THE JOURNAL OF BIOLOGKAL CHEMISTRY Vol. 265, No. 6. Issue of February 25, pp. 3340-3346, 1990 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

Differential Processing and Turnover of the Oncogenically Activated neu/erb B2 Gene Product and Its Normal Cellular Counterpart*

(Received for publication, May 17, 1989)

Shuan Shian HuangSB, Han A. Koh$, Yasuo Konishll, Lea Doerr Bullock11 , and Jung San Huangt: From the SE. A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri. the (IBiotechnoloev Research Institute. National Research Council, Canada, Montreal, Quebec, Canada H4P2R2, and the 11 Mokwzto Company,~Fhesterfield, Missouri 63198

In nontransformed DIIFR/G-8 cells (NIH 3T3 cells transfected with normal rat neu gene), the normal neu gene product was initially synthesized as a 170-kDa protein bearing endoglycosidase H-sensitive oligosac- charide chains and was then processed to a 175-kDa mature form with endoglycosidase H-resistant, endo- glycosidase F-sensitive oligosaccharide chains. Most of this 175-kDa mature form appeared on the cell surface 2 h following synthesis and showed a half-life of -3 h. In the presence of a growth factor(s) partially purified from bovine kidney, the half-life of this 175-kDa nor- mal neu gene product was shortened to less than 30 min.

In B104-l-l cells (NIH 3T3 cells transfected with neu gene activated oncogenically by a point mutation that changes a valine residue to a glutamic acid residue in the putative transmembrane region), the oncogeni- tally activated neu gene product was also synthesized as a 170~kDa precursor with endoglycosidase H-sen- sitive oligosaccharide chains. However, this 170-kDa precursor diminished very fast and was only partially processed to a 185-kDa mature form which exhibited a half-life of <30 min. The 185-kDa activated neu gene product possessed an unidentified post-translational modification in addition to N-linked oligosaccharide chains. Both the precursor and mature forms of the mutationally activated neu gene product showed in- creased tyrosine-specific phosphorylation as compared with those of their normal counterparts in DHFR/G-8 cells.

The mutationally activated neu gene product in B104-l-l cells shared several features which have been reported previously for the ligand-activated platelet-derived growth factor receptor in v-sis- or c- sis-transformed cells. These properties include: 1) ac- celerated turnover of the precursor and mature forms compared with the rates of turnover of its normal counterparts, 2) insensitivity of this rapid turnover to lysosomotropic amines, and 3) increased in viva tyro- sine-specific phosphorylation of both the precursor and mature forms. These findings suggest that the muta-

* This work was supported by the National Institutes of Health Grant CA-38808 and 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 “aduer- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: E. A. Doisy Dept. of Biochemistry and Molecular Biology, St. Louis University School of Medicine, 1402 South Grand Blvd., St. Louis, MO 63104. Tel.: 314- 577-8148.

tionally activated neu gene product may transform the cells by mimicking ligand-induced activation.

The neulerb B2 proto-oncogene encodes a receptor-like protein possessing an extracellular domain, a single mem- brane-spanning domain, and a tyrosine kinase domain (1, 2). The neulerb B2 proto-oncogene is closely related to, but distinct from, the EGF’ receptor gene (c-erb Bl) (l-6) and has been suggested to be a receptor for an unidentified growth factor(s) (1, 2, 4-6).

The neulerb B2 proto-oncogene was found to be activated in chemically induced neuroblastomas and glioblastomas of rats (7,s). The oncogeneic activation of the neu/erb B2 proto- oncogene in these carcinogen-induced tumors appears to be the result of a point mutation which changes a valine residue to a glutamic acid residue in the transmembrane region of the predicted amino acid sequence of the protein encoded by the neu/erb B2 gene (3). The mutationally activated neu/erb B2 gene shows a potent transforming activity which is >lOO times more active than the normal neu/erb B2 gene in transforming assays (9). The mutationally activated neu gene also induces mammary adenocarcinoma in transgenic mice (10).

The mechanism by which a point mutation in the trans- membrane domain increases the transforming activity of the neu gene is not known. However, the 185-kDa protein encoded by the mutationally activated neu gene was demonstrated to have increased protein tyrosine kinase activity in plasma membranes (9, ll), suggesting that activation of the kinase activity by this mutation may be the mechanism.

We have been investigating the mechanism of autocrine transformation by v-sis and c-sis. We found recently that the activation of precursor and mature forms of the PDGF recep- tor by the ligand, the sis gene product, resulted in increased tyrosine-specific phosphorylation and rapid turnover of these PDGF receptors (12). This persistent intracellular activation of the growth factor receptor may be a general mechanism for autocrine transformation by growth factors (12). Since the mutationally activated neu gene encodes a 185-kDa protein which constitutively expresses a protein tyrosin kinase activ- ity (9, ll), it was of interest to see the effect of activation on the processing and turnover of this molecule following syn- thesis. In this paper, we report that the precursor and mature forms of the mutationally activated neu gene product under-

’ The abbreviations used are: EGF, epidermal growth factor; DMEM, Dulbecco’s modified Eagle’s medium; SDS, sodium dodecyl sulfate; Endo H and F, endoglycosidases H and F; KDGF, kidney- derived growth factor; PDGF, platelet-derived growth factor; FGF, fibroblast growth factor; PBS, phosphate-buffered saline.

