coli, p509et, are autophosphorylated in vitro on tyrosine resi- dues. Using the immunocomplex kinase assay, p1404et activity was detected in various human ...
Proc. Natl. Acad. Sci. USA Vol. 85, pp. 21-25, January 1988 Biochemistry
Characterization of the TPR-MET oncogene p65 and the MET protooncogene p140 protein-tyrosine kinases (cell-surface receptor/phosphorylation/expression in tumor cell lines/expression in Escherichia cola)
M. GONZATTI-HACES, A. SETH, M. PARK, T. COPELAND, S. OROSZLAN, AND G. F. VANDE WOUDE Bionetics Research Inc.-Basic Research Program, National Cancer Institute-Frederick Cancer Research Facility, P.O. Box B, Frederick, MD 21701
Communicated by Howard L. Bachrach, September 8, 1987 (received for review June 16, 1987)
cDNA fragment containing the MET kinase domain was inserted in frame at the unique HindIII site of pJLA16 (7) to generate plasmid pAMET-2. A 50-kDa protein (p5soe,) was expressed in bacterial cells containing pAMET-2 upon induction at 420C. The p5Oet was purified as described (7) and analyzed by NaDodSO4/polyacrylamide gel electrophoresis followed by staining with Coomassie blue or assayed in vitro for kinase activity. Preparation of MET-Specific Antisera. Three peptides corresponding to the predicted 8-, 16-, and 28-amino acids at the C terminus of the MET protein (5) were constructed by solid-phase Merrifield procedures (8) as described (9). Peptide coupled to keyhole limpet hemocyanin (9, 10) was mixed with Freund's complete adjuvant and administered subcutaneously into rabbits. Sera were tested by the diazo-immobilized peptide assay, and positive antibody was purified by affinity chromotography (11). Purified peptide antiserum (anti-C28) prepared against the conjugated 28-mer antigen (C28) H2-Ala-Pro-Tyr-Pro-Ser-Leu-Leu-Ser-Ser-Glu-Asp-
The proteins encoded by the human TPRABSTRACT MET oncogene (p65tPr-mt) and the human MET protooncogene (p140O"') have been identified. The p65tPr-met and p44lt, as well as a truncated TPR-MET product expressed in Escherichia coli, p509et, are autophosphorylated in vitro on tyrosine residues. Using the immunocomplex kinase assay, p1404et activity was detected in various human tumor epithelial cell lines. In vivo, p65P'-pr- is phosphorylated on both serine and tyrosine residues, while pl4Ont is phosphorylated on serine and threonine. pl4et is labeled by cell-surface iodination procedures, suggesting that it is a receptor-like transmembrane proteintyrosine kinase.
The MET oncogene was identified in a N-methyl-N'-nitroN-nitrosoguanidine (MNNG)-treated human osteogenic sarcoma cell line, MNNG-HOS (1, 2), by using the murine NIH 3T3 cell transfection assay. We have shown that activation of the MET oncogene occurred via a chromosomal DNA rearrangement (3, 4). This rearrangement created a hybrid TPRMET gene with upstream sequences derived from a locus on chromosome 1 (designated TPR for translocated promoter region) fused to downstream sequences from the MET protooncogene locus located on chromosome 7, 7q21-31 (5). The MET protooncogene is predominantly expressed in human fibroblast and epithelial cell lines as a 9.0-kilobase (kb) RNA species, whereas the activated TPR-MET oncogene expresses a novel 5.0-kb TPR-MET hybrid RNA species (3). Nucleotide sequence analysis of the MET protooncogene cDNA revealed an open reading frame of 1408 amino acids with features characteristic of the growth factor receptor protein-tyrosine kinase family (6). The predicted primary structure contains a 926-amino acid external domain and a 435-amino acid cytoplasmic domain with homology to the protein-tyrosine kinase family of genes. We have used C-terminal anti-MET peptide antibodies to identify and characterize the TPR-MET oncogene and MET protooncogene protein products. Both p65tPr-met and p140met are protein-tyrosine kinases and undergo autophosphorylation in vitro.
