regulation of phosphorylation of the c-erbb-2/her2 gene ... · mouse immunoglobulins g (ram) was...

MOLECULAR AND CELLULAR BIOLOGY, Feb. 1991, p. 979-986 0270-7306/91/020979--08$02.00/0 Copyright © 1991, American Society for Microbiology

Vol. 11, No. 2

Regulation of Phosphorylation of the c-erbB-2/HER2 Gene Product by a Monoclonal Antibody and Serum Growth Factor(s)

in Human Mammary Carcinoma Cells RAK.ESH KUMAR,1* H. MICHAEL SHEPARD,2 AND JOHN MENDELSOHN1·3

Laboratory of Receptor Biology, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, New York 100211; Developmental Biology, Genentech Inc ., South San Francisco, California 9408a2;

and Cornell University Medical College, New York, New York 100213

Received 9 May 1990/Accepted 17 November 1990

Monoclonal antibody (MAb) 4DS was used to analyze the phosphorylation of p185HER2, the gene product of c-erbB-21HER2, in SK-BR-3 cells. Culture in the continuous presence of 4DS reduced the in vivo steady-state levels of p185HER2 phosphorylation by 80% in a dose-dependent manner, suggesting that MAb 4DS may have interfered with the activation of phosphorylation of p185HER2. The observed MAb-mediated reduction of p185HER2 phosphorylation could not be completely accounted for by down-regulation. When cultures were grown under serum-free conditions, the steady-state levels ofpl85HER2 phosphorylation were reduced by 56%, and addition of 4DS further inhibited phosphorylation to 20% of steady-state levels. With continuous exposure to increasing concentrations of newborn calf serum in these cultures, the.re was a linear increase in tyrosine-specific phosph~lation of p185HER2, reaching a 5.4-fold increase with 10% newborn calf serum. Phosphorylation of pl85H R2 in the presence of newborn calf serum was not attributable to stimulation of the epidenna1 growth factor receptor by epiderma1 growth factor or by transforming growth factor-a. Extension of these observations to two other mammary carcinoma ceU lines, MDA·MB-453 and BT-474, a1so demonstrated a significant capacity of serum to induce pl85HER2 phosphorylation. The demonstration of antibody· mediated partial inhibition of phosphorylation under serum-free conditions suggests that mammary carcinoma cells may also produce and secrete a factor or factors which may activate l185HER2. Our observation that growth-inhibitory MAb 4DS is able to reduce the phosphorylation of p185HB by newborn calf serum and by a cellular-derived factor(s) suggests the existence of a growth factor(s) which uses phosphorylation of p185HER2 as a signal transduction pathway to regulate ceU proliferation.

Proto-oncogenes are a group of normal genes which play important roles in the regulation of cell proliferation and function (2, 5). Abnormalities in the expression , structure, or activity of proto-oncogene products contribute to the development and maintenance of the malignant phenotype in complex but important ways (36, 37, 46). Evidence that the gene products of several activated proto-oncogenes are either growth factors or growth factor receptors has suggested a possible link between proto-oncogenes and growth factors (20). For example, the receptor for macrophage colony-stimulating factor is identical to the product of c-fms (35), and c-erbB-1 encodes the receptor for epidermal growth factor (13) and transforming growth factor-a (TGF-a) (43). Growth factor receptors encoded by proto-oncogenes are transmembrane glycoproteins with intrinsic tyrosine-kinase activity (22). Receptor tyrosine kinases are activated by binding of their respective ligands, the growth factors (48). This activity is thought to be an integral part of signal transduction processes involved in the regulation of cell proliferation (21). Overexpression of some growth factor receptors has been shown to induce transformed properties in recipient ceUs (11, 32), possibly because of excessive activation of signal transduction mechanisms. Furthermore, a number of tumor cells with increased expression of growth factor receptors also produce ligands for these receptors (10).

HER2 (also known as c-erbB-2 or c-neu), the human

• Corresponding author.

979

homolog of the rat proto-oncogene neu (9), encodes a 185-kDa transmembrane glycoprotein with intrinsic tyrosine kinase activity which is presumed to be the receptor for an as-yet-unidentified ligand (3, 39). pl85HERz also has homology to, but is distinct from, the epidermal growth factor receptor (EGF-R), which is the product of c-erbB-1. Both proteins have a cysteine-rich extracellular domain, a transmembrane domain, and an intracellular tyrosine kinase (4, 31 , 47). In spite of sequence homology between c-erbB-2 and c-erbB-1, EGF does not bind to pl85HERz (33). pl85HERz has been shown to be overexpressed or amplified or both in a number of human malignancies: breast (45), ovarian (38), thyroid (1), lung (7), salivary gland (34), and stomach (50). In addition, pl85HER2 is a potent oncogene capable of inducing transformation and tumorigenesis when overexpressed in NIH 3T3 ceUs (12, 19). Overexpression of pl85HER2 also induces tumor cell resistance to macrophage killing (15). Thus p185HERZ may have an important role in the development and maintenance of human tumors.