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went fast turnover with a t% of ~30 min when compared with those of the normal neu gene product (t’/z = -3 h). Both precursor and mature forms of the mutationally activated neu gene product showed much greater tyrosine-specific phos- phorylation compared with the normal neu gene products in the absence of ligand activation. We also report that the cell surface normal neu gene product could be down-regulated by incubation with a growth factor(s) partially purified from bovine kidney (kidney-derived growth factor). The mutation- ally activated neu gene product was resistant to this down- regulation.

EXPERIMENTAL PROCEDURES

Ma~eerials-[[35S]Methionine (>lOOO Ci/mmol) and ortho[3ZP]phos- phate (285 Cifmg) were obtained from ICN. [merhyl-3H]Thymidine (79.4 Ci/mmol) was purchased from Du Pont-New England Nuclear. Protein A-Sepharose was obtained from Pharmacia LKB Biotech- nology Inc. Suramin was purchased from Mobay Laboratory. Calf intestine alkaline phosphatase, endoglycosidase H (recombinant), endoglycosidase F (Flauobacterium meningosepticum), sialidase (Vi- brio cholerae), and 0-glycanse (Diplococcus pneumoniae) were ob- tained from Boehrineer Mannheim Biochemicals. Human PDGF was prepared from human platelet-rich plasma (12). Murine EGF and bovine basic fibroblast growth factor (bFGF) were obtained from Collaborative Research. Bovine acidic fibroblast growth factor (aFGF) and sulfated Sephadex G50 (Sulfadex) were prepared as described previously (13). Phosphotyramine was kindly provided by Dr. Kam Fok, Monsanto Company, St. Louis, MO. Bovine trypsin and soybean trypsin inhibitor were obtained from Sigma.

Cells-DHFR/G-8 cells and B104-1-l cells were NIH 3T3 cells transfected with normal rat neu proto-oncogene and oncogenic rat neu genes, which has a point mutation in the transmembrane domain, respectively (11,14). DHFR/G-8 cells were not transformed, whereas B104-l-1 cells were transformed and highly tumorigenic in athymic nude mice. B104-l-l and DHFR/G-8 cells were nrovided bv Dr. Mine- Chi Hung, Department of Turn& Biology, M. 6. Anderson Hospital, University of Texas, Houston.

Antisera-The neu gene product-derived peptide (lie7 PAGATLERPKTLSPGKNG”a4) was synthesized by the Merritield method using 9-fluorenylmethoxycarbonyl amino acids (15). The conjugate of this peptide with bovine thyroglobulin was prepared by the water-soluble carbodiimide procedure (16). The production of rabbit anti-neu gene product-derived peptide conjugate antiserum was carried out according to our published procedure (21). Anti- phosphotyrosine antibody was raised in rabbits using O-phosphotyr- amine-conjugated thyroglobulin and was purified from the sera by affinity chromatography on phosphotyramine-coupled Sepharose as described previously (16).

Metabolic Labeling-B104-l-l and DHFR/G-8 cells were grown to confluence in P-60 Petri dishes in Dulbecco’s modified Eagle’s me- dium (DMEM). The cells were nulse-labeled with 135Slmethionine (200 pCi/ml) in methionine-free medium for 30 min and chased with DMEM containing 10 mM methionine for various time intervals. After the chase period, the cells were extracted with 1 ml of RIPA buffer (1% sodium deoxycholate, 1% Nonidet P-40, 0.1% SDS, 25 mM Tris-HCl, pH 7.4, 0.15 M NaCl) and centrifuged. The protein content and radioactivity of each clarified extract was determined by the method of Lowry et al. (17) and scintillation counting procedure, respectively.

Immunoprecipitation-The immunoprecipitation of ?+labeled RIPA buffer extracts was carried out as described previously (12) using rabbit anti-neu gene product antiserum. The immunoprecipi- tates were analyzed with 7.5% SDS-polyacrylamide gel electropho- resis followed by fluorography.

Treatment of the Cell Surface neu Gene Product with Trypsin- Cells were pulse-labeled with [YSjmethionine for 30 min and chased for 2 h in DMEM containing 10 mM methionine. Following the chase, the cells were treated with trypsin (0.1 mg/ml) at 37 “C for 10 min. Soybean trypsin inhibitor (2 mg/ml) was added to stop the treatment. In the control cells, soybean trypsin inhibitor was added prior to addition of trypsin.