Asn-Ala-Asp-Asp-Glu-Val-Asp-Thr-Arg-Pro-Ala-Ser-PheTrp-Glu-Thr-Ser-COOH was used in these studies. Similar results were obtained with the other peptide antisera. Purified pS0met, resolved on a 12% NaDodSO4/polyacrylamide gel, was homogenized in 0.5% NaDodSO4 in phosphate-buffered saline, mixed with Freund's adjuvant, and injected intradermally into rabbits. Sera were tested by the immunocomplex kinase assay, and antibody was purified by protein A-Sepharose chromatography (Pharmacia). Cell Labeling and Immunoprecipitation. Cells were labeled with 0.5 mCi (1 Ci = 37 GBq) of [35S]methionine (New England Nuclear) for 2 hr at 37°C in media lacking methionine. Labeled cells were extracted with 3 ml of RIPA buffer (20 mM Hepes, pH 7.0/10 mM EDTA/0.1% NaDodSO4/1% Nonidet P-40/0.5% deoxycholate/2.5 mM phenylmethylsulfonyl fluoride/27 kallikrein units of aprotinin per ml). Clarified cell lysates (CC13COOH-precipitable material 7 x 106 cpm) were immunoprecipitated with purified anti-C28 MET antibody (0.020 ml) and preincubated in the presence or in the absence of excess peptide antigen (5 ,ug) for 20 min at 30°C. Immunoprecipitations were carried out at 4°C for 4-16 hr. Immunocomplexes were collected on protein A-Sepharose, washed extensively with RIPA buffer and high-salt buffers (50 mM Hepes, pH 7.0/0.5 M LiCl/10 mM EDTA/1% 2-mercaptoethanol/1.25 mM phenylmethylsulfonyl fluoride/ 27 kallikrein units of aprotinin per ml), and solubilized by boiling in NaDodSO4 sample buffer. Samples were analyzed by NaDodSO4/8% polyacrylamide gel electrophoresis followed by fluorography at -70°C. Metabolic labeling of cells with 0.5 mCi of [32P]orthophosphate (New England Nuclear) was performed as described above for [35S]methionine only with phosphate-free media. Cells lysates were prepared with RIPA buffer containing 100 ,uM Na3VO4 and immunoprecipitated with anti-C28 MET
MATERIALS AND METHODS Cell Lines. The human osteogenic sarcoma cell line HOS, 7,12-dimethylbenz[a]anthracene (DMBA)-treated HOS cells, and MNNG-treated HOS cells were obtained from J. S. Rhim. Other human cell lines were obtained from the American Type Culture Collection and grown according to their recommendations. Expression of the MET Kinase Domain in Escherichia coli. The plasmid pBR5a, containing 2.1-kb TPR-MET human cDNA (6) was digested with HindIlI, and the resulting 1.6-kb The publication costs of this article were defrayed in part by page charge
payment. This article must therefore be hereby marked "advertisement"
Abbreviations: TPR, translocated promoter region; MNNG, N-
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
methyl-N'-nitro-N-nitrosoguanidine. 21
22
Biochemistry: Gonzatti-Haces et al.
