 (0) and mutant F1173 (0) EGF receptor protein tyrosine kinase activity using varying concentrations of angiotensin II as substrate. Membranes containing equivalent amounts of EGF binding activity were first incubated with 290 nM EGF at 0 "C for 45 min followed by incubation at 22 "C for 15 min to activate the receptor fully. The membranes were then combined with the indicated antiogensin I1 concentrations before initiating the reaction with 10 p~ [T-~'P]ATP. The reactions were carried out a t 30 "C for 2 min and incorporation of phosphate into angiotensin I1 was measured as described previously (32, 34). Each point is the average of duplicate determinations and analogous results were obtained in two other experiments. Panel A , plot of the initial velocity of enzyme activity versus angiotensin I1 concentration; Panel B, double-reciprocal plot of control and mutant EGF receptor protein tyrosine kinase activity.

pendent of the presence of ATP (36), can act as competitive inhibitors of the basal and EGF-activated receptor/kinase activity.

Kinetics and Mechanism of F1173 EGF Receptor Self-phos- phorylatwn on Remaining Sites-As a further characterization of the integrity of the mutant EGF receptor, it was purified from B82L cells via immunoaffinity chromatography and the ATP dependence of its self-phosphorylation reaction on the remaining sites was assessed (Fig. 5). When the incorporation rate of the first mole of phosphate was studied under

TABLE I Comparison of the kinetics of Y1173 and F"73 EGF receptor-catalyzed

phosphorylation of two peptide substrates Membranes containing equivalent amounts of EGF binding activ-

ity were first incubated with 300 nM EGF at 0 "C for 45 min followed by incubation at 22 "C for 15 min to activate the receptor fully. The membranes were then combined with the various concentrations of angiotensin 11 (0.1-3 mM) or src-peptide (0.075-2 mM) before initiating the reaction with 10 p~ [y3'P]ATP. In some experiments, to assess if the calculated K,,, values were for fully phosphorylated enzyme, both receptor forms were first incubated with 15 p~ ATP at 30 "C for 1 min followed by a measurement of their K, for the src- peptide. The reactions were carried out at 30 "C for 2 min, and incorporation of phosphate into angiotensin I1 was measured as described under "Experimental Procedures." The kinetic constants were calculated using a nonlinear regression analysis (71), and similar results were obtained in two other experiments.

Peptide substrate K, of EGF receptor forms

mM

Angiotensin I1 0.93 f 0.04 2.63 f 0.20 u-src-Peptide 0.20 f 0.03 0.74 & 0.06 u-src-Peptide + first incubation 0.27 & 0.05 0.77 * 0.11

with 15 PM ATP

1 E. All = 0.2 mM

1 I I

100 200 300 400 600 [EGFI (nM)

FIG. 4. EGF-dependent activation of control Y1173 (0) and mutant F1IT3 (0) EGF receptor protein tyrosine kinase activity. Membranes containing control or mutant (B82L) EGF receptors and equal levels of EGF binding activity were assayed for EGF-dependent protein tyrosine kinase activity as described under "Experimental Procedures" using 1.5 mM (Panel A ) or 0.2 mM (Panel E ) antiotensin I1 (AZZ) as substrate. The membranes were incubated with the indicated concentrations of EGF for 10 min at 22 "C to ensure EGF binding as described under "Experimental Procedures." Assay tubes were then placed on ice, the kinase reaction components added, and the samples were incubated on ice for 1 min. The final concentrations of the kinase reaction components were 0.2 or 1.5 mM angiotensin 11, 4 mM MnCl', 200 p~ Na3V04, and 3 p~ [T-~'P]ATP. The data points are the average of triplicate measurements.

initial velocity conditions (30 s), low temperatures (0 "C), and low ATP levels (0.1-1.0 pM), a K,,, for ATP = 0.21 f 0.03 pM was calculated. This value is essentially identical to that obtained with the Y1173 EGF receptor (0.25 f 0.04 p ~ ) and is about 10-fold lower than that calculated for the phosphorylation of exogenous substrates (36).