Preparation of Bovine Kidney-derived Growth Factor (KDGFj- Fresh bovine kidney was homogenized in phosphate-buffered saline (PBS) at a ratio of 2 ml/g tissue. After centrifugation, the supernate was subjected to ammonium sulfate fractionation. The pellet obtained

by 35-70% saturation with ammonium sulfate was dissolved in PBS, dialyzed against PBS, and passed through a column (2.0 X 50 cm) of DE-52 (Whatman) which had been pre-equilibrated with PBS. The flow-through fractions were then pooled and applied onto a column (50-ml volume) of sulfated Sephadex G50 (Sulfadex) gel. The mito- genie activity toward DHFR/G-8 cells was‘eluted with a linear gra- dient of NaCl from 0.15 to 3 M NaCl and designated kidney-derived growth factor. The mitogenic activity toward DHFR/G-8 cells was assayed in serum-free DMEM as described previously (12). KDGF activity appeared to be acid-labile and trypsin-sensitive. KDGF also stimulated the phosphorylation of the neu gene product in intact cells and plasma membranes of DHFR/G-8 cells and SK-BR-3 cells (hu- man breast cancer cells). The detail of characterization of bovine kidney-derived growth factor will be published elsewhere.

Endoglycosidase and Exoglycosidase Digestion-The immunopre- cipitates of ?S-labeled cell extracts were treated with endoglycosidase H or endoglycosidase F as described previously (12). The digests were concentrated by immunoprecipitation with anti-neu gene product antiserum and analyzed by 7.5% SDS-polyacrylamide gel electropho- resis and fluorography. For the activated neu gene product, the endoglycosidase F digests were further hydrolyzed with sialidase (neuraminidase) (50 milliunits/ml) and 0-glycanase (endo-a-N-ace- tylgalactosaminidase) (25 milliunits/ml). No detectable protease ac- tivity was found in the preparations of these glycosidases. However, 1 mM of phenylmethanesulfonyl fluoride was included in the reaction mixtures.

Phosphorylation of Normal and Mutationally Activated neu Gene Products in B104-l-l and DHFRIG-8 Cells-Cells were labeled with ortho[32P]phosphate according to our published procedures (12). The “P-labeled neu gene products were immunoprecipitated with anti- neu gene product antiserum + protein A-Sepharose. The immunopre- cipitates were analyzed by 7.5% SDS-polyacrylamide gel electropho- resis followed by alkaline treatment of the gel with 1 N KOH (18) and autoradiography.

RESULTS

Specificity of Anti-neu Gene Product Antiserum-The pre- dicted amino acid sequence of the neu gene is colinear and 50% homologous to that of the EGF receptor (2). To obtain a highly specific antiserum to the neu gene product, an anti- serum was raised in rabbits using a synthetic peptide which corresponds to sequence from the nonhomologous cytoplasmic region of the neu gene product. The specificity of this anti- serum was tested by immunoprecipitation of 35S-labeled mu- tationally activated and normal neu gene products in NIH 3T3 cells transfected by mutationally activated and normal neu genes (B104-l-l and DHFR/G-8 cells, respectively). Cells were labeled with [35S]methionine for 30 min. The 35S-labeled cell lysates were immunoprecipitated with the antiserum and analyzed by 7.5% SDS-polyacrylamide gel electrophoresis, followed by fluorography. After the 30 min pulse, the 35S- labeled neu gene products in Bl04-1-l and DHFR/GS cells appeared as the 170-kDa precursor (Fig. 1, lanes 1 and 5). The immunoprecipitation of this 170-kDa protein was com- pletely blocked in the presence of peptide antigen (Fig. 1, lanes 2 and 6). Very little, if any, YS-labeled product was observed in the immunoprecipitates of cell lysates of untrans- fected NIH 3T3 cells (Fig. 1, lanes 3 and 4). These results indicate the specificity of antiserum for the neu gene product.

Processing and Turnover of Mutationally Activated and Normal neu Gene Products-To examine the processing and turnover of neu gene product, cells were pulse-labeled with [35S]methionine for 30 min and chased for various time pe- riods in 10 mM unlabeled methionine. As shown in Fig. 2A, in DHFR/G-8 cells the normal neu gene product appeared to be a 170-kDa precursor after the 30 min pulse (or zero time chase). During the chase periods, this 170-kDa precursor was converted to a 175-kDa mature form with a t% of -30 min (Fig. ZB). Very little conversion of the precursor form to mature form was observed in the 30 min chase period. After a 2 h chase, most newly synthesized neu gene product mole-

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3342 Processing and Turnover of neujerb B2 Oncogene Products

