The Tyrosine Kinase Encoded by the MET Proto-Oncogene Is ...

Vol. 11, No. 4

MOLECULAR AND CELLULAR BIOLOGY, Apr. 1991, p. 1793-1803 0270-7306/91/041793-11$02.00/0 Copyright © 1991, American Society for Microbiology

The Tyrosine Kinase Encoded by the MET Proto-Oncogene Is Activated by Autophosphorylation LUIGI NALDINI,* ELISA VIGNA, RICCARDO FERRACINI, PAOLA LONGATI, LUCIA GANDINO, MARIA PRAT, AND PAOLO M. COMOGLIO Department of Biomedical Sciences and Human Oncology, University of Turin Medical School, 10126 Turin, Italy Received 12 July 1990/Accepted 28 December 1990

Protein tyrosine kinases are crucially involved in the control of cell proliferation. Therefore, the regulation of their activity in both normal and neoplastic cells has been under intense scrutiny. The product of the MET oncogene is a transmembrane receptorlike tyrosine kinase with a unique disulfide-linked heterodimeric structure. Here we show that the tyrosine kinase activity of the MET-encoded protein is powerfully activated by tyrosine autophosphorylation. The enhancement of activity was quantitated with a phosphorylation assay of exogenous substrates. It involved an increase in the Vm. of the enzyme-catalyzed phosphotransfer reaction. No change was observed in the Km (substrate). A causal relationship between tyrosine autophosphorylation and activation of the kinase activity was proved by (i) the kinetic agreement between autophosphorylation and kinase activation, (ii) the overlapping dose-response relationship for ATP, (iii) the specificity for ATP of the activation process, (iv) the phosphorylation of tyrosine residues only, in the Met protein, in the activation step, (v) the linear dependence of the activation from the input of enzyme assayed, and (vi) the reversal of the active state by phosphatase treatment. Autophosphorylation occurred predominantly on a single tryptic peptide, most likely via an intermolecular reaction. The structural features responsible for this positive modulation of kinase activity were all contained in the 45-kDa intracellular moiety of the Met protein.

Tyrosine kinases are crucially involved in the transduction of growth-promoting stimuli to the cell interior. Transmembrane growth factor receptors such as the epidermal and platelet-derived growth factor, insulin, and colony-stimulating factor 1 receptors, are endowed with ligand-induced tyrosine kinase activity. Upon ligand binding, they display a short-lived pulse of activity leading to their autophosphorylation and to the phosphorylation of cellular substrates on tyrosine residues (for a review, see references 52 and 56). Membrane-associated tyrosine kinases of the src family are thought to be similarly involved in signal transduction operated by a distinct set of receptors. The most abundant class of oncogenes comprises the genes coding for both types of tyrosine kinases. Whatever the mechanism, activation of their transforming potential leads to the expression of a protein with nonregulated, often enhanced enzymatic activity (reviewed in references 28, 32, and 33). The enzymatic activity of tyrosine kinases can be modulated in several ways (reviewed in references 33, 52, and 56). Ligands are well-known activators of the receptors. Structural alterations such as N- and C-terminal truncation and point mutations are critical for the transforming proteins. In addition, phosphorylation is a ubiquitous way of transiently modulating tyrosine kinase activity. Tyrosine autophosphorylation has often been associated with activation. Protein kinase C-mediated serine or threonine phosphorylation has been shown to be inhibitory for some growth factor recep-

tyrosine autophosphorylation has been shown for the v-fps oncogene product (54) and the insulin receptor (35, 45, 57, 58). A moderate activation by autophosphorylation was also reported for the epidermal growth factor (EGF) receptor (2, 3, 31). Negative modulation of activity by protein kinase C-dependent phosphorylation has been shown for the EGF, insulin, and insulinlike growth factor-1 (IGF-1) receptors (reviewed in reference 52). Previous work by others and in this laboratory showed that the product of the MET oncogene is a transmembrane receptorlike tyrosine kinase (6, 11, 43). Its disulfide-linked heterodimeric structure with a 50-kDa a chain and a 145-kDa P chain is novel for a putative growth factor receptor (22, 24, 49). A N-terminal truncated version of the gene was originally identified as the transforming gene in a carcinogentreated human osteosarcoma cell line (8, 42). The transforming protein displayed unregulated tyrosine kinase activity (26, 48). In a human gastric carcinoma cell line, the MET locus was found to be amplified and overexpressed. The normal-sized gene product accumulated in the cells and exhibited abnormally high tyrosine kinase activity, possibly from autocrine stimulation (21, 23, 24). Negative modulation of its phosphotyrosine content was exerted by protein kinase C activation (20). Here we show that the kinase activity of the MET-encoded protein is powerfully activated by tyrosine autophosphorylation on a single tryptic peptide. The enhancement of activity was observed for both autophosphorylation and phosphorylation of exogenous substrates. The effect of tyrosine autophosphorylation was to increase the V1max of the enzyme-catalyzed phosphotransfer reaction without changing the Km (substrate). Furthermore, the structural features responsible for kinase activation were all contained in a catalytically active 45-kDa fragment from the intracellular moiety of the Met protein ,B subunit. Self-mediated enhancement of tyrosine kinase activity

tors.

However, a clear account of the influence of the phosphorylation status on enzymatic activity has only been provided for a few tyrosine kinases. Different tyrosine residues have been involved in positive or negative modulation of the activity of the src gene product (11, 27). Activation by *

Corresponding author. 1793

1794

NALDINI ET AL.

may contribute to the transforming potential of the MET oncogene.