Because the mechanism for the mutant EGF receptor self- phosphorylation of the remaining sites could proceed via an intramolecular (first order with respect to enzyme level) or an intermolecular (second order) process, the effect of enzyme concentration on the reaction was studied. Fig. 6A shows that self-phosphorylation exhibited a first order (linear) dependence on mutant (F1173) EGF receptor concentration over a 32- fold dilution range suggesting an intramolecular reaction. Also consistent with an intramolecular process is the fact that the amount of phosphate incorporated into the receptor varied by

EGF Receptor Self-phosphorylation Mutant 3615

EGF-RECEPTOR (F"73) SELF-PHOSPHORYL&UION .- ENZYME DEPENDENCE (32-FOLD DILUTION RANGE)

A )

Fl173 Km(ATP):0.21f0.03pM 8 -

00 ; A Ib I:

I/[ATP] (pM)"

B)

170 kDa-

FIG. 5. The effect of ATP concentration on the self-phosphorylation of the purified parental (Y1173) and mutant (F1173) EGF receptor/kinase. Panel A, determination of the K,,, for ATP for self-phosphorylation using the parental (0.9 pmol) or the mutant (0.6 pmol) EGF receptor. Self-phosphorylation was measured at 0 "C using a 30-s assay time and an ATP concentration range of 0.1-1.0 PM. The data points shown are the average of duplicate determinations, and similar results were obtained in two experiments. The kinetic data were analyzed by a nonlinear regression routine (71). Panel B, autoradiogram of the self-phosphorylated F1173 EGF receptor using the indicated levels of ATP.

only about %fold, even though receptor concentration varied by 32-fold (Fig. 6B). Finally, the van't Hoff plot of self- phosphorylation (log u uersus log [EGF receptor]) shown in Fig. 6C, whose slope is equal to the varied substrate's (enzyme's) order in the reaction (26, 58), yields a slope equal to 0.77, also indicating an intramolecular mechanism. These results do not appear to be the result of receptor aggregation because, as previously noted (34,59), immunoaffinity-purified EGF receptor exists as an active monomer.

Effects of Y1'73+F"73 on EGF-stimulated Cell Growth-To test the functional consequences of the TyrdPhe-1173 mutation, we determined its influence on EGF-stimulated prolif- eration of CHO cells. When the EGF-dependent growth of CHO cells expressing comparable levels of control or mutant EGF receptor was measured, we observed that the ability of the mutant receptor to mediate cell growth in response in EGF was reduced by approximately 50%, yet both receptors had a similar affinity (KD = 0.5 nM) and ED, (0.02 nM) for EGF (Fig. 7). These experiments were performed several times and the average increase in cell number for the control receptor at saturating EGF was 7.4-fold, whereas the cells contain-

z = I , I , I I , Q E '0 0.4 0 8 1.2 1.6 2.0 2.4

EGF RECEPTOR (F"") (pmol)

4 0 r

9 2.0

0" 1.0 SLOPE=0.77

J , '1.5 2 .O 2.5 3.0 3.5

LOG [EGF-RECEPTOR]

FIG. 6. The influence of enzyme concentration on the purified mutant EGF receptor self-phosphorylation. Immunoaffin- ity-purified F1"3 EGF receptor was assayed (see "Experimental Pro- cedures") for self-phosphorylating activity in the presence of 0.25 pM [-p3*P]ATP for 30 s at 0 "C. The enzyme concentration vaned from approximately 1.9 to 60 nM (0.75-2.4 pmol/assay). Each point is the average of duplicate determinations, and similar results were observed in two experiments. Panel A, plot of the initial velocity of self- phosphorylation Versus the amount of mutant EGF receptor protein per assay. Panel B, plot of the total amount of phosphate incorporated per receptor as a function of enzyme level. Panel C, a van't Hoff plot of the log of the initial velocity (femtomoles/min) Versus the log of enzyme concentration (nanomolar). Linear regression of the data points yielded a slope of 0.77 and a correlation coefficient of 0.96.

ing the mutant receptor averaged a 2.1-fold increase in cell number at saturating EGF. These results support the concept that that this self-phosphorylation region of the molecule is important in modulating its maximal biological activity.