DHFR/G 8 Cells

A chose Ill) D 05 10 152545

peptlde -+II--m

170kDa-. - -205 kDo

-1

- 116kDo - 97kDa

- 66 kDa

45 kDo

12345 6 FI(;. 1. Immunoprecipitation of normal and mutationally

activated neu gene products in DHFR/G-8 and B104-l-l cells by anti-neu gene product antiserum. Cells (DHFR/G-8, Bl04-l- 1, and NIH 3T3 cells) were labeled with [ “Slmethionine for 30 min in methionine-free DMEM. The “S-labeled cell extracts were im- munoprecipitated with anti-neu gene product antiserum in the pres- ence (+) and absence (-) of neu gene product-derived peptide (5 mg/ ml). The immunoprecipitates were then analyzed by 7.5% SDS- polyacrylamide gel electrophoresis and fluorography. Similar quantity of radioactivity of immunoprecipitates from DHFR/G-8 and B104-l- 1 cells was apphed onto SDS gel. The arrow: indicates the location of the precursors of normal and activated neu gene products.

cules appeared on the cell surface and were sensitive to trypsin treatment (data not shown). This cell surface 175-kDa neu gene product showed a half-life of -3 h. However, in the presence of a growth factor(s) partially purified from bovine kidney, the half-life of this 175kDa neu gene product was shortened to less than 30 min (Fig. 3). Incubation with epi- dermal growth factor did not influence the half-life of this 175kDa neu gene product (data not shown). Suramin (1 mM) appeared to block the down-regulation of the cell surface normal neu gene product induced by KDGF (Fig. 4, lane 5). At this concentration (1 mM), suramin was found to be a nonspecific inhibitor for PDGF, FGF, EGF, and transforming growth factor p (TGFB) activities (13, 19-21). Protamine (10 fig/ml) failed to have an effect on the KDGF-induced down- regulation of the normal neu gene product (Fig. 4, lane 6). Protamine was shown to be a specific inhibitor for FGF activities at the concentration of 10 pg/ml (13). These results are compatible with the suggestion that the neu gene product is a receptor for an unidentified growth factor(s) which can be isolated from bovine kidney.

In B104-1-l cells, the processing of the mutationally acti- vated neu gene product appeared to be different from that of its normal counterpart in DHFR/G-8 cells. A similar 170- kDa precursor was observed after a 30 min pulse (or zero time chase) (Fig. 2C). However, the intensity of 170-kDa precursor diminished very fast. About 25% of the 170-kDa precursor was converted to a 185.kDa mature form of the activated neu gene product (Fig. 20). This 185-kDa mature form had a short half-life of ~30 min. The growth factor(s) partially purified from bovine kidney appeared to have no significant effect on the turnover of this 185-kDa mutationally activated neu gene product (Fig. 3).

Characterization of Mutationally Activated and Normal neu Gene Products-The mature forms of mutationally activated and normal neu gene products differ in their molecular mass, whereas the precursors of mutationally activated and normal neu gene products show identical molecular mass. These results suggest a difference in post-translational modification

66 kDo

- 45kDa

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185 kDa - 205 kDa

170kDa : I) k, w na a”* -* (

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FIG. 2. Kinetics of processing and turnover of normal and mutationally activated neu gene products in DHFR/G-8 and B104-l-l cells. DHFR/G-8 cells (A and B) and B104-l-1 cells (C and D) were pulse-labeled for 30 min with [““Slmethionine and chased for various time periods in medium containing excess unlabeled methionine. Cell extracts were immunoprecipitated with anti-neu gene product antiserum and analyzed by 7.5% SDS-polyacrylamide gel electrophoresis and fluorography. The intensity of the bands corresponding to the 170-kDa precursors and the 175.kDa mature form in DHFR/G-8 cells (A) or the 185-kDa mature form in B104-l- 1 cells (C) was quantitated by densitometric scanning. The analysis is shown in B and L), respectively, with the relative intensity of the bands plotted against length of chase. The intensity of the 170.kDa precursor after a 30-min pulse (zero time chase) was taken as 100% either in DHFR/G-8 or in Bl04-1-l cells.

of the mature forms of the molecules. To examine the glyco- sylation, the mutationally activated and normal neu gene products were treated with either endoglycosidase H or F (Endo H or Endo F). As shown in Fig. 5A, Endo H digestion of the 170.kDa precursor of normal neu gene in DHFR/G-8 cells yielded a product with a molecular mass of 160 kDa (lane 4). The sensitivity to Endo H digestion indicates the presence of high mannose-type oligosaccharide chains in this precursor molecule. The 175-kDa mature form was resistant to Endo H-digestion but sensitive to Endo F hydrolysis (Fig. 5A, lanes 1 and 3), suggesting the presence of N-linked complex-type carbohydrate moieties in the mature form molecules. The molecular mass (160 kDa) of Endo F-hydrolyzed product of the 175.kDa mature form appeared to be identical with that of Endo H-digested product of the precursor (Fig. 5A, lanes 3 and 4). Since glycoproteins undergo N-linked glycosylation in the endoplasmic reticulum, followed by the processing of high mannose-type to Endo H-resistant complex-type carbo- hydrate in the Golgi apparatus (22), the 170-kDa precursor and 175.kDa mature forms may represent the endoplasmic reticulum and Golgi apparatus forms of the protein, respec- tively.