MATERIALS AND METHODS Reagents and cells. All reagents used were of analytical grade. Protease inhibitors, phosphoamino acid markers, myelin basic protein, and [Val5] angiotensin II were purchased from Sigma. 3-[(3-Cholamidopropyl)dimethylammonio-1-propanesulfonate] (CHAPS) and phospholipid preparations were from Fluka. Staphylococcus aureus protein A covalently coupled to Sepharose was purchased from Pharmacia. [_y-32P]ATP (specific activity, 7,000 Ci/mmol) and 125I-protein A were obtained from Amersham. Reagents for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and nitrocellulose filters were from Bio-Rad. High-pressure liquid chromatography (HPLC)-grade solvents were from Baker. 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) (Bethesda Research Laboratories). Phosphotyrosine antibodies were raised against p-aminobenzenephosphonate and affinity purified as described previously (7, 12, 19, 41). Anti-Met antibodies were raised in rabbits immunized against the synthetic peptide VDTRPA SFWETS, corresponding to the amino acid sequence at the C-terminal end of the predicted MET gene product as described before (48) and kindly provided by M. F. Di Renzo. Monoclonal anti-Met antibodies were used as the culture supernatant of a hybridoma screened from fusions of the spleen cells of mice immunized with GTL-16 cells (lOa). The GTL-16 cell line is a clonal cell line derived from a poorly differentiated gastric carcinoma cell line (40). Other cell lines were CALU-1 and A-549 lung carcinoma cells, HT-29 colon carcinoma cells, and KB oral carcinoma cells, all obtained from the American Type Culture Collection. Cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum and maintained at 37°C in a humidified atmosphere with 5% CO2. Immunoprecipitation and kinase assays. Subconfluent cell cultures were placed on ice and washed twice with cold phosphate-buffered saline (PBS). Cells were scraped with a rubber policeman into ice-cold PBS containing 1 mM EDTA and 1 mM phenylmethylsulfonyl fluoride (PMSF), pelleted at low speed at 4°C, and extracted with 1% CHAPS in HEPS buffer (25 mM HEPES [N-2-hydroxyethylpiperazine-N'-2ethanesulfonic acid]-NaOH [pH 7.4], 5 mM MgCl2, 1 mM EGTA [ethylene glycol tetraacetic acid], 100 mM NaCl, 10% [vol/vol] glycerol, and a cocktail of protease inhibitors including 1 mM PMSF, leupeptin [50 jig/ml], soybean trypsin inhibitor [100 ,ug/ml], aprotinin [10 ,ug/ml], and pepstatin [10 ,ug/ml]) for 20 min at 4°C with stirring. The cell lysates were cleared by centrifugation at 15,000 x g for 20 min at 4°C. Sepharose-protein A (30 ,ul of packed beads per 100 p.l of lysate) was preincubated with anti-Met peptide antiserum (3 ,ul/100 ,u1 of lysate), washed twice with buffer, and incubated with the lysates for 3 h at 4°C with stirring. When monoclonal antibodies were used, an additional incubation with affinity-purified goat anti-mouse immunoglobulin (Miles) was included. Bound proteins were washed three times with HEPS buffer and twice with kinase buffer (KB; 20 mM HEPES-NaOH [pH 7.1], 5 mM MgCl2, 100 mM NaCl) supplemented with 50 ,ug of egg yolk phosphatidylcholine at 40C. For preactivation experiments, the concentration of ATP

MOL. CELL. BIOL.

indicated in the figure legends was added to the buffer. When saturation was required, 40 ,uM ATP was used. The immunoprecipitates were then washed once with excess KB buffer and exposed to the concentration of [y-32P]ATP indicated in the figure legends in KB buffer. Usually 10 ,uCi of [_y-32P]ATP (specific activity, 7,000 Ci/mmol) per sample was diluted with the required concentration of unlabeled ATP. When a maximal rate of reaction was required, 20 p.M ATP was used for autophosphorylation experiments and 100 p.M ATP was used for peptide phosphorylation. For doseresponse experiments, the different concentrations of ATP were prepared by serial dilution, so the specific activity did not change. When substrate phosphorylation was assayed, the concentration of [Val5] angiotensin II or myelin basic protein indicated in the figure legends was added to the reaction buffer. The standard reaction time was 3 min for autophosphorylation and 15 min for substrate phosphorylation at 4°C, with continuous stirring. For the autophosphorylation and myelin basic protein phosphorylation assays, the reaction was stopped by adding concentrated boiling Laemmli buffer (37). The eluted proteins were subjected to 8% SDS-PAGE (autophosphorylation) or 5 to 15% polyacrylamide gradient SDS-PAGE (myelin basic protein phosphorylation), followed by autoradiography for 3 h at -70°C with intensifying screens and preflashed films. Phosphate incorporation was estimated by densitometric scanning of the film with an LKB 2202 Ultroscan laser densitometer and/or by scintillation counting of the excised labeled bands. For the peptide phosphorylation assay, the reactions were stopped by adding EDTA to a final concentration of 10 mM. The reaction supernatant was collected and cleared by precipitation with 5% trichloroacetic acid for 1 h at 4°C. Equal volumes were then spotted onto 3.5-cm2 phosphocellulose paper (P81 ion-exchange chromatography paper from Whatman), washed once in 30% acetic acid for 15 min, twice in 15% acetic acid for 15 min, and once in acetone for 5 min, air dried, and counted in a Packard scintillation counter with scintillation fluid for 32p, as described before (25). To assess dephosphorylation, samples of Met immunoprecipitate were incubated with [_y-32P]ATP and [Val5] angiotensin II in reaction buffer as described above. The reaction was stopped by adding EDTA to a final concentration of 10 mM, and the immunoprecipitates were further incubated in the same buffer at 4°C. At different times, samples were taken, and the phosphorylation level was evaluated for both the Met protein and the peptide substrate. Western immunoblotting. For blotting experiments, immunoprecipitates prepared as described above were eluted with Laemmli buffer, subjected to SDS-PAGE, and transferred to nitrocellulose paper (Bio-Rad) by high-intensity wet blotting as described before (4, 51). Blots were probed with 10 ,ug of purified antiphosphotyrosine or anti-Met antiserum per ml, followed by 251I-labeled protein A. Filters were subjected to autoradiography overnight at -70°C with intensifying screens. FPLC molecular sieving chromatography. For fast protein liquid chromatography (FPLC), cell lysate was prepared as above in HEPS buffer, cleared by ultracentrifugation at 150,000 x g for 45 min at 4°C, loaded on a column of Superose 6 (Pharmacia) equilibrated in HEPS buffer containing 0.1% CHAPS and 5% glycerol, and resolved at a flow rate of 0.33 ml/min at 4°C. Fractions (0.5 ml) were collected. Void volume corresponded to fraction 5. The peak elution of molecular size markers was as follows: thyroglobulin (669 kDa), fraction 12; ,-amylase (200 kDa), fraction 15; and