These observations suggest that receptor-associated tyrosine kinase activity of overexpressed proto-oncogene protein products is important for the regulation of cell growth. We have developed a panel of monoclonal antibodies (MAbs) reactive with domains of the human EGF-R (23) and p185HER2 (17, 18) in intact ceUs and have demonstrated antiproliferative effects of these antibodies in vitro (16, 18, 23) and in vivo (29). Antibody 405 , which is specifically directed against pl85HERZ, exhibits strong antiproliferative activity on cultured human breast tumor cell lines which overexpress pl85HER2 (18). Since p185H.ER2 is a receptor

1 of 8 Celltrion, Inc., Exhibit 1088

980 KUMAR ET AL.

with intrinsic tyrosine kinase activity, we investigated the modula'.i-Jn of p185HERi phosphorylation by MAb 405. We report here that activation of phosphorylation of p185HERi by serum was reduced in the presence of an excess of MAb 405 and that MAb-sensitive phosphorylation was mediated by a growth factor or factors other than TGF-o: or EGF. Furthermore, SK-BR-3 cell-conditioned medium contained a factor(s) that could activate pl85HERi phosphorylation and was partially inhibited by MAb 405 .

MATERIALS AND METHODS

Materials. MAbs 4D5 (18) and 906 (44) were raised against human p185HER2

• MAbs 528 and 225 bind to the human EGF-R \23). Antiphosphotyrosine MAb PY-69 was obtained from ICN Biochemicals, Inc. Rabbit immunoglobulin to mouse immunoglobulins G (RAM) was supplied by Accurate Chemicals, Westbury, N.Y. 32P; (carrier free ; 28.5 Ci/nmol) and 35S-labeled L-cysteine (1,030 Ci/mmol) were purchased from New England Nuclear, Boston, Mass.

Cell lines and cell culture. Human breast tumor cell lines SK-BR-3, BT-474, and MDA-MB-453 were obtained from the American Type Culture Collection. The A431 human epidermal carcinoma cell line was originally supplied by Gordon Sato. All cell lines except MDA-MB-453 (which was grown in L-15 medium) were maintained in Ham F-12-Dulbecco modified Eagle medium (1:1, vol/vol) (F-12/ DMEM) supplemented with 10% fetal bovine serum.

Labeling of p185HER2 with 32P1 and [35S)cysteine. Cells (3 x 105

) were plated in F-12/DMEM in each well of a six-well dish. Twenty-four hours later, cultures were washed with phosphate-free medium and incubated for up to 15 h in phosphate-free F-12/DMEM containing 0.4 mCi of 32P; per ml in the presence or absence of MAb and newborn calf serum. At desi.red times, cells were harvested in 400 µI of lysis buffer (20 mM HEPES [N-2-hydroxyethylpiperazineN' -2-ethanesulfonic acid; pH 7.5), 1% Triton X-100, 10% glycerol, 1.5 mM magnesium chloride, 1 mM ethyleneglycol bis-N,N,N' ,N'-tetraacetic acid, 0.1 mM phenylmethylsulfonyl fluoride , 10 µg of leupeptine per ml , 2 mM sodium orthovanadate) at 4°C for 20 min. The lysate was centrifuged at 10,000 rpm in an Eppendorf microfuge for 10 min, and then 60 µl of Pansorbin was added as described elsewhere (42). For labeling with [35S]cysteine, the cells were washed with cysteine-free medium and refed with cysteine-free F-12/DMEM containing 0.15 mCi of [35S]cysteine per ml with or without 5% newborn calf serum.

lmmunoprecipitation and SDS-polyacrylamide gel electrophoresis. Aliquots (350 µI) of the cell lysates (or equal amounts of trichloroacetic acid-precipitable counts per minute) containing 32P-labeled or [35S]cysteine-labeled p185HER2 were subjected to immunoprecipitation with 10 µg of MAb 906, 528, or PY-69 at 4°C for 2 h. Immune complexes were collected by absorption to RAM-protein A-Sepharose beads at 4°C for 1 h. Beads were washed three times with 1 ml of buffer (20 mM HEPES [pH 7 .5], 150 mM NaCl, 0.1% Triton X-100, 10% glycerol, 2 mM sodium orthovanadate). Washed pellets were mixed with 40 µI of sample loading buffer (10 mM Tris HCl [pH 6.8], 1% sodium dodecyl sulfate [SOS], 0.2% 2-~-mercaptoethanol, 10% glycerol, 0.001% bromophenol blue), heated at 95°C for 5 min, and resolved on a 7% SDS-polyacrylamide slab gel (26). The efficiency of precipitating labeled receptor with MAb 906 is 80 to 90% when this procedure is used. Low-molecular-mass colored markers (Amersham Corp.) were used as standards.

Phosphoamino acid analysis. The band corresponding to

PETITIONER'S EXHIBITS

MOL. CELL. BIOL.