In B104-1-l cells, Endo H digestion also converted the 170. kDa precursor of the mutationally activated neu gene product to a product with a molecular mass of 160 kDa (Fig. 5B, lane

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8104. I- I DHFR/G-8 A B Chase

(h) I 0.5 I I 0.5 I DHFR/G-BCells BlO4-I-1 Cells

Chase 1 I I I

(h) 22200 22200 185kDa - n(I)- - 175 kDa

KDGF - ++ - ++

FIG. :3. Effect of a growth factor(s) partially purified from bovine kidney on the turnover of cell surface normal and mutationally activated neu gene products. DHFR/G-8 or B104- l-l cells were pulse-labeled with [%]methlonine for 30 min and chased for 2 h in medium containing excess unlabeled methionine. The cells were then further chased in the presence (+) and absence (-) of 100 nr/ml KDGF Dartiallv ourified from bovine kidnev for 0.5 _ . and 1 h. Cell lysates were lmmunoprecipitated with anti-neu gene product antiserum and analyzed by 7.5% SDS-polyacylanude gel electrophoresls and fluorography. Arrows mdlcate the locations of 175.kDa mature form of the normal neu gene product in DHFR/G-8 cells and of 185.kDa mature form of the mutationally activated neu gene product m B104-1-l cells.

KDGF - + + 4

Suramln - - (1 mM)

+ - + -

Protamine + - + (10 w/ml)

FIG. 4. Effects of suramin and protamine on the down-reg- ulation of cell surface normal neu gene product by a growth factor(s) partially purified from bovine kidney. DHFR/G-8 cells were pulse-labeled with [““Slmethionine and chased in excess unlabeled methionine as described in the legend of Fig. 3. The cells were then further chased in the presence (+) and absence (-) of 100 rig/ml KDGF and with or without suramin (1 mM) or protamine (10 &ml) for 1 h. Cell lysates were immunoprecipitated with anti-neu gene product antiserum and analyzed by 7.5% SDS-polyacrylamide gel electrophoresis and fluorography. The arrou: indicates the location of 175-kDa mature form of normal neu gene product.

nose-type oligosaccharide chains. The 185.kDa mature form of mutationally activated neu gene product in B104-l-1 cells was resistant to digestion with Endo H (Fig. 5B, lane 6). However, this 185-kDa mature form could be converted to a product with a molecular mass of 170 kDa following the digestion with Endo F (Fig. 5B, lane 8). This loss of a molecular mass of 15 kDa was equivalent to that observed after hydrolysis of the 175.kDa mature form of normal neu product with Endo F. These results raised the possibility that the higher molecular weight (185 kDa) of mature form of mutationally activated neu gene product, compared with that of the mature form of normal neu gene product, may be due to a second distinct post-translational modification such as O-linked glycosylation in addition to N-linked glycosylation. To test this possibility, the Endo F-hydrolyzed mature form of mutationally activated neu product was further digested sequentially with sialidase and 0-glycanase. Treatment of the 185-kDa mature form with Endo F followed by digestion with sialidase and 0-glycanase gave a discrete band with a molec- ular mass of 170 kDa (data not shown). This 170-kDa product is indistinguishable from the product digested with Endo F alone (Fig. 5B, lane 8), suggesting no apparent O-linked glycosylation in the 180-kDa mature form molecule. To ex- amine whether the apparent molecular mass difference of the mature forms of normal and mutationally activated neu gene products is due to differential phosphorylation, the 185.kDa mature form of mutationally activated neu gene product was treated with calf intestine alkaline phosphatase according to the procedure of Ludlow et al. (23) and then analyzed by SDS- polyacrylamide gel electrophoresis and fluorography. The re- sult of this experiment indicated that the phosphatase treat- ment did not alter the mobility of “S-labeled 185-kDa mature form of mutationally activated neu gene product on SDS- polyacrylamide gel (data not shown). In the control experi- ment, phosphatase treatment was able to dephosphorylate the “P-labeled neu gene product. From these results, we conclude that the 185.kDa mature form of mutationally activated neu gene product and 175-kDa mature form of normal neu gene

9). This is similar to the result observed with the normal neu gene product in DHFR/G-8 cells (Fig. 5A, lane 4), suggesting that the precursors of both the mutationally activated and the normal neu gene products possess very similar high man-

Endo H -l--+- +--+- Endo F --+-- --+--

I 2 3 4 5 67 B 9 IO

FIG. 5. Endo H and Endo F digestions of normal and muta- tionally activated neu gene products in DHFR/G-8 and B104- l-l cells. DHFR/G-8 (A) and B104-l-1 (H) were pulse-labeled with [ “Slmethionine for 30 min and chased for 2 h in medium containing excess unlabeled methionine. Cells were lysed and incubated with anti-neu gene product antiserum. The immunoprecipitates were di- gested with (+) or without (6) Endo H or Endo F and analyzed by “ , rc, ,..I /O SDS-polyacrylamide gel electrophoresis followed by fluorogra- pk.