VOL . 1

l,

1991

Met TYROSINE KINASE ACTIVATED BY AUTOPHOSPHORYLATION

bovine serum albumin (66 kDa), fraction 18. The bulk of the extracted proteins were in fractions 17 to 21. Phosphoamino acid analysis and phosphopeptide tryptic mapping. For phosphoamino acid analysis, 32P-labeled proteins were subjected to 8% SDS-PAGE, electrotransferred to hydrophobic polyvinylidene difluoride (PVDF; Immobilon; Millipore), and exposed for autoradiography. The labeled p145 bands were excised and hydrolysed in 6 N HCl (constant boiling; Pierce) at 110°C for 1 h. Collected hydrolysates were washed in water, lyophilized, and resuspended in 5% acetic acid-0.5% pirydin-5 mM EDTA, pH 3.5 (running buffer) (9). Unlabeled phosphoserine, phosphothreonine, and phosphotyrosine (1 mg/ml each) were included as external standards. Samples were run on a silica gel plate (Merck) for 90 min at 1,500 V in running buffer with cooling. The plate was then dried in a fume hood, and phosphoamino acid standards were localized by spraying the plate with 0.25% ninhydrin in acetone. The dried plate was then subjected to autoradiography at -70°C with an intensifying screen for 24 h. For phosphopeptide mapping, immunoprecipitates of Met proteins were labeled with 500,uCi of [y-32P]ATP at 10,uM. Labeled bands were excised from the polyacrylamide gel, washed twice with 10% methanol, minced, and dried in a lyophilizer. The gel pieces were rehydrated with 50 mM NH4CO3 (pH 7.8) containing 50,ug of trypsin treated with L- (tosylamido-2-phenyl)ethyl chloromethyl ketone (Worthington) and incubated for 2 h at 37°C. The trypsin digestion was repeated once, and the gel pieces were further eluted with 50 mM NH4CO3 (pH 7.8) (31). Pooled eluates were lyophilized in a Savant vacuum dessicator, redissolved in 70% formic acid containing 100 mg of cyanogen bromide per ml, and incubated for 1 h at room temperature. The reaction was stopped by diluting with H20 and three cycles of

lyophilization. The digest

was

resuspended in 50 mM

NH4CO3 (pH 7.8) containing 10 p.g of trypsin-TPCK (Worthington) and further digested for 2 h at 37°C. The final peptide mixture was lyophilized and resuspended in HPLC buffer A (0.1% trifluoroacetic acid in H20). The phosphopeptides were analyzed on a reverse-phase C2-C18 Superpack Pep-S column (Pharmacia-LKB) resolved with a gradient of acetonitrile in buffer A of 0.5%/min at a flow rate of 1 ml/min. The eluted radioactivity was monitored on line with a Radiomatic A-100 radioactive flow detector. RESULTS MET-encoded tyrosine kinase displays an ATP-dependent activation step. The product of the MET gene was immunoprecipitated from several human cell lines with an antiserum directed against a synthetic peptide derived from the C-terminal sequence of the protein. A kinase assay was performed on the immunoprecipitate with [_y-32P]ATP and MgCl2. As shown previously, the major protein labeled in the immunoprecipitate was the product of the MET gene. The prominent 32P-labeled band of 145 kDa corresponded to the reduced 3-chain of the protein phosphorylated on tyrosine, most likely via autophosphorylation (24) (Fig.1A). The kinetics of phosphorylation of the Met protein and of the peptide substrate [Val5] angiotensinII were compared at 4°C for the protein immunoprecipitated from GTL-16 cell extracts. The phosphorylation of the Met protein increased with first-order kinetics, reaching a plateau after approxi4°C (Fig.1B). The kinetics of substrate mately 20 min at phosphorylation displayed a biphasic curve. An initial, linear region with a slower rate of product accumulation gradually

1795

A '160 0

-

12v

do

_~~~~~~~~~~~~~~~~~r CL

c

4: 6.

3 -0 2

0'

t:me~ min..,n ,:rze. ~

CL

C

0

20

3C

time, min

FIG. 1. Kinetics of autophosphorylation of the Met p145 1 subunit and of phosphorylation of the peptide substrate [Val5] angiotensin II. The reactions were performed on immunoprecipitates made with anti-Met peptide antiserum from GTL-16 cell extracts with 40F.M [-y-32P]ATP and 3 mM [Val'] angiotensin 11 (C) as described in the Materials and Methods section. Autophosphorylation followed first-order kinetics. Substrate phosphorylation was biphasic, with ATP acting both as substrate and as activator of the kinase. In panel B the optical density, in arbitrary units (A.U.), of the p145 band from the autoradiogram shown in panel A is plotted against the time of reaction. In panel C the accumulation of the phosphorylated peptide substrate [Val5] angiotensin II is shown for the untreated (solid circles) and ATP-preincubated (open circles) samples. A representative experiment out of three performed is shown. The migration of molecular mass standards (in kilodaltons) and the position of the p145 Met 1 subunit are shown in panel A.

turned-between 10 and 20 min-in a steeper region with a

much faster rate (Fig.1C). To test whether the acceleration in the accumulation of product reflected an ATP-dependent enzyme activation step, the immunoprecipitate of the Met protein was preincubated in the presence or absence of excess unlabeled ATP, washed adequately, and assayed again for substrate phosphorylation with [.y-32P]ATP. The amount of peptide phosphorylated by the ATP-incubated sample increased linearly throughout the assay. The accumulation rate was as fast as that observed in the steeper portion of the kinetic curve of the untreated sample (Fig.1C, open circles). Thus, ATP could function both as a substrate and as an activator of the enzyme. Identical results were obtained with Met proteins immunoprecipitated from different cell lines, including CALU-1 and A-549 lung carcinoma cells, HT-29 colon carcinoma cells, and KB oral carcinoma cells (not shown). Met protein autophosphorylates on tyrosine residues during the activation step. To investigate the nature of the activation step, samples of Met immunoprecipitate were incubated as above with or without ATP and then subjected to Western blotting analysis with either anti-Met antiserum or purified antiphosphotyrosine antibodies. While the amount of Met protein in the immunoprecipitate was virtually the same, its phosphotyrosine content was remarkably different. No phosphotyrosine was detectable in the control samples,

1796

NALDINI ET AL. A

MOL. CELL. BIOL. B

A

C

FIG. 2. Kinase activation correlates with the phosphotyrosine content of the immunoprecipitated Met protein. Immunoprecipitation was done either with monoclonal antibodies against the extracellular domain of the Met protein (lanes 1 and 2) or with anti-Cterminal Met peptide antiserum (lanes 3 and 4) from GTL-16 cell extracts. Parallel samples were preincubated either with 40 ,uM ATP for 30 min at 4°C (lanes 2 and 4) or with buffer only (lanes 1 and 3) and treated as follows. (A and B) The immunoprecipitates were eluted with Laemmli buffer, subjected to SDS-PAGE, transferred to nitrocellulose, and probed either with anti-Met antiserum (A) or with purified antiphosphotyrosine antibodies (B). (C) A peptide phosphorylation assay was performed on the immunoprecipitates with 40 pLM [_y-32P]ATP and 1.5 mM [Val5] angiotensin II as described in the Materials and Methods section. The migration of molecular mass standards (in kilodaltons) and the position of the p145 Met p subunit are shown.