2 3 4 5

FIG. 1. Effect of MAb 405 on steady-state levels of pl85HERZ phosphorylation in SK-BR-3 cells. Subconfluent cultures were labeled with 32P; (400 µCi in 1 ml of phosphate free F-12/DMEM supplemented with 5% newborn calf serum) in the continuous presence of different amounts of antibody for 15 h. Detergent extracts were made, and pl85HER2 was immunoprecipitated by using MAb 9G6 and then resolved by 7% SDS-polyacrylamide gel electrophoresis (Materials and Methods). An autoradiogram resulting from 16 h of exposure of the dried gel is shown here. The arrow indicates the position of 32P-labeled pl85HER2 • Lane 1, Control cells; lanes 2 to 4, cells treated with MAb 405 at 30, 150, and 300 nM, respectively; lane 5, cells treated with 400 nM F(ab) fragment of MAb 405. The amounts (in counts per minute) of pl85HER2 in each lane were 4,453 (lane 1), 1,967 (lane 2), 1,785 (lane 3), 1,040 (lane 4). and 335 (lane 5). Counts were corrected by subtracting the background of 60 cpm. The results shown are representative of results in six different experiments.

the 185-kDa HER2 protein, resolved as described above, was excised out of the gel. 32P-labeled pl85HERz in a gel slice was partially hydrolyzed with 200 µI of 6 N HCI at ll0°C for 1 h. Two portions (10 µleach) of the hydrolysate were taken for measurement of radioactivity in a liquid scintillation counter to determine the total incorporation of 32P into the pl85HER2 receptor. The rest of the hydrolysate was dried, suspended in distilled water, and applied to a Dowex AG1-X8 column. The column was washed with distil.led water, and the absorbed 32P-labeled materials were eluted with 0.5 N HCI and lyophilized. The recovery of radioactivity by this procedure was 78 to 85%. 32P-phosphoamino acids mixed with unlabeled carrier phosphoamino acids (phosphoserine, phosphoth.reonine, and phosphotyrosine [1:1:1)) were analyzed by thin-layer electrophoresis as described elsewhere (8).

RESULTS

MAb 405 reduces amount of 32P-labeled pl8SH£R2• MAb

4D5 was used to investigate the regulation of phosphorylation. SK-BR-3 cells, which have an amplified c-erbB-2 gene (45), were cultured for 15 h in medium containing 32P; in the continuous presence of various concentrations of MAb 4D5. The pl85/fERi from these cells was immunoprecipitated with another anti-p185H£Ri MAb, 906, which recognizes a distinct epitope of p185HERz, and resolved by SOS-polyacrylamide gel electrophoresis. Results of such an experiment are shown in Fig. 1. Treatment of cells with 4D5 reduced in vivo steady-state levels of 32P-labeled p185HERi up to 80% in a dose-dependent manner (lanes 2 through 4). There was 49% ± 8% reduction in phosphorylation by 150 nM MAb 405 in eight different experiments. When the F(ab) fragment of 4D5 was used instead of intact antibody, comparable or greater reduction of 32P-labeled p185HER2 was observed (lane 5). As a control, SK-BR-3 cells were incubated with another MAb, 225 lgGI, specifically directed against the EGF-R, and there was no effect on the amount of 3 2P-labeled pl85He:Ri (unpublished data). The reduction in steady-state levels of 32P-labeled p185H£RZ was not due to interference by 405 with MAb 906 during the immunoprecipitation reaction, as immunoprecipitation performed with another polyclonal an-

Exhibit 1088 of 8 2 of 8 Celltrion, Inc., Exhibit 1088

VOL. 11, 1991

Abs P185 EGF-R

2 31 l' 21 31

I ____ ... --

FIG. 2. Specificity of the reduction of 32P-labeled pl85H£Rl by MAb in SK-BR-3 cells in the presence or absence of MAb 405. Subconfluent cells were labeled with 32Pi for 15 h. The cells were lysed in 600 µJ of extraction buffer and divided into two equal pans of 250 µ.I each. lmmunoprecipitation was performed with anti-pl85 MAb (lanes l to 3) or with anti-EGF-R MAb 528 (lanes l ' to 3'). An autoradiogram of a dried gel is shown here. Lane l and l '. Control: lanes 2 and 2'. 30 nM MAb 405: lanes 3 and 3'. 150 nM MAb 405. Counts per minute: lane 1. 5.985; lane 2, 3,798; lane 3, 3,120; lane l '. 853; lane 2', 779; lane 3'. 932. Abs, Antibodies.

tibody (18) recognizing the carboxy-terminal 17 amino acids of pl85HER2 gave similar results (unpublished data).

Next , we examined the possibility of general inhibitory effects of MAb 405 on the steady-state levels of other 32P-labeled receptor proteins by analyzing the amount of 32P-labeled pl85HER.Z and 32P-labeled EGF-R in the same experiment (Fig. 2). These results indicated that there was no reduction of 32P-labeled EGF-R during 15 h of treatment of SK-BR-J cells with 150 nM MAb 405. which had reduced the amount of 32P-labeled pl85HER by 48%.

