Kinase Encoded by the MET Proto-Oncogene ... - Europe PMC

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Sep 22, 1991 - The MET proto-oncogene encodes a 190-kDa disulfide-linked heterodimeric ... Proto-oncogenes encoding tyrosine kinase receptors, or.
Vol. 11, No. 12

MOLECULAR AND CELLULAR BIOLOGY, Dec. 1991, p. 6084-6092 0270-7306/91/126084-09$02.00/0

Defective Posttranslational Processing Activates the Tyrosine Kinase Encoded by the MET Proto-Oncogene (Hepatocyte Growth Factor Receptor) ANNA MONDINO, SILVIA GIORDANO, AND PAOLO M. COMOGLIO* Department of Biomedical Sciences & Oncology, University of Torino Medical School, 10126 Torino, Italy Received 10 June 1991/Accepted 22 September 1991

The MET proto-oncogene encodes a 190-kDa disulfide-linked heterodimeric receptor (pl90aI) whose tyrosine kinase activity is triggered by the hepatocyte growth factor. The mature receptor is made of two subunits: an a chain of 50 kDa and a a chain of 145 kDa, arising from proteolytic cleavage of a single-chain precursor of 170 kDa (prl70). In a colon carcinoma cell line (LoVo), the precursor is not cleaved and the Met protein is exposed at the cell surface as a single-chain polypeptide of 190 kDa (pl9ONC). The expression of the uncleaved Met protein is due to defective posttranslational processing, since in this cell line (i) the proteolytic cleavage site Lys-303-Arg-Lys-Lys-Arg-Ser-308 is present in the precursor, (ii) pl9ONC is sensitive to mild trypsin digestion of the cell surface, generating a and j8 chains of the correct size, and (iii) the 205-kDa insulin receptor precursor is not cleaved as well. pl9oNC is a functional tyrosine kinase in vitro and is activated in vivo, as shown by constitutive autophosphorylation on tyrosine. The MET gene is neither amplified nor rearranged in LoVo cells. Overlapping cDNA clones selected from a library derived from LoVo mRNA were sequenced. No mutations were present in the MET-coding region. These data indicate that the tyrosine kinase encoded by the MET proto-oncogene can be activated as a consequence of a posttranslational defect. structure of the Met protein is indistinguishable from that found in other normal cells, indicating that overexpression alone is most probably sufficient for the activation of the Met kinase (43). The MET proto-oncogene encodes a 190-kDa transmembrane receptor (pl90O) composed of two disulfide-linked chains of 50 (ox) and 145 (,B) kDa. The N-terminal domains of the a chain and the at chain are exposed at the cell surface (18, 20). The C-terminal part of the P chain is cytoplasmic and contains the tyrosine kinase domain (7, 12, 21, 54). The receptor is synthesized as a precursor of 170 kDa, which undergoes cotranslational glycosylation and is then cleaved to yield the mature pl90%' heterodimer (19). Two C-terminal-truncated Met proteins have been recently described

There are many genetic alterations that could conceivably convert a proto-oncogene into an activated oncogene, and several different examples of mechanisms of activation have been described. A gene may be altered by point mutation, chromosomal translocation, or insertion of mobile genetic elements, such as a retrovirus. The change can occur in the protein-coding region so as to yield a hyperactive product, or it can occur in the adjacent control regions so that the gene is overexpressed and/or amplified (for reviews, see references 2 and 3). Some proto-oncogenes have been implicated in the genesis of human malignancies. The same protooncogenes can be activated through different mechanisms (3, 28). Proto-oncogenes encoding tyrosine kinase receptors, or receptorlike molecules, play a key role in the complex signalling network governing cell growth (27, 29, 56, 62). For this reason, genetic alterations leading to deregulated tyrosine kinase activity have a high oncogenic potential (62). 5' or 3' deletions activate v-erbB (15), v-kit (45), v-fms (59), and neu (1). Point mutations activate v-fis (59), HER21neu (1), and TRK (11). Amplifications activate HER21neu (13, 26) and MET (20). Rearrangements have been found to activate the proto-oncogenes TRK (32), RET (51), and MET (10, 41). The MET proto-oncogene was originally identified as a transforming gene, activated after in vitro treatment of a human osteosarcoma cell line with a chemical carcinogen. In this cell line, the 3' region of MET is rearranged with the 5' region of TPR (translocation promoter region) (41). The resulting hybrid transcript is translated into a protein lacking the first 1,026 amino acids of the Met protein and endowed with constitutive tyrosine kinase activity (12, 21, 53). Moreover, the MET proto-oncogene has been found amplified and overexpressed, and the protein has been found activated in a gastric carcinoma cell line (20). In this cell line, the primary

*

(44).