while the phosphotyrosine antibodies strongly labeled the ATP-treated samples (Fig. 2). In order to set the zero the assay of kinase activity with respect to the phosphorylation status of the protein, no phosphotyrosyl-phosphatase inhibitors were included in the cell lysis buffer. Proteins were thus exposed to phosphatase action during immunoprecipitation, and even though the phosphotyrosine content of the Met proteins in intact cells varied among the cell lines tested, none of it remained detectable after immunoprecipitation. Met proteins immunoprecipitated from different cell sources displayed comparable basal activity and extent of activation with ATP. We then attempted to establish whether the tyrosine phosphorylation of the Met protein occurred via autophosphorylation. Nonspecific association of a different kinase to the immunoprecipitated Met had to be ruled out. We first analyzed immunoprecipitates obtained with different antisera and with a panel of monoclonal antibodies directed against the extracellular domain of the Met I subunit. The phosphorylation and the extent of activation of the kinase by ATP did not differ with the various antibodies (Fig. 2). Then, an aliquot of the cell extract was size-fractionated by molecular sieving chromatography on a Superose 6 FPLC column equilibrated in lysis buffer. The elution volume of the Met protein was determined by probing aliquots from the collected fractions with anti-Met antiserum in a Western blot. It corresponded to a high molecular weight (approximately 600,000), as expected for a detergent-solubilized transmembrane molecule possibly existing in oligomers (Fig. 3A). This early elution of the Met kinase provided substantial purification from the bulk of the contaminating cellular proteins. From these high-molecular-weight fractions, the protein was immunoprecipitated and assayed for ATP-activated kinase activity. Again, no difference in phosphorylation and activation behavior was found from the immunoprecipitate made from unfractionated cell extracts. A 45-kDa proteolytic fragment of the Met protein, recognized by the anti-Met antiserum and endowed with kinase activity (see below),

B

C

FIG. 3. Autophosphorylation and activation of kinase activity are displayed by the partially purified Met protein and by a 45-kDa fragment from its intracellular moiety. (A) GTL-16 cell extract (0.5 ml) was fractionated by FPLC molecular sieving chromatography on a Superose 6 column as described in the Materials and Methods section. Aliquots from the collected fractions were subjected to SDS-PAGE and probed in Western blots with anti-C-terminal Met peptide antiserum. Fraction numbers are indicated above the lanes. The migration of molecular mass standards (in kilodaltons) and the position of the p145 Met P subunit are shown on the left. The bulk of the extracted proteins was in fractions 17 to 21. A few proteins cross-reacting with the antipeptide antiserum were also detected. The catalytically active 45-kDa Met fragment elutes in fraction 20 and migrates as multiple bands due to differences in phosphorylation content. Immunoprecipitation with anti-C-terminal Met peptide antiserum was performed from fractions 10 (lanes 1 and 2) and 20 (lanes 3 and 4). The immunoprecipitates were split into equal samples, which were preincubated either with 40 ,uM ATP for 30 min at 4°C (lanes 2 and 4) or with buffer only (lanes 1 and 3) and treated as follows. (B) Autophosphorylation was assayed. (C) Substrate phosphorylation was assayed with 20 ,uM [_y-32P]ATP and 1.5 mM [Val5] angiotensin II, as described in the Materials and Methods

section.

was not responsible for the phosphorylation of the intact Met protein. In fact, this polypeptide and the full-length Met protein eluted with different volumes and could be assayed separately in different fractions (Fig. 3B and C). Hence, either there is a specific association of a catalytic amount of a distinct tyrosine kinase to the Met protein or the activity responsible for the tyrosine phosphorylation of the Met protein must reside in its own molecule. As shown in Fig. 7, the basal enzyme activity increased linearly with the amount of immunoprecipitate assayed. However, if the concentration of enzyme in the immunoprecipitate was changed together with the amount of enzyme assayed (i.e., immunoprecipitating increasing volume of

VOL . 1l,

1991


extract with the same amount of immobilized antibodies), an exponential increase in enzyme activity was noted (data not shown). This suggests an intermolecular mechanism of au-

tophosphorylation for the kinase (46). Considering the immobilized status of the enzyme in the assay and its likely occurrence as oligomer-as suggested by the elution volume of the soluble Met molecules in molecular sieving chromatography-oligomerization may be a prerequisite for activation. On the other hand, ATP activation was also displayed by the 45-kDa intracellular fragment of the Met protein, which eluted as a monomer in molecular sieving chromatography. Tyrosine autophosphorylation of the Met protein is responsible for kinase activation. [y-32P]ATP was used for the kinase activation step to perform phosphoamino acid analysis on the 32P-labeled Met ,B subunit. Phosphotyrosine was the only labeled amino acid (Fig. 4A). Thus, all the detectable phosphate transfer occurring onto the Met protein in the activation step was transferred to tyrosine residues. This almost ruled out the unlikely contribution of a distinct serine or threonine kinase to the activation of the Met tyrosine kinase. Phosphopeptide mapping by reverse-phase HPLC was also performed on the isolated 32P-labeled 145-kDa band from Met immunoprecipitates made with anti-N-terminus and anti-C-terminus antibodies. Sequential digestions with trypsin, cyanogen bromide, and trypsin again were used to ensure complete proteolysis. The same major phosphopeptide was resolved above a number of minor labeled species on both chromatograms. The major peak represented a single phosphopeptide, as supported by further characterization with other techniques. The minor peaks could be ascribed to low-stoichiometry phosphorylation and/or partial digestion of different sites (17a). No significant differences were observed with the two kinds of antibodies (Fig. 4B and C). We next attempted to derive a causal relationship between the tyrosine autophosphorylation of the Met protein and the activation of its kinase activity. There was good agreement between autophosphorylation kinetics and the time required for maximal kinase activation at a given concentration of ATP (see above). The dependence on ATP concentration were then compared for MET tyrosine autophosphorylation and kinase activation. To measure the dependence of the autophosphorylation on ATP concentration, a short incubation time of 3 min at 4°C with [-y-32P]ATP was chosen. Phosphate incorporation then represented the initial rate of reaction and could be plotted against ATP concentration according to Michaelis-Menten kinetics. The Km [ATP] for autophosphorylation was found to be about 4 ,uM at 4°C (Fig. 5, solid squares. A parallel set of samples were incubated with the same concentrations of unlabeled ATP as above for 3 min. Kinase activity was then assayed as usual with [_y-32P]ATP and [Val5] angiotensin II. Half-maximal activation was obtained with an ATP concentration significantly similar to that at which half-maximal autophosphorylation occurred (Fig. 5, open circles). The dose-response for ATP of both processes could be described by the same mathematical relationship. Furthermore, ATP was the only nucleotide effective for the activation. Preincubation of the Met immunoprecipitate with the nonhydrolyzable ATP analog 5'-adenylylimidodiphosphate or with GTP did not affect the enzyme, which displayed only basal tyrosine kinase activity (Fig. 6). The dependence of the extent of activation on the amount of enzyme input was also investigated. A linear dependence