Analysis of reduction of pl8SH£Rz phosphorylation. The reduction of steady-state levels of 32P-labeled pl85HERi by MAb 405, shown in Fig. 1 and 2, could result from downregulation of pl85HER2 and/or interference in the activation of pl85HER2 phosphorylation by a direct or indirect m1:chanism(s). In initial studies to explore these possibilities. parallel cultures of cells were metabolically labeled with [35S)cysteine or 32P;. During 11 h of concurrent incubation with MAb 405 , there was a 45% reduction in 32P-labeled pl85HERZ (Fig. JA) and only a 14% reduction in JSS-labeled pl85HERZ (Fig. JB). This suggests that the reduced 32P label in pl85HERZ in the presence of MAb 405 can only partially be attributed to reduced pl8511ERZ content. Next, we performed a similar experiment comparing the capacities of the monovalent F(ab) fragment of MAb 405 and an intact MAb 405 to affect the reduction of 3~S-labeled pl85HERi . There was no change in 35S-labeled pl85HERi in the presence of F(ab), but there was a 26% reduction caused by MAb 405 (Fig. JC, lanes J and 2, respectively). The results obtained in the immunoprecipitation experiments documented in Fig. JA through C were confirmed by immunoblotting (0 ). lmmunoblotting of the 32P-labeled SK-BR-J cell extracts used in Fig. 2 demonstrated only a marginal reduction in the content of pl85HERz protein when cells were cultured in the presence of MAb 405 but a substantial reduction in the amount of 32P-labeled pl85HERZ (Fig. 2). The expression ofEGF-R was not affected. Immunoblotting of similar unlabeled SK-BR-J extracts also demonstrated very little reduction in the content of pl85HERi by MAb 405 (Fig. J D, experiment 2). These findings indicate that increased receptor catabolism induced by a MAb cannot fully account for the observed reduction in 32P labeling and show £with F(ab)) that reduced labeling is dissociated from reduced content of pl85HER~ .

Next we addressed the possibility that the reduction in 32P-labeled pl85HER.Z associated with exposure to MAb 405 could be related to a change in the level of expression of pl85HERi on the plasma membrane or to the extent of


MODULATION OF PHOSPHORYLATION OF pl85mlU

A. 8.

405 +

0. EXP 1

Abs P185 EGF-R

-405 - + +

+

C.

EXP. 2

Pl85

+

2

981

3

FIG. 3. Analysis of the reduction ofpl85H£Ri phosphorylation in SK-BR-3 cells treated with MAb 405. Cells were labeled with 3~Pi (A) or [3jS]cysteine (Bl in the presence or absence of MAb 40 5 (150 nM) for 11 h. Samples were prepared and separated as described in Materials and Methods. The autoradiogram shown here was obtained by 6 h of exp0sure. (Cl Cells were labeled with [35S]cv~teine for 11 h in the presence of MAb 405 (150 nM. lane 2) or F(ab) <400 nM . lane 3) or with culture medium (lane 1). Samples were prepared and immunoprecipitation was carried out as described in Materials and Methods. An autoradiogram of a dried gel is shown here. Quantitation of the p185H£Rl bands was obtained by densitometric scanning (A through C) or by determining radioactivity associated with bands (A and 8 ). Quantitation by determining the radioactivity associated with pl85H£R1 bands in panels A and B gave results similar to tho~e with densitometric scanning, and there was a 27% :!: 3% additional red11c1infl in 32P-labeled pl85H£Rl compared with "S-labeled pl85H£Hl . ID) lmmunoblotting of pl85H£Rl and EGF-R proteins. In experiment 1 (Exp. I). 32P-labeled SK-BR-3 cell extracts (50 µ.g of pMteinl used in Fig. 2. lanes 1 and 2. were resolved on a 7"K SDS-p0lyacrylamide gel and then immunoblotted with anti-Pl85 M:\b 9G6 or aati-EGF-R p0lyclonal antibody RK-11. Experiment 2 ~hows the immunoblotting of unlabeled SK-BR-3 cell extracts µrepared following culture for 15 h with or without 30 nM MAb 405. Since some of the extracts used here were radiolabeled. immunoblotted membranes were visualized by using a protein A-gold ..:11ru1ni:ement kit i30). Abs. AntibvJu:s.

down-regulation of receptor protein. First, we determined what fraction of the 35S-labeled pl8511ERz is present on the cell surface at J7°C (Fig. 4A). In these experiments. pl8511£Rz expressed on the plasma membrane was identified by its capacity to bind MAb 405 prior to cell lysis. The results indicate that 19% ± 4% (average from three different experiments) of total 35S-labeled pl 8511ER2 is expressed on the cell surface under these experimental conditions; thus pl8511£Rz is available for down-regulation by MAb 405. Down-regulation of EGF-R has been shown to be dependent on temperature (41). To confirm that down-regulation of surface pi85HERZ also is reduced at 4°C, experiments were performed to analyze the effect of temperature on the abundance of 35S-labeled pl85HERi on the cell surface. Results indicated that at 4°C the amount of total 35S-labeled pl8511£Ri expressed on the surface increased to J5% ± J% (data not shown) compared with 19% ± 4% of total 35Slabeled pl8511ER~ at J7°C.

In order to defi ne the contribution of down-regulation to MAb-induced reduction in pl8511£Rz phosphorylation, we


982 KUMAR ET AL.

A. EXP. 1 EXP. 2

B.