DISCUSSION

The present study indicates that tyrosine 1173 in the EGF receptor, which is the kinetically preferred in uiuo self-phosphorylation site, can act as a competitive/altemate substrate for the enzyme, and that substitution of a phenylalanine residue in this location compromises the ability of the receptor to mediate cell growth in response to EGF. Several studies on the insulin receptor (17,18,39) and the transforming proteins of the Rous (19, 20) and Fujinami sarcoma viruses (21) have shown that self-phosphorylation enhances their protein tyrosine kinase activity, although the exact mechanism of this activation is unknown. Recent evidence on the biochemical and biological properties of pp60-c-src has shown that mutation of its carboxyl-terminal sites of tyrosine phosphorylation can serve to activate or suppress its transforming properties (60-62), indicating that this process is an important regulatory step.

In the case of the EGF receptor, self-phosphorylation appears to remove a competitive constraint so that the enzyme

3616 EGF Receptor Self-phosphorylation Mutant

6- 200 yii73

$ 50 - 1 0

0 -13 -12 -11 -10 -9 -8

log IEGF, MI FIG. 7. Comparison of the effect of EGF on the growth of

CHO cells containing Y11'3 versus F"'3 EGF receptors. Equal numbers of cells (2 X lo' cells/well) were plated in 12-well Costar dishes in DME/F-12 medium containing 1% calf serum. After attach- ment overnight, the dishes were washed with DME/F-12 medium three times at 37 'C and placed in DME/F-12 medium containing 200 rg/ml bovine serum albumin and the indicated concentrations of EGF. These completely serum-free, defined conditions were necessary to achieve a slow growth rate of untreated CHO cells. Cell numbers of triplicate dishes were determined on day 6. In three separate experiments, the plating efficiencies of CHO cells containing Y1173 and F"'3 EGF receptors averaged 75 and 64%, respectively, with an overlap in range. Assuming an average plating efficiency of 70% and in the presence of 1 nM EGF, the CHO cells containing Y11'3 EGF receptors underwent 3.5 population doublings (41 h/doubling), whereas cells containing F"'3 EGF receptors underwent 2.7 population doublings (53 h/doubling). These values are in good agreement with the population doubling time of 36 h determined from growth curves of CHO cells containing Y1173 EGF receptors (42), and a doubling time of 52 h determined from growth curves of CHO cells containing F11'3 EGF receptors (data not shown).

active site can be more freely accessible to certain exogenous substrates. In addition to this effect, Livneh et al. (63) have found that a carboxyl-terminal truncated EGF receptor de- void of two of the major self-phosphorylation sites also lacks high affinity EGF binding. These findings support the concept that the carboxyl terminus, which contains the major self- phosphorylation sites, can modulate receptor function. Alter- ations in this regulatory region of the molecule can result in altered receptor activity. Because erb B lacks the EGF binding domain and the major carboxyl-terminal self-phosphorylation site of the proto-oncogene EGF receptor (2, 3), it would not be influenced by these regulatory domains. Analyses of the effects of viruses recovered following avian leukosis and Rous- associated virus type 1 infection indicate a functional role for the carboxyl-terminal domain of the EGF receptor, ix. the region that is truncated in the erb B gene product. The avian leukosis virus results in the production of an erb B product via insertional mutagenesis which consistently truncates the amino-terminal EGF binding domain but variably deletes the carboxyl-terminal region (64, 65). Following infection with the recovered viruses, erythroblastosis has been found to occur independent of the degree of carboxyl-terminal truncation, but the generation of sarcomas is only detected with mutants lacking the carboxyl-terminal region (66).