1797

of the ATP-activated enzymatic activity from the amount of Met immunoprecipitate assayed was observed (Fig. 7). This renders it unlikely that an ATP-driven intermolecular reorganization, i.e., oligomerization, is the mechanism of activation, also given the immobilized status of the enzyme in the assay. Furthermore, the active state of the Met tyrosine kinase could be reversed by alkaline phosphatase treatment. Incubating the immunoprecipitates with alkaline phosphatase removed the ATP-induced tyrosine phosphorylation of the Met protein and returned its tyrosine kinase activity to the basal level (Fig. 8). To establish autophosphorylation as an independent mechanism of activation of the Met kinase, we had to rule out the contribution of the yet unknown Met ligand. A ligand could remain bound to the Met protein during its purification, although it would be expressed in all cell lines tested regardless of the intracellular phosphorylation status of the Met kinase. Monolayers of GTL-16 and CALU-1 cells were subjected to an acid wash (pH 4.0) prior to cell lysis to strip any potentially bound ligand (23). In other experiments, Met immunoprecipitates were washed with a milder acidic buffer (pH 5.5) prior to the kinase assay. In both cases, no difference was found in the extent of Met kinase activation by ATP from untreated control samples (data not shown). Tyrosine autophosphorylation of the Met kinase increases the V.. of substrate phosphorylation without changing the Km [substrate]. The effects of ATP activation on the kinetics of substrate phosphorylation by the Met tyrosine kinase were then analyzed. Experimental conditions were chosen to ensure steady-state phosphorylation. As shown in Fig. 1, the phosphorylation of a peptide substrate by the control and ATP-activated immunoprecipitate remained linear for up to 30 min at 4°C. The slower slope of the reaction for the untreated sample was maintained until approximately 15 min. We chose this time of reaction as representative of the two enzyme states. Serial dilution of the Met immunoprecipitate indicated that the assay conditions fell in the range of linear dependence of the phosphorylation rate from the enzyme input (Fig. 7). The dephosphorylation of both the 32P-labeled peptide substrate and the Met protein in the immunoprecipitate was also examined. No dephosphorylation of either molecule was apparent over the time of the assay (data not shown; see Materials and Methods section). Thus, the phosphorylation level of the peptide substrate represented real increments of phosphate incorporation under steady-state conditions rather than increased phosphate turnover. The kinetic parameters of the phosphorylation of the peptide substrate [Val5] angiotensin II by the Met protein were varied by activation of the enzyme as follows. There was a severalfold increase in the Vm. of the phosphorylation reaction. Because the absolute content of Met protein in the immunoprecipitate could not be estimated, the enzyme activity is expressed in relative terms. The Km [ATP] was not affected by the activation remaining at the value of 36 ,uM for both samples (Fig. 9A). The Km [peptide] displayed a slight increase with activation, from 1.4 mM [Val5] angiotensin II for the untreated sample to 2.5 mM for the activated one (Fig. 9B). With 40 ,uM ATP and 1.5 mM peptide, there was an approximately fivefold increase in the Vmax of the reaction. The low affinity of [Val5] angiotensin II for the Met kinase prompted us to examine the effects of ATP activation on the enzyme kinetics with another substrate having a lower Km

1798

NALDINI ET AL.

MOL. CELL. BIOL.

0)

--------

a{en.

m 1

'N

-0

Hoc

4

)

.4

C' ..4

-02

I

hOT

.4

I

.4 .4

.

S

.

t

c.: .!rS

e

Nw

-0 4


VOL . 1 l, 1991 100

1

W

-v

0

80

0.8

1799

o a,

E 4.0 .56 0 E 3.2 CL

-

SW.

60 .

0

8-

0 a

.-r *;

2.4

o

0

.Z-

c

40

0

0 CL

20

0.24 0

3 30

0

0

P

5

0

10

15

20

25

ATP concentration, pM FIG. 5. Dependence on ATP concentration is the same for autophosphorylation and kinase activation of the Met protein. Immunoprecipitates of the Met protein were prepared as above from GTL-16 cells. For measuring autophosphorylation, the immunoprecipitates were exposed to the indicated concentrations of [y-32P] ATP for 3 min at 4°C, eluted with Laemmli buffer, and subjected to SDS-PAGE and autoradiography. The optical density of the p145 band is plotted in arbitrary units (A.U.) (solid squares). For measuring kinase activation, a parallel set of samples were exposed to the indicated concentrations of unlabeled ATP for 3 min at 4°C as above, washed with excess buffer, and assayed for kinase activity with 3 mM [Val5] angiotensin II and 40 ,uM [y-32P]ATP for 15 min at 40C, as described in the Materials and Methods section. Plotted values are the means of duplicate determinations from one of two similar experiments.

c

E 0

E

0.60

08A

csa

0.36

c00 a

[

024 , 0.12

WI 0o00

.

I1 IFFI 0.

IL

0. ,

0.8

_.

S'

IL

z

FIG. 6. Specificity of the activation of the Met tyrosine kinase by ATP. Preincubation of Met immunoprecipitates with a 100 ,uM concentration of either GTP or the nonhydrolyzable ATP analog

5'-adenylylimidodiphosphate (AMP-PNP) did not affect kinase activity over the control level (-). Only ATP induced activation of the kinase. Kinase activity was measured by the phosphorylation of 1.5 mM [Val5] angiotensin II with 10 _FMy-32P]ATP as described in the Materials and Methods section. Plotted values are from a representative experiment.