405

I 1 2 1 I 3 4 1

37°C 4°C 2 3

114 5

- + - + FIG. 4. (A) Quantitation of surface expression of ' 5S-labeled

pl85HERZ. Cells were labeled with C' 5S]cysteine for 11 h. At the end of incubation, some cultures were lysed in 500 µ.I of lysis buffer for the determination of total 35S-labeled p185HERZ by immunoprecipitation with 10 µ.g of 405 (Materials and Methods). For measuring the surface expression of 35S-labeled p185HERZ. cultures were washed with phosphate-buffered saline and further incubated with F-12/ OMEM-20 mM HEPES (pH 7 .5) containing 20 µ.g of high-affinity MAb 405 per ml for 1 hat 4°C. The cultures were washed. lysed in 500 µ.I of extraction buffer, and processed for immunoprecipitation by adding RAM-protein A-Sepharose beads but no more MAb 405 during the immunoprecipitation procedure. The results of two representative experiments are shown here. Lanes 1 and 3, Total 35S-labeled pl85H£RZ; lanes 2 and 4, 35S-labeled pl85H£Ri on the cell surface. (B) Effect of MAb 405 on surface expression of pl85HERZ in a temperature shift experiment. SK-BR-3 cells were first equilibrated with 32P; for 4 h at 37°C (lane 1) and then further incubated with or without MAb 405 for an additional 11 h (in the continuous presence of 32P;) either at 37°C (lanes 2 and 3) or at 4°C {lanes 4 and 5). The autoradiogram shown here was obtained by exposing lanes 1 to 3 for 24 h and lanes 4 and 5 for% h. Quantitation of the amount of n p associated with pl85HERZ bands was obtained by densitometric scanning of the autoradiogram and by determining the radioactivity associated with p185HERZ bands (A and B).

analyzed the effect of incubation with MAb 405 at 4°C. In these studies, cells were first equilibrated with 32P1 for 4 h at 37°C (Fig. 4B, lane 1) and then maintained at 37°C (lanes 2 and 3) or shifted to 4°C (lanes 4 and 5) for an additional 11 h , with or without MAb 405. A comparison of the labeled material in lanes 2 and 4 in Fig. 4B (ftuorographs exposed for 24 and % h, respectively) showed a significant 85% reduction in 32P labeling of pl85HERi during 11 h at 4°C compared with labeling at 37°C. However, incubation of cells at 4°C did not prevent a further substantial MAb-mediated reduction in steady-state levels of pl85HERi phosphorylation: there was a 34% decrease at 4°C (compare lanes 4 and 5) and a 51% decrease at 37°C (compare lanes 2 and 3). Taken together, these observations indicate that MAb-induced reduction in pl85HER phosphorylation cannot be completely accounted for by down-regulation.

Experiments were performed to determine whether the F(ab) fragment might have the capacity to act as an agonist by activating tyrosine phosphorylation. The results in Fig. 5 indicate that the addition of F(ab) for 15 min sli_ghtly stimulated in vivo tyrosine phosphorylation of pl85 E R i in cultures labeled with 32P (Fig. 5, lane 2'). However, there was no activation in cultures exposed to F(ab) for a longer treatment of 60 min (Fig. 5, lane 3 '). The observation that the F(ab) fragment of 405 does not down-regulate the 35S-


Antibody P185

1 2 3 - .. -

MOL. CELL. BIOL.

P-Tyr

11

21

311

FIG. 5. Partial agonist nature of F(ab). Subconftuent SK-BR-3 cells were labeled with 32P; for 15 h. Some cultures were treated with 400 nM F(ab) for the indicated times. The cells were lysed in 800 µ.I of extraction buffer. The lysates were divided into two equal parts of 350 µ.I each and then immunoprecipitated with MAb 906 (lanes 1 to 3) or with antiphosphotyrosine MAb PY-69 (lanes 1' to 3'). An autoradiogram resulting from a 1-h exposure of dried gel is shown here. Lane 1, Control; lane 2. F(ab) incubation for 15 min; lane 3, F(ab) incubation for 60 min.

labeled pl85HERi but can act for a short time as a partial agonist is interesting; however, we have not attempted to further characterize these properties in the present study.

Activation of phosphorylation of p18SHER2 in presence or absence of newborn calf serum. Next, we investigated the possible source of the factor(s) that might stimulate pl85HERi phosphorylation. As shown in Fig. 6A, culturing the cells in serum-free medium resulted in a steady-state level of phosphorylation of p18511ER2 reduced 56% (lane 1) compared with that observed in the continuous presence of newborn calf serum (lane 3). The addition of MAb 405 in serum-free culture conditions further reduced pl85HERi

A. 2 3 4 B.

- ..... Serum + + + +

405 + +

C. • 2 3

FIG. 6. (A) Detection of newborn calf serum-mediated phosphorylation of pl85HERi. Subconfluent SK-BR-3 cells were labeled with 32P; in the culture medium without (lanes 1 and 2) or with (lanes 3 and 4) 5% newborn calf serum for 15 h. Cultures analyzed in lanes 2 and 4 also were continuously exposed to 150 nM MAb 405. Samples were prepared and immunoprecipitated for assaying the amount of pl85HERi as described in the Materials and Methods. Quantitation of the pl85 bands was obtained by densitometric scanning of the autoradiogram. (B) Control experiment showing effect of serum on the 32P; labeling of EGF-Rs for 15 h in SK-BR-3 cell cultures. Cell extracts were immunoprecipitated with antiEGF-R MAb 528, which recognizes one distinct band with an approximate molecular mass of 170 kDa (arrow). (C) Two-dimensional thin-layer electrophoresis pattern of 32P-phosphoamino acids in a hydrolysate of the p185HERZ immunoprecipitated in panel A. S, Phosphoserine; T . phosphothreonine; Y. phosphotyrosine. Number at lower left of each autoradiogram indicates the following culture conditions: l , with no serum; 2, with serum; 3, with serum and MAb 405. Tyrosine phosphorylation in control cells was visualized faintly on the autoradiogram but reproduces poorly.