Under physiological conditions, the kinase activity is dependent on the binding of the mature heterodimer to the specific ligand. MET-encoded pl90a1 has been identified as the receptor for the hepatocyte growth factor (HGF) (4, 38). HGF is a dimeric protein that behaves as a powerful mitogen for hepatocytes and other epithelial cells (33, 35, 52, 63). Recently, we found that pl9OaP binds to scatter factor and to HGF with identical affinity. We have also demonstrated that these molecules are structurally and functionally interchangeable (39). The scatter factor is known to dissociate epithelial cells, increasing mobility and invasiveness (50, 58). In this paper, we describe an altered Met protein exposed at the cell surface of a colon carcinoma cell line. The protein is a single polypeptide with an apparent molecular mass of 190 kDa, resulting from the 170-kDa precursor which undergoes glycosylation but is not cleaved. This uncleaved molecule (pl90NC) is phosphorylated on tyrosine in vivo, indicating constitutive activation, and has tyrosine kinase activity in vitro. Analysis of cDNAs derived from LoVo transcripts revealed no differences between the primary sequences of pl90O and pl9oNC. We infer that pl90Nc results from

Corresponding author. 6084

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defective processing and suggest that the tyrosine kinase encoded by the MET oncogene can be activated by a defect occurring at the level of posttranslational processing.

EGTA [ethylene glycol-bis(P-aminoethyl ether)-N,N,N',N'tetraacidic acid], 1% Triton, and protease inhibitors. Extracts were clarified at 12,000 x g for 15 min and immunoprecipitated with the specific antibodies. The immunocomplexes were collected on protein A-Sepharose, washed, and eluted in Laemmli buffer with or without ,-mercaptoethanol. All the steps were carried out at 4°C. Proteins were subjected to SDS-PAGE, and the gels were fluorographed, dried, and exposed to Amersham Hyperfilm for autoradiography. Immunoprecipitation and kinase assays. Subconfluent cell cultures were placed on ice and washed twice with cold PBS. Monolayers were extracted for 20 min at 4°C with 1% CHAPS {3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate} in HEPS buffer (25 mM HEPES [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid]-NaOH [pH 7.4], 5 mM MgCl2, 1 mM EGTA, 100 mM NaCl, 10% glycerol [vol/vol], 1 mM PMSF [phenylmethylsulfonyl fluoride], 50 ,ug of leupeptin, 100 ,ug of soybean trypsin inhibitor, 10 ,ug of aprotinin, 10 ,ug of pepstatin per ml). The cell lysates were cleared by centrifugation at 15,000 x g for 20 min at 4°C. Sepharose-protein A (30 ,lI of packed beads per 100 ,ul of lysate) was preincubated with anti-Met peptide antiserum (3 ,u/100 ,ul of lysate), washed twice with buffer, and incubated with the lysates for 3 h at 4°C with stirring. Bound proteins were washed three times with HEPS buffer and twice with kinase buffer (20 mM HEPES-NaOH [pH 7.1], 5 mM MgCl2, 100 mM NaCl). The immunoprecipitate was washed once with excess kinase buffer and exposed to [_y-32P]ATP in kinase buffer. Usually, 10 p.Ci of [-y-32P]ATP per sample (specific activity, 7,000 Ci/mmol) was diluted with different concentrations of unlabeled ATP. The standard reaction time was 3 min for autophosphorylation at 4°C, with continuous stirring. The reaction was stopped by the addition of concentrated boiling Laemmli buffer (31). The eluted proteins were subjected to SDS-8% PAGE followed by autoradiography for 3 h at 70°C by using intensifying screens. Southern and Northern blotting. Techniques of chromosomal DNA isolation, restriction enzyme digestion, and Southern blotting are described in Sambrook et al. (46). RNA was prepared by the guanidine monothiocyanate-LiCl method, described by Cathala et al. (6). Formaldehydeagarose gel electrophoresis, Northern transfer, and hybridization were performed according to the procedures of Wahl et al. (57). Probes were labelled with the Multiprime DNA labelling system of Amersham Corp. (Arlington Heights, Ill.). The specific probes used were a full-length MET cDNA cloned from a GTL-16 library (17a) and a full-length HGF cDNA obtained by polymerase chain reaction (PCR) amplification (39). Reverse transcription and PCR. mRNA, prepared as described above, was used for reverse transcription (RT). The reaction mixture (40 ,ul) contained the enzyme buffer as supplied by Bethesda Research Laboratories, 10 jig of mRNA, 1 U of RNasin per ml (Promega, Madison, Wis.), 50 pmol of the 3' PCR primer (see below), 1 mM (each) deoxynucleotide triphosphate, and 10 U of Moloney murine leukemia virus reverse transcriptase per ml from Bethesda Research Laboratories. The reaction was incubated at 37°C for 1 h, the enzyme was then denatured for 3 min at 95°C, and the product was kept at -20°C. PCR was carried out on the products of the RT reactions as follows. In a final volume of 100 ,ul, the reaction mixture contained 10 jIl of the products of the RT reaction as the source of a template, 50 mM Tris (pH 8.4), 50 mM KCl, 25