0.0

0 7.5

15.0

30.0

45.0

60

MET immunoprecipitate, pi FIG. 7. Dependence of Met kinase activity and its extent of activation by ATP on the amount of enzyme assayed. The phosphorylation rate for both the untreated and the activated samples increased linearly with the amount of Met immunoprecipitate input. An immunoprecipitate of the Met protein was prepared as above from GTL-16 cells and serially diluted with kinase buffer. Parallel samples were incubated either with 40 pLM ATP (open circles) or with buffer only (solid circles) for 30 min at 4°C and assayed for phosphorylation of 2 mM [Val5] angiotensin II with 100 ,M [.y-32P]ATP as described in the Materials and Methods section. Plotted values are the means of duplicate determinations from a representative experiment out of three performed.

for phosphorylation. We then assayed ATP-treated and control samples of Met immunoprecipitate with myelin basic protein, a phosphorylation substrate with a Km of approximately 4 ,uM. Again there was an obvious increase in the Vmax of the reaction without significant change in the Km for the substrate (Fig. 10). Phosphoamino acid analysis of the 32P-labeled myelin basic protein showed that all label incorporation occurred onto tyrosine residues (not shown). The ratio between the active and control enzyme activity was affected by the concentrations of ATP and substrate used to assay the enzyme. The ATP was both a substrate and an activator for the kinase, and the peptide/protein substrate inhibited enzyme autophosphorylation (data not shown), a well-known effect for the EGF receptor (31, 53) and insulin receptor (39). Thus, the activity displayed by the control sample reflected the phosphorylation level reached by the control enzyme during the time of the assay. The effects of ATP activation were also examined for the autophosphorylation of the Met kinase. The autophosphorylation rate was faster for the ATP-activated enzyme, although it reached a lower plateau, most likely as a consequence of the previous occupancy of phosphorylation sites in the activation step (not shown). In this case, however, no accurate assay could be developed because of the low concentration of phosphorylation sites available, similar to that of the enzyme, and their partial occupancy after preactivation. Activation by ATP is manifest in a 45-kDa catalytically active fragment from the ,-chain of the Met protein. A polypeptide of 45 kDa recognized by the anti-C-terminus Met antiserum was highly phosphorylated in the immunoprecipitate kinase assay. It could be separately assayed for kinase activity in the low-molecular-weight chromatographic fractions shown in Fig. 3. This polypeptide most likely originated by postlytic proteolysis of the intact Met protein, presumably at a stretch of four basic amino acid immediately next to the putative transmembrane domain. In fact, the

1800

NALDINI ET AL.

MOL. CELL. BIOL.

E h,

A

I

E E 0

200

ia

-

p,45

--

CI

97

-

68

-

a

11

"

-,

::>

-M

-

-

+

+

t

AkFh Y

-

vC4

-f

-

AH : ASkh TP

+

AIkYl.

-

t

_ _

-+

FIG. 8. Alkaline phosphatase (Alk.Ph.) treatment reverses the activation of the Met tyrosine kinase induced by autophosphorylation. Met proteins were immunoprecipitated with anti-C-terminal Met peptide antiserum from GTL-16 cell extracts. Parallel samples were preincubated either with 40 ,uM ATP for 30 min at 4°C (ATP +) or with buffer only (ATP -), washed, and further incubated in 100 mM Tris-HCl (pH 8.0)-10 mM MgCl2-0.1 mM ZnCl2-100 mM NaCI-0.2 mg of bovine serum albumin per ml, with or without 20 U of calf intestinal alkaline phosphatase (Promega) per ml for 1 h at 15°C, in the presence or absence of 1 mM sodium orthovanadate (VO43-). The samples were then washed with excess kinase buffer. (A) The immunoprecipitates were eluted with Laemmli buffer, subjected to SDS-PAGE, transferred to nitrocellulose, and probed with purified antiphosphotyrosine antibodies. (B) A peptide phosphorylation assay was performed on the immunoprecipitates with 40 ,uM [-y-32P]ATP and 2 mM [Val5] angiotensin II as described in the Materials and Methods section. The migration of molecular mass standards (in kilodaltons) and the position of the p145 Met subunit are shown.

45-kDa polypeptide could not be detected in extracts of cells directly lysed in boiling SDS. Its amount in nonionic detergent extracts was influenced by the concentration and type of protease inhibitors added to the buffer. Furthermore, a 32P-labeled 45-kDa fragment could be generated from intact Met protein by mild trypsin treatment of immunoprecipitates, as described for the EGF receptor by Basu et al. (1) (data not shown). Phosphopeptide mapping of the 45-kDa fragment gave the same elution profile as the intact 145-kDa Met protein (data not shown). The 45-kDa polypeptide exhibited an ATP-activated tyrosine kinase activity similar to that of the intact Met protein (Fig. 3C). Furthermore, it contained the sites of tyrosine phosphorylation used "in vitro" by the Met tyrosine kinase. Hence, whatever the mechanism of activation of the Met kinase by autophosphorylation may be, it appears to be contained entirely in the 45-kDa intracellular domain of the protein. DISCUSSION Autophosphorylation on tyrosine residues is the bestdocumented result of the activation of a tyrosine kinase (reviewed in references 28, 32, 33, 52, and 56). It may affect the interaction of the molecule with other components (effectors or regulators) and/or direct it to a particular intracellular routing. Furthermore, it may have important functional consequences on the enzymatic activity of the molecule. Here we showed that the tyrosine kinase activity of the MET-encoded protein is powerfully activated by tyrosine autophosphorylation. As no ligand was present, the net effect of autophosphorylation on enzyme activity was observed. The increase in phosphorylation rate was due to a severalfold increase in the Vmax of the enzyme-catalyzed phosphotransfer reaction. The Km for the substrates was not

affected. The calculated enzymatic parameters for the Met tyrosine kinase were similar to those reported for other tyrosine kinases. By analogy with the EGF receptor kinase, a 10-fold difference was observed between the Km [ATP] for autophosphorylation and substrate phosphorylation (3, 53). This could reflect a higher affinity of the enzyme for the endogenous phosphorylation sites than for the exogenous substrates and an ordered bi-bi mechanism for the reaction, with substrate binding preceding ATP binding. A causal relationship between tyrosine autophosphorylation and activation of the kinase activity was established as follows. (i) There was a good kinetic agreement between the two processes. (ii) The dose-response relationships for ATP of autophosphorylation and kinase activation were superimposable. (iii) The activation process was specific for ATP, and a nonhydrolyzable ATP analog was ineffective. (iv) The only amino acid residue of the Met protein phosphorylated to a detectable level during the activation step was tyrosine. (v) The dependence of the extent of activation from the input of enzyme was linear. (vi) The activation was reversed by treatment of the immunoprecipitates with alkaline phosphatase. A 45-kDa fragment corresponding to the intracellular moiety of the Met protein contained all the structural features responsible for this positive modulation of the kinase activity. The generation of a catalytically active fragment by trypsin treatment was previously reported for the EGF and insulin receptors (1, 47). Partial proteolysis of a receptor tyrosine kinase may thus release the catalytic domain from an inhibitory constraint exerted by the extracellular regulatory domain. This would lead to the expression of an enhanced, ligand-independent enzymatic activity. Modulation of the activity of a tyrosine kinase by its phosphorylation status has been well substantiated for the following molecules. In the pp6O product of the src gene, different tyrosine residues are clearly involved in both