Another line of evidence with suggests that the carboxyl- terminal region of the EGF receptor is important in regulating its biological properties comes from the analysis of a chimeric, ligand-binding u-erb BIEGF receptor which still retains its transforming potential (67). In that study, the ligand binding domain of the EGF receptor was coupled to the u-erb B transforming protein, thereby creating a protein that was no longer truncated at the amino terminus but still lacked the 32-amino acid carboxyl-terminus which contains tyrosine 1173. The chimeric protein was transforming in the absence of EGF, but its transforming potential could be enhanced via the addition of EGF. Because this chimeric protein was still

capable of transformation even after putting it under EGF control, these investigations suggested that the carboxyl-terminal region was also critical in modulating enzyme activity (671, although other differences between the intracellular domain of the EGF receptor and u-erb B may prove important as well. In contrast, the holo-EGF receptor, without or with EGF, was not transforming (67).

The results presented here support the idea that this region of the EGF receptor is important in regulating its biological activity. It is unclear whether the reduction in receptor me- diated cell growth seen with the mutant (F1173) protein is a result of decreased intracellular phosphorylation of critical substrates (since they are presumably nonsaturating within the cell) and/or a result of failure of the receptor to interact with other cellular proteins or pathways, i.e. because it lacks the appropriate carboxyl-terminal tyrosine phosphate. It is conceivable that, due to the reduced tyrosine kinase activity of the mutant receptor at nonsaturating substrate levels, the rate of phosphate incorporation into important substrates is not sufficiently above endogenous phosphotyrosyl phospha- tase activities to allow for rapid growth. In this regard, EGF receptor protein tyrosine kinase activity has recently been shown to be essential for both immediate and delayed cellular responses to EGF (42).

I t is also uncertain if self-phosphorylation plays a role in modulating the specificity or activity of the receptor toward various types of substrates. Recent studies with the Ca2+- and phospholipid-activated protein kinase C indicate that certain substrates can impart differing Ca2+ and phospholipid require- ments toward the stimulation of kinase activity (68). Some substrates (e.g. histone) required the presence of both activators, some substrates (myelin basic protein) required only the presence of phospholipid while others, such as protamine, required no activators at all. In the EGF receptor studies described by us here and previously (32, 33), and in those by Downward et al. (16), only a few small peptide substrates were used to assess the influence of self-phosphorylation on receptor kinase activity. Thus, it is possible that other changes in receptor activity/specificity induced by self-phosphorylation may have gone undetected using these peptide substrates.

In summary, the present studies indicate that the major EGF receptor self-phosphorylation site may serve as a competitive inhibitor uersus exogenous substrates, explaining why self-phosphorylation can enhance receptor tyrosine kinase activity. The inhibition of the maximal response to EGF by the mutant receptor (F'"') suggests that the carboxyl terminus may have a functional/regulatory role in uiuo, but that it is quantitatively smaller than that of EGF itself. Because self- phosphorylation of tyrosines 1148 and 1068 also occurs by an intramolecular process, these residues may also act as effective competitive inhibitors with exogenous substrates, e.g. there is a redundancy in the regulatory function of the carboxyl terminus. This possibility can also be tested by converting these residues to phenylalanines.

Self-phosphorylation accounts for only a fraction of total EGF receptor phosphorylation in uiuo (6, ll), and it appears that other protein kinases, such as the CAMP-dependent protein kinase (69, 70), can phosphorylate the receptor. The function of these other phosphorylations is unclear, although they may directly exert additional controls on receptor activity or may function indirectly by targeting the receptor for interaction with other intracellular components or pathways.

Acknowledgments-We thank Dr. Claude Cochet for advice and contributions to the kinetic experiments; we also thank Charles Nelson for tissue culture work and Gary Heisermann for HPLC analysis.

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EGF Receptor Self-p

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