MATERIALS AND METHODS Reagents and cells. All reagents used were analytical grade. Protease inhibitors were purchased from Sigma. Staphylococcus aureus protein A covalently coupled to Sepharose was purchased from Pharmacia [-y-32P]ATP (specific activity, 7,000 Ci/mmol) and '25I-labelled protein A were obtained from Amersham. Reagents for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and nitrocellulose filters were from Bio-Rad. The molecular mass markers used in SDS-PAGE were prestained myosin (200 kDa), phosphorylase b (92 kDa), bovine serum albumin (69 kDa), egg albumin (46 kDa), and carbonic anhydrase (30 kDa) from Bethesda Research Laboratories, Inc. Phosphotyrosine antibodies were raised against p-aminobenzene-phosphonate and affinity purified as previously described (9, 14, 36). Anti-Met antibodies were raised in rabbits immunized against the synthetic peptide VDTRPAS FWETS corresponding to the amino acid sequence at the C-terminal end of the predicted c-MET gene product as described previously (53). GTL-16 is a clonal cell line derived from a poorly differentiated gastric carcinoma line (18, 34). LoVo is a cell line established from a colon carcinoma, CALU-1 and A549 cell lines were derived from lung carcinomas, and MRC-5 cells are human embryonal fibroblasts of the lung. LoVo, CALU-1, A549, and MRC-5 lines were obtained from the American Type Culture Collection. Cells were grown in RPMI 1640 or in Ham's F-12 medium containing 10% fetal bovine serum and maintained at 37°C in a humidified atmosphere with 5% CO2. Western blotting. For Western blotting (immunoblotting) experiments, subconfluent cell monolayers were washed and solubilized in boiling Laemmli buffer (31) in the presence or in the absence of ,B-mercaptoethanol. Samples were adjusted to a protein concentration of 300 ,ug per well, subjected to SDS-PAGE, and transferred to nitrocellulose paper (BioRad) by the high-intensity wet-blotting technique, as described previously (5, 55). Blots were probed with 10 ,ug of affinity-purified anti-phosphotyrosine antibodies per ml and then with 1251I-labelled protein A. Filters were subjected to autoradiography for 24 h by using intensifying screens. Proteolytic digestion. Mild trypsin digestion was performed as follows. Cells were carefully washed with phosphatebuffered saline (PBS) and incubated in serum-free medium with increasing trypsin concentrations (from 10 to 100 jig/ ml). Digestion was carried out at 37°C for times between 10 and 20 min. The reaction was stopped by a 100-fold excess of soybean inhibitor. Metabolic labelling, cell surface iodination, and immunoprecipitation. Cells were treated with 3H-glucosamine (100 ,uCi/ml) for 18 h. For pulse-chase experiments, cells were incubated in methionine-free medium for 30 min and pulsed with 100 ,uCi of [35S]methionine per ml for 15 min. Cells were then washed twice with complete medium and chased for different times. Cell surface iodination was performed with the lactoperoxidase-H202-catalyzed radioiodination procedure as previously described (18). After being labelled, cells were extracted with ice-cold DIM (detergent insoluble matrix) buffer containing 10 mM PIPES [piperazine-N,N'-bis(2-ethanesulfonic acid)] (pH 6.8), 100 mM NaCl, 5 mM MgCl2, 300 mM sucrose, 5 mM