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3344 Processing and Turnover of neulerb R2 Oncogene Products

product, differ mainly in an unidentified post-translational modification.

To investigate the effect of N-linked glycosylation on the subsequent unidentified post-translational modification dur- ing the processing of mutationally activated neu gene product, H104-1-l cells were pulse-labeled and chased in the presence and absence of tunicamycin, an inhibitor for N-linked glyco- sylation (24), and the mutationally activated neu gene product was then analyzed with SDS-polyacrylamide gel electropho- resis and fluorography. As shown in Fig. 6, the molecular mass for normal and mutationally activated neu gene products was 160 kDa in tunicamycin-treated DHFR/G-8 and B104-l- 1 cells (2nnes 4-6 and 10-12). The failure to see the difference in molecular mass between the normal and mutationally activated neu gene products in the presence of tunicamycin suggests that N-linked glycosylation of the activated neu gene product is necessary for the unidentified post-translational modification or for transport to the site of the unidentified post-translational modification (possibly the Golgi appara- tus). It is noteworthy that this 160-kDa product in B104-1-l cells but not in DHFR/G-8 cells still underwent rapid turn- over in the presence of tunicamycin (Fig. 6, lanes 4-6).

1 2

FIG. 7. Phosphorylation of the normal and mutationally ac- tivated neu gene products in DHFR/G-8 and B104-l-l cells. DHFR/G-8 cells and B104-1-l cells were labeled with ortho[“P] phosphate at 37 “C for 3 h. The extracts of “Plabeled cells were &~&oprecipitated with anti-nru gene product antiserum and ana- lvzed hv 5.5% SDS-uolvacrvlamide gel electroghoresis and autoradi- dgraphy. Z?ar\ indicate the’ locations of “I’-l&eled polypeptides of 130 and 88 kDa which are possibly degradation product\ of the neu gene products.

In Vito Phosphor$ation of Mutationally Activated and Nor- mal neu Gene Products-Since autophosphorylation is a bio- chemical characteristic of activated growth factor receptors with protein tyrosine kinase activity (25,26), it was of interest to investigate the in situ phosphorylation status of the mu- tationally activated and normal neu gene products. B104-1-l and DHFR/G-8 cells were labeled with “P for 3 h. The “P- labeled cell lysates were then immunoprecipitated with anti- neu gene product antiserum and analyzed by SDS-polyacryl- amide gel electrophoresis followed by autoradiography. In B104-1-l cells, both precursor and mature forms of the mu- tationally activated neu gene product appeared to be phos- phorylated, although the former showed less “P-labeling than the latter (Fig. 7, lane 1). Two “P-labeled bands of low molecular weight (88 and 130 kDa) were also seen. These may represent degradation products of mutationally activated neu gene product. The “P-labeling of the mutationally activated neu gene products was found to be resistant to alkaline

8104 -I- I Cells DHFR/G-8 Cells Chase 1 II 3

ih) 01 20120120l2

Tun~camyc~n - - - + + + - - - + + +

I 2 3 4 5 6 7 8 3 IO II I2

FIG: (i Effect of tunicamycin treatment on the processing of normal and mutationally activated neu gene products in DHFRIG-8 and B104-l-l cells. Follo\ving r)reincuhation with (+I or \vclthout i-1 tunlcarnkcm (:’ ~g/ml) for :10 mm. cells were pulse- labeled alth [ “S]methlonme and chased in medium contaming excess unl~~heled methlonlne 111 the I,resence of tunicam>cm for 1 and 2 h. The cell Iycates were then lmmunoprecipltated \\lth antl-nru gene product nntlserum and anal? zed IX 7 .j’r SDS-pal? acr! lamlde gel clectrophoresls and fluorograph>.

treatment (1 N KOH) (18), suggesting a tyrosine-specific phosphorylation of the mutationally activated rzeu gene prod- ucts. This suggestion has been supported by the observation that these ‘“P-labeled species were immunoprecipitated by anti-phosphotyrosine antibody (data not shown). Much less “‘P-labeled normal neu gene product was found in DHFR/G- 8 cells (Fig. 7, lane 2). It is interesting to note that very little, if any, “‘P-labeled precursor of normal neu gene product was observed in DHFR/G-8 cells. These results suggest an in- creased tyrosine-specific phosphorylation of both precursor and mature forms of mutationally activated neu gene product when compared with their normal counterparts (normal neu gene product in DHFR/G-8 cells).