VOL . 1 l, 1991 c

16

E

E

0.35

E

0.28

1801

0

0

ECL

12

CL o

._0

8

0.21

0.

00.

,

._

0.14

c a

4 *0.07

0 0

25

c

100 ATP concentration, pM 50

75

125

6

12

18

24

30

Myslin Basic Protein, pM

25 ._

20

0

0

15 .a c

0

0

FIG. 10. Effect of ATP activation on the phosphorylation kinetics of the protein substrate myelin basic protein by the Met tyrosine kinase. The Vmax of the reaction is increased approximately threefold while the Km for the protein substrate is unchanged. Immunoprecipitates of the Met protein were prepared from GTL-16 cells, and their kinase activity was assayed by myelin basic protein phosphorylation as described in the Materials and Methods section. The dependence on myelin basic protein concentration is shown for untreated (solid circles) and activated (open circles) samples. Plotted values are the means of duplicate determinations from a representative experiment out of three performed.

CL

E

0.00

10

.2

S 0 0

2

1

[NaO51

3

4

5

angiotensin 11, mM

FIG. 9. Effect of ATP activation on the phosphorylation kinetics of the peptide substrate [Val5] angiotensin II by the Met tyrosine kinase. The Vmax of the reaction is increased approximately fivefold while the Km for ATP is unchanged and the Km for the peptide substrate is slightly increased. Immunoprecipitates of the Met protein were prepared from GTL-16 cells, and their kinase activity was assayed by [Val5] angiotensin II phosphorylation as described in the Materials and Methods section. (A) The dependence on ATP concentration is shown for untreated (solid circles) and activated (open circles) samples. (B) The dependence on the concentration of the peptide substrate [Val5] angiotensin II is shown for the same set of samples. Plotted values are the means of duplicate determinations from a representative experiment out of three performed.

positive and negative modulation of enzyme activity (10, 27). Positive regulation by tyrosine autophosphorylation has also been shown for the v-fps oncogene product (54) and the insulin receptor (35, 45, 57, 58). Furthermore, the kinase activity of pp6csrc (5, 17, 36, 44) of ppl20gag-fPs (38) and of the insulin receptor (15) was greatly diminished upon substitution of a consensus tyrosine in the kinase domain by a phenylalanine residue. For the insulin receptor, the role of tyrosine autophosphorylation as an independently acting mechanism of activation of the receptor kinase was better elucidated. In fact, an autophosphorylation-dependent enhancement of kinase activity could be distinguished from that induced by ligand binding per se, although it was observed only in the presence of the ligand (39). The net increase in phosphorylation rate was mainly due to an increase in the Vm., of the reaction, without a change in the Km for the substrates (45). In the case of the EGF receptor, a number of earlier reports both showed and denied a role for autophosphorylation in the activation of the tyrosine kinase activity of the molecule (3, 13). The presently accepted view is that the C-terminal tail of the molecule acts as a pseu-

dosubstrate, exerting a competitive-type inhibition on the kinase. Autophosphorylation on C-terminal tyrosine residues removes the inhibition and induces moderate activation by a decrease in the Km for the substrates (2, 14, 30, 31). It thus appears that there are at least two independent mechanisms for enhancing the activity of a receptor tyrosine kinase. Ligand-dependent activation would be the most powerful, increasing the Vmax of the reaction, possibly by recruiting more enzyme into an active oligomeric state (16, 55). Autophosphorylation-dependent enhancement of activity would be an additional amplification mechanism available to some kinases. It may operate in two distinct ways. It may remove an inhibitory constraint from the C-terminal tail, thus decreasing the Km of the reaction, as with the EGF receptor. Alternatively, it may more effectively activate the enzyme by the phosphorylation of a tyrosine(s) embedded in the kinase domain in a position corresponding to the tyrosine 416 of pp60Src, a well-known activating site (28). The functional consequence would be an increase in the Vm. of the reaction, possibly by driving the enzyme into an active conformation, as with the insulin receptor (18, 29, 50). Here we showed evidence for the latter mechanism for the Met tyrosine kinase. The similarity with the insulin receptor adds to that already known about the structure of the two molecules. While a ligand for the Met protein is presently unknown, it could further enhance the enzyme activity as insulin does for its receptor. Final proof of the proposed activation mechanism will require identification of the relevant tyrosine phosphorylation sites and their site-specific in vitro mutagenesis. Such regulatory features are expected to be crucial in growth factor receptors, transmembrane allosteric molecules whose enzymatic moiety is affected by several noncatalytic domains. Furthermore, the structural basis for this modulation could be hot spots for mutations activating the transforming potential of the kinase.

1802

NALDINI ET AL.