Proc. Natl. Acad. Sci. USA 85
antibody. Labeled samples were analyzed by NaDodSO4/ polyacrylamide gel electrophoresis and autoradiography at -700C. In Vitro Kinase Assay. Subconfluent cultures (=5 x 106 cells) were extracted with kinase-lysis buffer (1% Triton X100/0.05% NaDodSO4/10 mM NaH2PO4/Na2HPO4, pH 7.0/ 150 mM NaCl/5 mM EDTA/100 ,uM Na3VO4/2.5 mM phenylmethylsulfonyl fluoride) as described (12). Equivalent amounts of protein (0.7 mg) as determined by the Bradford assay (13) were immunoprecipitated with either anti-C28 or anti-p50reI antibody (0.030 ml). Immunocomplexes, washed with kinase-lysis buffer without NaDodSO4 and with 50 mM Hepes (pH 7.0), were resuspended in 50 mM Hepes, pH 7.0/10 mM MnCl2/5 mM MgC12/1 dithiothreitol/10 ,uM ATP (5 uCi per assay) and were incubated at 300C for 20 min. Reactions were terminated with NaDodSO4 sample buffer and analyzed by NaDodSO4/8% polyacrylamide gel electrophoresis and autoradiography. Two-dimensional phosphoaminoacid analysis of pl40met and p65tPr-met was carried out as described (14). Surface lodination. Subconfluent cultures of HOS and MNNG-HOS cells (=2 x 106) were labeled with Na251I (New England Nuclear) and lodo-Gen (Pierce) as described (15). Clarified cell extracts were subjected to immunoprecipitation with anti-C28 antibody (0.03 ml). Labeled proteins were resolved by NaDodSO4/polyacrylamide gel electrophoresis and visualized by fluorography at -700C.
(1988)
RESULTS
Immunoprecipitation of MET Proteins with Anti-C28 Antibody. A human osteosarcoma cell line, HOS, its tumorigenic derivative, MNNG-HOS, and three MET-containing NIH
3T3 transformants were examined for MET protein expression by immunoprecipitation with anti-C28 antibody (Fig. lA). A protein that migrated electrophoretically as a diffuse band with an average molecular mass of 65 kDa (p65) was immunoprecipitated from the four cell lines containing the activated TPR-MET oncogene. Immunoprecipitation of p65 was effectively blocked by preincubation of the serum with the C28 peptide. These analyses show that p65 is the TPRMET oncogene product encoded by the 5.0-kb hybrid TPRMET RNA transcript (3). In addition to p65tPrmet, three polypeptides of molecular masses 160, 140, and 110 kDa (p160, p140, and p110) were immunoprecipitated from HOS and MNNG-HOS cell extracts with anti-C28 antiserum. Immunoprecipitation of these proteins was blocked in the presence of the C28 peptide. One (9.0 kb) or two (9.0 and 7.0 kb) MET protooncogene transcripts have been detected in fibroblastic and epithelial human cell lines, whereas three MET transcripts (9.0, 7.0, and 6.0 kb) are expressed in HOS and MNNG-HOS cells (3). The p140 was observed in all these cell lines despite the different MET transcripts expressed (Fig. 1B). Low levels of p140 were observed in immunoprecipitates from NC-37 cells
A
B
200
|-160 -140
160140-
-110
97.4
110-
65-
:.
::::..:
.2_ __,_
....
_: 3 _!!'--i T_.
-
+
+
_
+
HOS MNNG- NIH HOS 3T3
-
+
- +
-
221c
2212b
MT113
+
Peptide
_
Cell Line
NC-37
+
-
..
...
tt!!!||wRt-
+
DLD-1
-
_
+
Calu 1
FIG. 1. Immunoprecipitation of TPR-MET oncogene- and MET protooncogene-encoded proteins with anti-C28 antibody. (A) Extracts from HOS and MNNG-HOS cell lines, NIH 3T3, and MET transformants 221c, 2212b, and MT113 were labeled with [35S]methionine. Labeled cell extracts were immunoprecipitated with anti-C28 MET antibody in the presence (lanes +) or absence (lanes -) of excess peptide. (B) Similar analyses were carried out with the NC-37, DLD-1, and Calu-1 cell lines. Immunoprecipitated proteins were resolved by NaDodSO4/8% polyacrylamide electrophoresis and visualized by fluorography.
Biochemistry: Gonzatti-Haces et al.