VOL. 11, 1991

phosphorylation (Fig. 6A, lane 2) to 20% of the steady-state levels achieved in the absence of newborn calf serum (Fig. 6A, Jane 1). Experiments were done to examine the capacity of newborn calf serum to stimulate tyrosine phosphorylation ofp185H£Rl in short-term ~eatment. There was no increased activation of phosphorylation when serum-free cultures were supplemented with newborn calf serum for 30 min at 37 or 4°C (data not shown).

To determine the specificity of the ca~city of newborn calf serum to stimulate activation of p185 .ERZ phosphorylation in SK-8R-3 cells, we investigated the potential for serum activation of another closely related molecule, the EGF-R. There was no potentiating effect of newborn calf serum on phosphorylation of the EGF-R in SK-8R-3 cells (Fig. 68). Ravi~ shown an increase in the steady-state levels of

p185H£ phosphorylation induced by newborn calf serum and its reduction by MAb 405, we determined the phosphoamino acid content of p185H£Ri under these conditions by two-dimensional thin-layer electrophoresis (Fig. 6C). p185HER2 from Cells Cultured in the absence Of newborn Calf serum contained predominantly pbosphoserine and phosphotlireonine with little phosphotyrosine (Fig. 6C, blot 1). The presence of some phosphorylatio~ on tyrosine can be demonstrated by longer exposure of the autoradiogram but is not visualized well in the figure shown. Quantitation of the relative amount of label in each amino acid was obtained by scraping the ninhydrin-ideiltified spots from the thin-layer plate for liquid scintillation counting. Activation by 5% newborn calf serum increased the total phosphoamino acid content 2.2-fold, while for phosphotyrosine, the increase was 3.9-fold (Fig. 6C, blot 2). Inhibition of newborn calf serum-mediated stimulation ofpl85H£Ri phosphorylation by MAb 405 resulted in a parallel reduction in the content of all three phosphoamioo acids (Fig. 6C, blot 3).

Tyrosine phosphorylation or pl858ER2 . To further quanti

tate tlie relative increase in the steady-state phosphotyrosine content of pl85H£RZ induced by newborn calf serum, cells were metabolically labeled with [35S]cysteine and assayed for the steady:state phosphotyrosine content of p185HERl by using antiphosphotyrosine MAb PY-69. MAb PY-69 was specific for pbospbotyrosine in the immunoprecipitation reaction; i.e. , we were able to show competion for binding with cold phosphotyrosine and not with phosphoserine in experiments with 35S-labeled EGF-R (data not shown). As illustrated in Fig. 7 A, lanes 1' to 4', the amount of phosphorylation of p185HERi on tyrosine increased with the concentration of newborn calf serum in the culture medium. The level of activation of tyrosine phosphorylation in serum-free medium was 18% of that observed in 10% newborn calf serum , and in medium containing 2.5% serum, tyrosine phosphorylation was 35% of that observed with a serum concentration of 10%. As a control, equal amounts of labeled cell extracts were immunoprecipitated with anti-pl85HER2 MAb 9G6 (Fig. 7 A, lanes I through 4). There was no significant effect of serum on the levels of 35S-labeled pl85H£RZ in the cells. To quantitate these results, the ratios of phosphotyrosine-associated counts to total counts associated with p1858 £R2 are presented in Fig. 78, which shows a dose-dependent increase of up to 5.4-fold with 10% newborn calf serum in the culture medium.

Partial depletion of activating factor from newborn calf serum. Since our results indicated the presence of some activating factor(s) for p185H£R2 phosphorylation in newborn calf serum, we sought confirmation of this observation by determining whether newborn calf serum could be de-


MODULATION OF PHOSPHORYLATION OF pl85HER1 983

A. Antibody Pl85 P-Tyr

2 3 4 ,. 2' 3' 4'

- -% Serum O 2.5 5 10 0 2 .5 5 10

B. 500 • C . 1 2 3 4 1()-

/ ro E a:c ...... 0 300 -_u ~a d.~ /

100

0 5 10 Percent Serum, VIV

FIG. 7. (A) Detection of tyrosine-specific phosphorylat.ion of pl85HERz by newborn calf serum. Subconfluent SK-BR-3 cells were metabolically labeled with [35S]cysteine iii the presence of dilferent concentrations of serum for 15 h. Cells were lysed in 650 µI of extraction bu1fer. The lysates were divided into two equal parts of 300 µ.I each and then immunoprecipitated with anti-pl85HER1 MAb 9G6 (lanes 1 to 4) or antiphosphotyrosine MAb PY-69 (lanes l ' to 4'). Other details of the assay were as described in the legend to Fig. 5. To detect the phosphotyrosine (P-Tyr) signal in cells cultured in the absence of serum (lane l '), it was necessary to expose the autoradiogram for 20 h, which resulted in overexposure of lanes 1 to 4. (B) To quantitate the data in panel A, the radioactivity associated with p185HER1 was determined by counting the excised bands in a liquid scintillation counter. The ratios of counts in pl85H£Rz phosphotyrosine over total counts in pl85H~ were plotted as a percentage of control with 0% newborn calf serum against the concentration of newborn calf serum used in the culture medium. (C) Depletion of the activator(s) of p18SHER1 phosphorylation in serum. SK-BR-3 cells were labeled with 32P1 in the absence or presence of serum for 15 h. Lane l, Control without serum; lane 2, medium with 5% newborn calf serum; lane 3, medium with 5% newborn calf serum depleted of factor(s) by three repetitive adsorptions of 4 h each on SK-BR-3 cells at 4"C; lane 4, control medium adsorbed on A431 cells. Cell extracts were prepared and p185HER1 was assayed as described in Materials and Methods.