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,ug of bovine serum albumin per ml (Bethesda Research Laboratories), 200 ,uM deoxynucleotide triphosphate, 50 pmol of the 3' PCR primer, 50 pmol of the 5' PCR primer, and a variable concentration of MgC12. The final concentration of MgCl2 ranged between 1.5 and 6.5 mM and was selected by the testing of different concentrations for each pair of primers. Taq polymerase (5 U; Promega) was added, and 100 ,u1 of mineral oil was overlaid on the reaction mixture. Thirty cycles of denaturation, annealing, and extension were then performed with a Biostar Violet thermal cycler. Denaturation was at 92°C for 5 min for the first cycle and for 1 min for subsequent cycles. Annealing temperatures varied between 45 and 63°C according to the base composition of the primers; the annealing time was 1.5 min. The extension temperature was 72°C. The extension time was calculated with an assumed rate of extension of 1,000 bases per min, according to the predicted length of the amplified product. The products of the PCR reactions were run in 1% agarose ge's, amplified bands were excised, and DNA was recovered by using Qiagen (catalog no. 20020). The oligomers used for PCR amplifications were designed on the cDNA sequence of c-MET as follows: pair 1, sense oligomer corresponding to the nucleotides from -20 to 25 (5'-CCGAAAGATAAACCTCTCATAATGAA-3') and antisense oligomer corresponding to the nucleotides 1257 to 1278 (5'-CGCGCTGCAAAGCTGTGGTAA-3'); pair 2, sense oligomer corresponding to the nucleotides 894 to 921 (5'-ATTCTCACAGAAAAGAGAAAAAAGAG-3') and antisense oligomer corresponding to the nucleotides 2037 to 2061 (5'-TCTTAAGGGTGACAAATCCATTAAA-3'); pair 3, sense oligomer corresponding to the nucleotides 1818 to 1838 (5'-AATGAGAGCTGCACCTTGAC-3') and antisense oligomer corresponding to the nucleotides 2343 to 2364 (5'-CTTCCTATGACTTCATTGAA-3'). The oligomers used for PCR amplifications of the HGF sequence were the following: sense oligomer corresponding to the nucleotides 967 to 985 (5'-GAATTCCATGTCAGCGTTGG-3') and antisense oligomer corresponding to the nucleotides 1603 to 1625 (5'-TAGAAGTCTCGAGAAGGGAAACA-3'). Positive controls were mRNAs prepared from the HGF-producing MRC-5 cell line. Preparation and screening of the LoVo MET cDNA library. Poly(A) mRNA was purified from LoVo cells and used to construct a cDNA library. RT was started from two METspecific oligonucleotides. The first one (5'-GGGACCAAGC TTCTGGTTCTGATGC-3') matched a sequence in the 3'untranslated region of the 9-kb MET mRNA; the second one (5'-GGATCTTCACGGTAACTGAA-3') matched a sequence in the middle of the coding region. The doublestranded cDNA was prepared according to the Amersham cDNA Synthesis System (RPN 1256Y/Z) and cloned in Agtll vector as indicated by the Amersham cDNA Cloning System (RPN 1280). For the screening of the library, replica filters were prepared and hybridized according to Sambrook et al. (46), with a probe encompassing the entire MET-coding region. Positive clones were subcloned in the M13 Bluescript plasmid and sequenced. Nucleotide sequencing. Double-stranded DNA sequencing was carried out by the dideoxynucleotide method (46), with Sequenase (U.S. Biochemicals). The annealing mixture (final volume, 10 ,ul) contained 1 pmol of double-stranded DNA, 50 pmol of the oligomer used as a primer, 1 ,ul of dimethyl sulfoxide, and lx Sequenase annealing buffer. The mixture was boiled for 3 min and then quenched in liquid nitrogen. Labelling and termination reactions were

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FIG. 1. LoVo cells express a 190-kDa single-polypeptide chain reacting with anti-Met antibodies. Detergent-solubilized proteins were separated by SDS-PAGE under reducing (A) or nonreducing (B) conditions, transferred to nitrocellulose paper, and stained with specific antibodies directed against the C terminus of the Met ,B chain. Different lanes contain proteins solubilized from different cell lines. Under nonreducing conditions, l8 chains obtained from CALU-1 or GTL-16 cells shifted from 145 to 190 kDa because of their associations with the a chains.

done according to the Sequenase protocol. Both strands were sequenced. Transfection of MET cDNA in COS 7 cells. The expression vector used for these studies was based on the plasmid pMT2 containing the major late adenovirus promoter. The inserted 4.3-kb cDNA was obtained by ligation of the fragments (see Fig. 8D). The plasmid was transfected into COS 7 cells by the lipofection procedure. Three days after transfection, the expression and the processing of the Met protein were analyzed by Western blot. Nucleotide sequence accession number. The sequence number of the protein encoded by the MET gene refers to the cDNA sequence revised by Ponzetto et al. (43). The sequence has been filed with the EMBL Data Bank under code number X54559. RESULTS Detection of a single-chain 190-kDa protein by anti-Met antibodies in a colon carcinoma cell line. A Western blot screening of several human cell lines expressing the MET gene was performed by using antibodies directed against a synthetic peptide derived from the C-terminal sequence. In control cell lines (CALU-1 and GTL-16) under reducing conditions, the antibodies detected the mature Met 145-kDa p subunit (p1450) and a small amount of the 170-kDa Met precursor (prl70). In a colon carcinoma cell line (LoVo), the major immunologically reactive protein detected showed an abnormal Mr of 190 (p190NC) (Fig. 1A). The prl70 was barely detectable, because of its migration close to pl9ONC, and the mature p1450 was present only in trace amounts. The apparent Mr of p190NC did not change when electrophoresis was performed under nonreducing conditions, while in control cells, p145P shifted to the apparent Mr of 190 because of its association with the 50-kDa ot chain (Fig. 1B). P19ONC is

VOL . 1 l, 1991

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