Proc. Nati. Acad. Sci. USA 85 (1988)
in which no MET transcripts have been detected by blothybridization analyses (3). In Vitro Phosphorylation of MET Polypeptides. Nucleotide sequence homology has suggested that MET is a member of the protein-tyrosine kinase family of oncogenes (5). Thus, the TPR-MET and MET protooncogene products were tested for kinase activity in vitro by using the immunocomplex assay. The p65tPr-met from MNNG-HOS cells and from three METcontaining transformants was readily phosphorylated in vitro (Fig. 2A). Neither kinase activity nor labeled p65tPr-met was detected by using anti-C28 antiserum preincubated with competing C28 peptide. Upon longer exposure, phosphorylation of p140 was observed in extracts of HOS and MNNGHOS cells (exposure not shown). A large variation in MET kinase activity was observed in the human tumor cell lines tested (Fig. 2B and Table 1). For example, no phosphorylation of p140 was detected in immunoprecipitates from MIA-PAC-a2, SW480, and J82 cells. Low levels of p140 phosphorylation were detected in SKMES-1, Panc-1, and A549 cells, and high levels were observed in KB and Calu-1 cells, as well as in DLD-1, Calu-6, and C-41 cells. Immunoblot analyses indicate that the level of p140 protein in HOS, MNNG-HOS, and Calu-1 cell extracts is approximately the same (data not shown). Thus, the high level of p140 kinase activity in vitro in Calu-1 cells does not correlate with increased levels of protein but rather reflects an increase in the specific activity of the protein kinase. Expression of a Portion of TPR -MET Oncogene in E. coli. Several members of the oncogene tyrosine kinase family have been shown to be enzymatically active when expressed in bacteria (16, 17). The above analyses show that p6tPr-met and p140 are phosphorylated in vitro in the immunocomplex assay. To determine whether this resulted from autophosphorylation activity, the kinase domain of the TPR-MET oncogene derived from cDNA (6) was expressed in E. coli. A 50-kDa protein, p50me, was detected in extracts of induced cells containing pAMET-2 (Fig. 3A, lane 2) but not in control cells (Fig. 3A, lane 1). The partially purified p5Omet was A
Table 1. In vitro kinase assay Human cell lines Colon SW480 SW620 DLD-1 Pancreas Capan-1,-2, Panc-1
23
p140 phosphorylation ++ +++
++
Lung Calu-1,6 A549, SK-MES1, SK-LU-1
+++ ++
Cervix C-41 HeLa
+++ ++
Exponentially growing cultures (2-4 x 106 cells) were extracted with kinase-lysis buffer. Cell extracts (0.8 mg of protein) were immunoprecipitated with anti-C28, assayed for kinase activity in vitro, and analyzed by NaDodSO4/polyacrylamide gel electrophoresis followed by autoradiography; + + + denotes the highest level of p140 phosphorylation (see Fig. 2B).
phosphorylated in vitro (Fig. 3B, lane 1) on tyrosine residues
(data not shown). The anti-C28 antiserum recognized p6sPr-me' and p140 as the predominant products in the immunocomplex kinase assay (Fig. 2). To determine whether antibody directed against different MET epitopes would also recognize these phosphoproteins, anti-p50met antibody was tested in the immunocomplex kinase assay with MNNG-HOS cell extracts (Fig. 3C). Phosphorylation of p6SPr-met and p140 was observed with both antisera, although the anti-p50meI antibody partially inhibited the phosphorylation reaction. In Vivo Phosphorylation of Met Polypeptides. To determine whether p65tPr-met and p140 are phosphorylated in vivo, cell lysates from 32P-labeled MET-transformed NIH 3T3 cells (2212b) and Calu-1 cells were immunoprecipitated with anti-C28 antibody. Immunoprecipitates prepared from cells
B
200
m
S
p140
-t
97.4 ,68 f.
O*
p65-.