pleted of such a factor(s). In these experiments, phosphatefree medium containing 5% newborn calf serum was treated by repetitive absorption with SK-8R-3 cells (three times, for 4 h each time, at 4°C). Culture medium treated in an identical manner by adsorption with A431 cells, which do not express high levels of pl85"£R2, was used as control. Figure 7C shows that there was a 53% reduction in p1858 £R2 phosphorylation in SK-8R-3 cells cultured in the presence of medium preadsorbed with SK-8R-3 cells (lane 3) compared with untreated medium (lane 2), and there was only a 16% reduction in p185HERi phosphorylation when SK-8R-3 cell cultures were supplemented with medium preadsorbed with A431 cells (Fig. 7C, lane 4).

Activation factor(s) In newborn calf serum was not TGF-o or EGF. pl85H£Ri has been shown to be phosphorylated when EGF-Rs are activated by ex~sure to EGF or TGF-a, which are not ligands for pl8588 (24, 40). To determine whether the newborn calf serum-mediated 2.2-fold enhancement of pl85HER2 phosphorylation resulted from the activity ofTGF-a or EGF, we used anti-EGF-R MAb 528, which bas


984 KUMAR ET AL.

"' "E ::>

~5 Zi ~

~ 4 Q.

!2 !: 3 -0 ... ~2 ... 0 u c:

d.. ~

Serum + + + + + 405 + + 528 + + TGF-a + +

FIG. 8. Activation of pl85HERl by newborn calf serum was not due to TGF-a or EGF. Subconfluent SK-BR-3 cells were labeled with 32P1 for 15 h in the presence or absence of 5% newborn calf serum, 150 oM MAb 405, and 300 nM MAb 528. As a positive control, some cultures received 40 nM TGF-a either alone (column 6) or with 300 nM MAb 528 (column 7) during the last 20 min of labeling. Cells were lysed and then immunoprecipitated with MAb 9G6 to assay pl85HERJ. The insert displays the autoradiogram resulting from a 3-h exposure of the dried gel. To quantitate the amounts of pl85H~, protein bands were excised for determining the radioactivity, which is represented here on an arbitrary scale. These experiments were performed three times.

been shown to block the binding of EGF and TGF-a to their receptors (23, 25). The experiment whose results are shown in Fig. 8 demonstrates that coincubation of SK-BR-3 with a saturating amount of anti-EGF-R MAb 528 did not significantly alter the ca)Jacity of newborn calf serum to increase the levels of pl85 ERJ phosphorylation. As a positive control, some cultures received TGF-a with or without MAb 528 during the last 20 min of labeling before cells were harvested , and MAb 528 completely blocked the predicted and observed increases in phosphorylation of pl85HERi induced by TGF-a (Fig. 8, column 6 and 7).

Observations with other mammary carcinoma cells. In order to determine whether activation of p185HER2 phosphorylation by newborn calf serum and its inhibition by MAb 405 are phenomena restricted to SK-BR-3 cells or whether they can be demonstrated with other breast tumor cells overexpressing pl85HER2

, we extended our investigation to two other lines: MOA-MB-453 and BT-474. The results in Fig. 9 indicate a significant capacity of newborn calf serum to activate p185HER2 phosphorylation and show that the addition of MAb 405 could reduce the steady-state levels of p185HER2 phosphorylation both in the presence of newborn calf serum and in serum-free cultures.

DISCUSSION

Activation of kinase activity associated with receptor tyrosine kinase by ligand binding is thought to be the common mechanism by which transmembrane receptors for


MOL. CELL. BIOL.

A. 2 3 4..·;

-e. -

Serum + + 405 - + - +

FIG. 9. Effects of MAb 405 and newborn calf serum on steadystate levels of pl85HERZ phosphorylation in MDA-MB-453 mammary carcinoma cell line (A) and BT-474 mammary carcinoma cell line (B). Cells (4 x 10') from each cell line were cultured with 32P1 for 15 h in the presence or absence of 5% serum and/or MAb 405 (150 nM). Cell lysates were prepared and processed for the determination of radioactivity in pl85HERZ as described in Materials and Methods. The amounts of radioactivity in pl85HERJ (in counts per minute) in panel A were 420 (lane 1), 263 (lane 2), 1,237 (lane 3), and 804 (lane 4). In panel B. the counts per minute were 322 (lane 1), 268 (lane 2), 508 (lane 3), and 225 (lane 4).

growth factors transduce signals that activate cell proliferation (6, 22, 27, 48). Support for this model is provided by our experiments demonstrating growth inhib ition of cells overexpressing EGF-Rs by MAbs 225 and 528, which recognize these transmembrane receptors on intact cells, prevent binding of the growth factor-ligand, and block ligand-induced phosphorylation of EGF-Rs (16, 23, 29). A similar antiproliferative effect has been observed when SK-BR-3 cells, which overexpress p185RERi, are exposed to MAb 405 (17, 18). The present work was undertaken to explore the modulation of phosphorylation of pl85HeRz by an antipl85HERZ MAb, 405, and by newborn calf serum.