Mechanism of Rapid Turnover of Mutationally Activated neu Gene Products-As previously noted, both precursor and mature forms of the mutationally activated neu gene product turned over very fast when compared with their normal coun- terparts. In order to see whether lysosomes were involved in the rapid turnover of the mutationally activated neu gene product, the effects of lysosomotropic agents (12) on this process were examined. As shown in Fig. 8A, treatment of cells with ammonium chloride failed to have an inhibitory effect on the turnover of mutationally activated neu products. In the presence of 20 mM of ammonium chloride, the kinetics of processing of activated neu gene product were similar to those observed in the absence of ammonium chloride. Chlo- roquine, another lysosomotropic agent, at a concentration of 20 pM also did not exhibit any inhibitory effect on the fast turnover of mutationally activated neu gene product in B104- 1 cells (data not shown). These results suggest that lysosomes are not involved in the fast turnover of activated neu gene product. However, it is still possible t,hat removal of the C- terminal cytoplasmic domain of the protein which is recog- nized by anti-neu gene product antibodies is independent of lysosomal degradation but that lysosomes may be involved in degradation of remainder of the protein.

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n “% o 0.5 I I 5 2 2.5 .

B Chase

(h) 005 I 15 2 25

FIG. 8. Effects of ammonium chloride and suramin on the processing and turnover of the mutationally activated neu gene product in B104-l-l cells. Cells were pulse-labeled for 30 min with [““Slmethionine in the presence of 10 mM ammonium chloride (A) or 0.1 mM suramin (B) and chased in medium cont,aining unlabeled methionine and these reagents for various time periods. The cell lysates were then analyzed by immunoprecipitation with anti-neu gene product antiserum, followed by 73% SDS-polyacryl- amide gel electrophoresis and fluorography.

Suramin, a polysulfonate compound, was shown previously to block the rapid turnover of the PDGF receptor following its activation by ligands such as v-sis and c-sis gene products (12). In this case, suramin appeared to block the rapid turn- over of the PDGF receptor by inhibiting the interaction of newly synthesized PDGF receptor and its ligands, the newly synthesized sis gene products (12). Since the mutationally activated neu gene product in B104-1-l cells resembles the PDGF receptor in s&transformed cells with respect to in- creased protein tyrosin kinase activity and rapid turnover, but its mutational activation does not depend on ligand bind- ing, it was expected that suramin should have no effect on the rapid turnover of activated neu gene product. To test this prediction, B104-1-l cells were pulsed with [““Slmethionine and chased for various time periods in the presence of 0.1 mM suramin. The ““S-labeled cell lysates were immunoprecipi- tated and analyzed with 7.5% SDS-polyacrylamide gel elec- trophoresis and fluorography. As shown in Fig. 8B, the kinet- ics of processing of mutationally activated neu gene product in the presence of suramin was very similar to that observed in the absence of suramin (Fig. 2, D and E). This result indicates that suramin had no significant influence on the rapid turnover of mutationally activated rzeu gene product.

DISCUSSION

The mechanisms whereby proto-oncogenes are activated to oncogenes include point mutations, deletion, translocation, and amplification (27,28). The activation of the growth factor receptor family of proto-oncogenes mainly involves point mutations and deletion (3, 27-34). Among these receptor genes, the oncogenic activation of neu gene appears to be unique. The single point mutation in the putative transmem- brane domain of neu gene endows it with a highly transform- ing potential, as demonstrated in transfection experiments (9) and in transgenic mice (10). The molecular mechanism by which the expression of the mutated neu product causes the transformation is unknown. However, the increased protein tyrosine kinase activity associated with the mutated neu gene product suggests that the tyrosine kinase activity is closely involved in neoplastic transformation by mutationally acti- vated neu gene product (9-11).

Transmembrane domains are known to be essential for membrane anchoring of many proteins and may also play an important role in mediating signal transduction across mem-

branes following ligand binding to the cell surface domains (35). For growth factor receptors, the transmembrane do- mains are believed to be involved in the negative regulation of the cytoplasmic protein tyrosine kinase by the cell surface domain (36, 37). Ligand binding to the cell surface domain is suggested to cause a conformational change resulting in in- creased protein tyrosine kinase activity of the cytoplasmic portion of the receptor. The expression of protein tyrosine kinase activity, in turn, generates signals leading to DNA synthesis. Since the neu gene product is very likely a receptor for an unidentified growth factor(s), the specific amino acid change at the transmembrane domain might induce the acti- vation of cytoplasmic protein tyrosine kinase by mimicking ligand-induced activation.