4
_l_
43
25.7
+
-
+-+
HOS MNNG- NIH HOS 3T3
-+-
277a
+
221c
+
Peptide
2212b
Cell Line
-
-
+
-
MIA Calu PAC-A 2
+ I
+
+
SW 480
SKMES-1
-
+
KB
J82
A549
Panc -1
FIG. 2. Phosphorylation of p65tPr-me and p140 in vitro. Cell extracts (0.7 mg of protein) were immunoprecipitated with anti-C28 antibody in the presence (lanes +) or absence (lanes -) of competing peptide. Immunoprecipitates were washed and incubated with [y-32P]ATP, as described. Samples were analyzed by NaDodSO4/polyacrylamide gel electrophoresis and autoradiography for 6 hr at -70°C with an intensifying screen.
24
Biochemistry: Gonzatti-Haces et al.
A
B
Proc. Natl. Acad. Sci. USA 85 (1988)
A
C
B 2212b
Calu 1
--I
:~'rnf:
P5 mt
p50met
p1 40met
-
.,
41-
:z140e
ill.
p65tpr-met 0 1
2
1
, D
2
p65tPr-met-.D.
1
ap
2
FIG. 3. Expression of a bacterial p50met product. Proteins from induced bacteria carrying control plasmid (pJLA16) (A, lane 1) and plasmid pAMET-2 (A, lane 2) were purified by guanidine hydrochloride extraction, analyzed on a NaDodSO4/12% polyacrylamide gel, and visualized by Coomassie blue staining. Purified pSOre, was phosphorylated and immunoprecipitated with anti-C28, preincubated in the absence (B, lane 1) or in the presence (B, lane 2) of competing peptide. Extracts from MNNG-HOS cells immunoprecipitated with anti-p50ret (C, lane 1) or anti-C28 antibody (C, lane 2) were tested in the immunocomplex kinase assay. Samples were analyzed by NaDodSO4/polyacrylamide gel electrophoresis and subjected to autoradiography for 2 days at room temperature.
labeled in vivo with [35S]methionine or in vitro in the immunocomplex kinase assay were examined as well for comparison (Fig. 4 A and B). These analyses show that p65tPr-met present in 2212b cells and p140 present in Calu-1 cells are phosphorylated in vivo. Phosphorylation of p65tPrmet and p140 in vitro was on tyrosine residues (Fig. 4 C and D). Tyrosine and serine residues were phosphorylated in p65tPrmet in vivo (Fig. 4E). In contrast, phosphoserine and phosphothreonine were detected on p140 labeled in vivo (from both Calu-1 and HOS cells) (Fig. 4F). p140 Is Located on the Cell Surface. Collectively, the preceding analyses indicate that p140 is a MET protooncogene product. In addition to being recognized by both anti-C28 and anti-p50ret antibodies, p140 has been detected by electrophoretic immunoblot transfer analyses using antiC28 antibody (data not shown). Since the predicted protein encoded by the MET protooncogene cDNA is a member of the growth factor receptor tyrosine kinase family (6, 18, 19), we examined whether p140 is exposed on the cell surface. The p140 precipitated by anti-C28 antiserum from HOS and MNNG-HOS cells was labeled by 1251 (Fig. 5), and this immunoprecipitation was efficiently blocked competitively by the peptide antigen. The p65tPrmeI present in MNNG-HOS cells was not labeled by the cell-surface iodination procedure. We conclude that pl40met is located on the cell surface, possesses tyrosine kinase activity, and is the MET protooncogene product. DISCUSSION
Activation of the MET oncogene in MNNG-HOS cells is similar to the gene rearrangement responsible for the activation of the TRK oncogene in which 5' sequences derived from a nonmuscle tropomyosin were joined to a truncated receptor protein-tyrosine kinase (20). Oncogenic activation of MET is also reminiscent of the Philadelphia chromosomal translocation in chronic myelogenous leukemias that generated the hybrid BCR-ABL tyrosine kinase p210 (21-24). Unlike the v-erbB protein, which is a truncated version of the epidermal growth factor receptor containing a transmembrane domain and extracellular sequences (25-27), the p6StPrmet product is
1
2
3
1
2
FIG. 4. Phosphorylation of p65tPr-me' and p140 in vivo and in vitro. A MET NIH 3T3 transformant, 2212b (A), and a human carcinoma cell line, Calu-1 (B), were metabolically labeled with [35S]methionine (A, lane 1) or with (32P)orthophosphate (A and B, lanes 2). Immunoprecipitates prepared from labeled cell extracts were analyzed along with cell lysates tested for in vitro kinase activity (A, lane 3). Labeled proteins were resolved by NaDodSO4/8% polyacrylamide electrophoresis followed by autoradiography. (C-F) Phosphoaminoacid analyses of p65tPr-mc from 2212b cells labeled in vitro (C) and in vivo (E) and of p140 from Calu-1 cells labeled in vitro (D) and in vivo (F).