The results presented here indicate that treatment of SK-BR-3 cells with MAb 405 significantly reduced the steady-state levels of phospho~lation of the c-erbB-21 HER2/ c-neu gene product p185H£R on serine, threonine, and tyrosine residues. Anti-p185H£Ri MAb has been shown to cause enhanced down-regulation of pl85HERi from the surface of human and murine cells by reducing the half-life of pl85HERZ (14, 18). Our conclusion that MAb 405-mediated inhibition of the steady-state levels of pl85HER2 phosphorylation was only in part attributable to the down-regulation of p185H£R2 is supported by the following lines of evidence: (i) the reduction of phosphorylation was far in excess of the reduced content of 32S-labeled protein measured by immunoprecipitation and also by immunoblotting; {ii) inhibition of phosphorylation was observed at 4°C under conditions in which MAb-induced down-regulation of p185HERz wouJd be limited; (iii) the inhibitory effect of MAb 405 on pl85HER2 phosphorylation could be mimicked by F(ab) fragment, which does not down-regulate pl85HER2. The last observation is similar to findings with the F(ab) fragment of another MAb-specific murine p185HERJ (14). Taken together, these data provide strong indirect evidence that MAb 405 may have interfered with the activation of phosphorylation of pl85HER2 by a growth factor(s).

The observation that newborn calf serum contains an activator(s) of phosphorylation of pl85HER2 opens a new area of investigation for identifying an additional factor(s)


VOL. 11, 1991

which might stimulate pl85HERZ phosphorylation. This capacity of newborn calf serum to activate phosphorylation was specific for p185HER2; it did not affect the phosphorylation of EGF-Rs in SK-BR-3 cells. The fact that newborn calf serum was not able to stimulate p185HER2 phosphorylation in short-term treatment raises the possibi.lity of some indirect mechanism of p18511ER2 activation by newborn calf serum, or it could be a matter of quantity of the responsible factor(s). The evidence that newborn calf serum has the capacity to stimulate tyrosine-specific phosphorylation preferentially in p185HER2 (in addition to serine and threonine phosphorylation) is of special interest, as it strongly suggests that this serum probably contains a growth factor(s) capable of activating transduction of a known growth-regulating signal in cells.

Data from the literature and from this work suggest that p185HER2 may also be phosphorylated by exposing cells to TGF-a or EGF, probably as a result of activation of EGF-R tyrosine kinase (24, 40). However, interaction with EGF-Rs was not responsible for the observed newborn calf serummediated activation of p18511ER2 phosphorylation, since addition of MAb 528 did not alter the activation potential of newborn calf serum, although it blocked TGF-a-induced increase in p185HER2 phosphorylation. Moreover, our finding that serum was able to activate the phosphorylation of p185HER2 in MDA-MB-453 provides additional evidence against possible involvement of TGF-a or EGF in the observed serum-mediated activation of pl85HERz phosphorylation, since these cells do not have receptors that bind labeled EGF (49) and since p185HER2 phosphorylation is not activated in these cells by EGF (24, 49).

The reduced phosphorylation of p185HERz observed when MAb 405 was added to serum-free cultures of SK-BR-3, MDA-MB-453, and BT-474 cells also suggests that under these conditions it may have blocked activation of p18511ERZ phosphorylation by a cellular-derived factor(s) produced by these human mammary carcinoma cells. This view is further supported by our preliminary observations (data not shown) that under the appropriate conditions, SK-BR-3-conditioned medium can inhibit the binding of the growth-inhibitory MAb 405 but not the binding of another MAb, 6E9, which recognizes a distinct epitope on the extracellular domain of the receptor and has no effect on tumor cell growth (18). Additional evidence for a cellular-derived factor is provided by Lupu et al. (28), who have recently identified a polypeptide which is constitutively secreted in small amounts by SK-BR-3 cells and competes with MAb 405 for binding sites on the surface of SK-BR-3 cells.

The observation that MAb 405, which reduces the proliferation of cells bearing pl85HERZ, is able to inhibit the capacity of both newborn calf serum and a constitutive activity to increase levels of pl85HERz phosphorylation suggests the existence of a growth factor or factors for p185HERZ which may use receptor phosphorylation as a signal transduction pathway to regulate cell growth.

ACKNOWLEDGMENTS

We are grateful to M. J. Van de Vijver for providing anti-pl85HER2 MAb 9G6 and to J . Schlessinger for providing anti-EGF-R polyclonal antibody RK-ll. The excellent secretarial assistance of Frantzie Paul is gratefully acknowledged.

This study was supported by Public Health Service grants CA42060 and CA37641 from the National Institutes of Health.

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regulation of phosphorylation of the c-erbb-2/her2 gene ... · mouse immunoglobulins g (ram) was...

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