In this paper, we demonstrated that the mutationally acti- vated neu gene product in B104-l-1 cells shares several fea- tures with the ligand-activated PDGF receptor in sis-trans- formed cells which we described previously (12). These in- clude: 1) accelerated turnover; the newly synthesized mutationally activated neu gene product in B104-1-l cells underwent faster turnover than the normal product in DHFR/ G-S cells. The precursor of the mutationally activated neu gene product diminished very fast and was partially converted to the mature form, whereas the mature form showed a short half-life of <30 min. In s&transformed cells, the majority of the newly synthesized PDGF receptor failed to reach the cell surface and was rapidly degraded. This rapid turnover (t% <30 min) appeared to result from interaction of the sis gene product with the PDGF receptor in the endoplasmic reticulum and/or Golgi apparatus (12). 2) Tyrosine-specific phosphoryl- ation; both precursor and mature forms of mutationally acti- vated neu gene product were found to be tyrosine-specific phosphorylated. In sis-transformed cells, the precursor and mature forms of the PDGF receptor were also seen to be tyrosine-specific phosphorylated (12). 3) Resistance to lyso- somotropic amines; lysosomotropic agents such as ammonium chloride and chloroquine failed to block the rapid turnover of the newly synthesized mutationally activated neu gene prod- uct in Bl04-1-l cells and of PDGF receptor in sis-transformed cells (12).

Differences between the mutationally activated neu gene product in B104-1-l cells and the ligand-activated PDGF receptor in sis-transformed cells reported previously (12) are: 1) in s&transformed cells, both the precursor and mature forms of the PDGF receptor possess N-linked oligosaccharide chains similar to those of the PDGF receptor in normal cells. In contrast an unidentified post-translational modification, in addition to N-linked glycosylation, was observed in the mature form of mutationally activated neu gene product but not in that of normal neu gene product. This additional post- translational modification may reflect a conformational change resulting from the single amino acid change in the transmembrane domain and leading to exposure of potential modification site(s). This conformational change may be com- parable to that induced following ligand binding (25). 2) In sis-transformed cells, suramin appeared to reverse the fast turnover of the PDGF receptor by blocking the PDGF recep- tor-sis gene product interaction (12). However, suramin did not exert any influence on the rapid turnover of the muta- tionally activated neu gene product in Bl04-1-l cells. The inability of suramin to reverse the rapid turnover of the mutationally activated neu gene product is compatible with the fact that no ligand is involved in the activation of muta- tionally activated neu gene product.

Recently, Stern and his co-workers (38) reported a short half-life of mutationally activated neu gene product as com-

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3346 Processing and Turnover of neulerb B2 Oncogene Products

pared with that of normal neu gene product. However, the half-lives of the neu gene products that they reported (7 h for the normal neu gene product and 1.5 h for the mutationally activated neu gene product) are much longer than these we described in this paper. This discrepancy of half-lives of neu gene products is very likely due to different labeling conditions and the gel system. In our experiments, the cells were pulse- labeled with [35S]methionine for 30 min and chased in the presence of unlabeled methionine. In contrast, Stern and his co-workers (38) labeled the cells with a very low concentration (33 nM or 33 &i/ml) of [35S]cysteine overnight and then chased in the presence of 2 mM cysteine for various time periods. The depletion of cysteine in the medium overnight may have affected the synthesis of proteins or the protein turnover and explained the prolonged half-lives of neu gene products observed by Stern and his co-workers (38). Further- more, their long labeling conditions and their gel system did not resolve precursor and mature forms of ncu gene products, complicating the accurate estimation of half-lives of neu gene products.

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We proposed previously that the intracellular activation of the PDGF receptor by sis gene products in the endoplasmic reticulum and/or Golgi apparatus is important for the trans- formation by sis genes (v-sis and c-sis) (12). The resemblance of the activated neu gene product in B104-l-l cells to the ligand-activated PDGF receptor in s&transformed cells, with respect to increased protein tyrosine kinase activity and rapid turnover of the precursor, as well as, the mature form, suggests that the intracellular functional expression of mutationally activated neu gene product in the endoplasmic reticulum and/ or Golgi apparatus may also play an important role in the transformation induced by mutationally activated neu gene product.

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Aclznowledgments-We thank Drs. William S. Sly and Catherine M. Nolan for critical review of the manuscript, Yi-Feng Chen for technical assistance, and Kimberly Short and Mitchell Pollack for preparing the manuscript. We also wish to thank Dr. Ming-Chi Hung, University of Texas for his generous gifts of B104-1-l and DHFR/G- 8 cells and Dr. Kam Fok, Monsanto Company, St. Louis, MO, for his generous gift of phosphotyramine.

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S S Huang, H A Koh, Y Konish, L D Bullock and J S Huanggene product and its normal cellular counterpart.

Differential processing and turnover of the oncogenically activated neu/erb B2

1990, 265:3340-3346.J. Biol. Chem. 

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