truncated downstream from the transmembrane domain (6) and is not detected on the cell surface. Several characteristics of pl40met indicate that it is the product of MET protooncogene and is encoded by the 9.0-kb MET transcript. pl40me, is recognized by the anti-C28 and the anti-p50met antibodies. Moreover, its size is consistent with the estimated molecular weight of the putative product encoded by the MET protooncogene cDNA (6), provided that it is not extensively modified. Our preliminary analysis indicates that pl40met is not extensively glycosylated (M.G.-H., unpublished results). As predicted from the MET protooncogene cDNA sequence, p140met is located on the cell surface and possesses tyrosine kinase activity in vitro. Since both pl40met and p6tPr-met are phosphorylated on tyrosine residues in vitro and only p65tPrmt is phosphorylated on tyrosine in vivo, p65tPrmet may represent a constitutively activated form of the pl40met protein-tyrosine kinase. Ligand binding
Biochemistry: Gonzatti-Haces et al. Kd -200 pl 4Qmet
*Maw
-97.4
t
}=
--~~68 -43
'
+
HOS
Elm. + Peptide MNNG-HOS
FIG. 5. Cell-surface iodination of p140. HOS and MNNG-HOS cells were surface-labeled with Na1251 and extracted with RIPA buffer. Proteins were immunoprecipitated with anti-C28 antibody preincubated with (lanes +) or without (lanes -) competing peptide. Samples were analyzed by NaDodSO4/8% polyacrylamide gel electrophoresis and fluorography.
stimulates the intrinsic enzymatic activity of the growthfactor-receptor tyrosine kinases (28-31). If p140met functions as a receptor protein-tyrosine kinase for an unidentified growth factor, ligand binding may also be required to stimulate the autophosphorylation of pl40met in vivo. The variation in the levels of pl40met kinase activity in human tumor epithelial cell lines may reflect cell-specific differences in the overall regulation of protein phosphorylation. Alternatively, the pl40met activity could be directly influenced by an autocrine loop or by activation of MET as an oncogene. However, even if MET were activated by a point mutation, because of the large size of the MET protooncogene locus (100 kb) (32), it might not be detected in DNA transfection assays. Very different levels of pl40met kinase activity were observed in immunoprecipitates from two cell lines, SW480 and SW620, derived from the same patient. The SW480 cell line was derived from a primary colon adenocarcinoma, whereas the SW620 cell line was established a year later from a metastatic lymph node. The difference in the p140met kinase activity could be due to the selection of cell variants during explantation and propagation of the tumor cells in vitro. It may also indicate that p140met plays some role in tumor progression. We thank Beth Cassell and Young Kim for technical assistance, Stephen Hughes and Friedrich Propst for helpful comments, and Fusan Kelly for preparation of this manuscript. This research was Sponsored by the National Cancer Institute under Contract NO1'CO-23909 with Bionetics Research, Inc. 1. Cooper, C. S., Blair, D. G., Oskarsson, M. K., Tainsky, M. A., Eader, L. A. & Vande Woude, G. F. (1984) Cancer Res. 44, 1-10.
Proc. Natl. Acad. Sci. USA 85 (1988)
25
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