ACKNOWLEDGMENTS We thank Tiziana Crepaldi for kindly providing some anti-Met monoclonal antibodies and Paolo Dalla Zonca for help with the exogenous substrate kinase assay. We also thank Ottavio Cremona for help with data analysis and plotting. Critical discussions with Maria Flavia Di Renzo and Gianni Gaudino during the course of these studies are acknowledged. This work was supported by grants from the Associazione Italiana Ricerche Cancro and the Italian National Research Council (PFBiotecnologie no. 89.00145) to P.M.C. REFERENCES 1. Basu, M., R. Biswas, and M. Das. 1984. 42,000-molecular weight EGF receptor has protein kinase activity. Nature (London) 311:477-480. 2. Bertics, P. J., W. S. Chen, L. Hubler, C. S. Lazar, M. G. Rosenfeld, and G. N. GM. 1988. Alteration of epidermal growth factor receptor activity by mutations of its primary carboxyterminal site of self-phosphorylation. J. Biol. Chem. 263:36103617. 3. Bertics, P. J., and G. N. Gill. 1985. Self-phosphorylation enhances the protein-tyrosine kinase activity of the epidermal growth factor receptor. J. Biol. Chem. 260:14642-14647. 4. Burnette, W. N. 1981. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal. Biochem. 112:195-204. 5. Cartwright, C. A., W. Eckhardt, S. Simon, and P. L. Kaplan. 1987. Cell transformation by pp60csrc mutated in the carboxyterminal regulatory domain. Cell 49:83-91. 6. Chan, A. M. L., H. W. S. King, E. A. Deaking, P. R. Tempest, J. Hilkens, V. Kroezen, D. R. Edwards, A. J. Wills, P. Brookes, and C. S. Cooper. 1988. Characterization of the mouse met proto-oncogene. Oncogene 2:593-600. 7. Comoglio, P. M., M. F. Di Renzo, G. Tarone, F. Giancotti, L. Naldini, and P. C. Marchisio. 1984. Detection of phosphotyrosine containing proteins in the detergent-insoluble fraction of RSV-transformed fibroblasts by azobenzylphosphonate antibodies. EMBO J. 3:483-489. 8. Cooper, C. S., M. Park, D. Blair, M. A. Tainsky, K. Huebner, C. M. Croce, and G. F. Vande Woude. 1984. Molecular cloning of a new transforming gene from a chemically transformed human cell line. Nature (London) 311:29-33. 9. Cooper, J. A., B. M. Sefton, and T. Hunter. 1983. Detection and quantification of phosphotyrosine in proteins. Methods En-

zymol. 99:387-402. 10. Courtneidge, S. A. 1985. Activation of pp60csrc kinase by middle T antigen binding or by dephosphorylation. EMBO J. 4:14711477. 10a.Crepaldi, T., and M. Prat. Unpublished data. 11. Dean, M., M. Park, M. M. Le Beau, T. S. Robins, M. 0. Diaz, J. D. Rowley, D. G. Blair, and G. F. Vande Woude. 1985. The human met oncogene is related to the tyrosine kinase oncogenes. Nature (London) 318:385-388. 12. Di Renzo, M. F., R. Ferracini, L. Naidini, S. Giordano, and P. M. Comoglio. 1986. Immunological detection of proteins phosphorylated at tyrosine in cells stimulated by growth factors or transformed by retroviral-oncogene-encoded proteins. Eur. J. Biochem. 158:383-391. 13. Downward, J., M. D. Waterfield, and P. J. Parker. 1985. Autophosphorylation and protein kinase C phosphorylation of the epidermal growth factor receptor. J. Biol. Chem. 260:1453814546. 14. Downward, J., Y. Yarden, E. Moyes, G. Scrace, N. Totty, P. Stockwell, A. Ullrich, J. Schlessinger, and M. D. Waterfield. 1984. Close similarity of epidermal growth factor receptor and v-erb-B oncogene protein sequences. Nature (London) 307:521527. 15. Ellis, L., E. Clauser, D. 0. Morgan, M. Edery, R. A. Roth, and W. J. Rutter. 1985. Replacement of insulin receptor tyrosine residues 1162 and 1163 compromises insulin-stimulated kinase activity and uptake of 2-deoxyglucose. Cell 45:721-732.

MOL. CELL. BIOL. 16. Erneux, C., S. Cohen, and D. L. Garbers. 1983. The kinetics of tyrosine phosphorylation by the purified epidermal growth factor receptor kinase of A-431 cells. J. Biol. Chem. 258:41374142. 17. Ferracini, R., and J. Brugge. 1990. Analysis of mutant forms of the c-src gene product containing a phenylalanine substitution for tyrosine 416. Oncogene Res. 5:205-219. 17a.Ferracini, R., et al. Unpublished data. 18. Flores-Riveros, J. R., E. Sibley, T. Kastelic, and M. D. Lane. 1989. Substrate phosphorylation catalyzed by the insulin receptor tyrosine kinase. J. Biol. Chem. 264:21557-21572. 19. Frackleton, A. R., H. R. Alonzo, and H. N. Eisen. 1983. Characterization and use of monoclonal antibodies for the

20.

21.

22. 23.

24.

25. 26.

27. 28.

29. 30.

31.

32. 33. 34.

35. 36.

37.

isolation of phosphotyrosyl proteins from retrovirus-transformed cells and growth factor receptors. Mol. Cell. Biol. 3:1342-1352. Gandino, L., M. F. Di Renzo, S. Giordano, F. Bussolino, and P. M. Comoglio. 1990. Protein kinase-C activation inhibits tyrosine phosphorylation of the c-met protein. Oncogene 5:721725. Giordano, S., M. F. Di Renzo, R. Ferracini, L. Chiado'Piat, and P. M. Comoglio. 1988. P145, a protein with associated tyrosine kinase activity in a human gastric carcinoma cell line. Mol. Cell. Biol. 8:3510-3517. Giordano, S., M. F. Di Renzo, R. Narsimhan, C. S. Cooper, C. Rosa, and P. M. Comoglio. 1989. Biosynthesis of the protein encoded by the c-met protooncogene. Oncogene 4:1383-1388. Giordano, S., M. F. Di Renzo, R. Narsimhan, L. Tamagnone, E. V. Gerbaudo, L. Chiado'-Piat, and P. M. Comoglio. 1988. Evidence for autocrine activation of a tyrosine kinase in a human gastric carcinoma cell line. J. Cell. Biochem. 38:229-236. Giordano, S., C. Ponzetto, M. F. Di Renzo, C. S. Cooper, and P. M. Comoglio. 1989. Tyrosine kinase receptor indistinguishable from the c-met protein. Nature (London) 339:155-156. Glass, D. B., R. A. Masaracchia, J. R. Feramisco, and B. E. Kemp. 1978. Isolation of phosphorylated peptides and proteins on ion exchange papers. Anal. Biochem. 87:566-575. Gonzatti-Haces, M., A. Seth, M. Park, T. Copeland, S. Oroszlan, and G. F. Vande Woude. 1988. Characterization of the TPRMET oncogene p65 and the MET protooncogene p140 proteintyrosine kinases. Proc. Natl. Acad. Sci. USA 85:21-25. Graziani, F., E. Erikson, and R. L. Erikson. 1983. Characterization of the Rous sarcoma virus transforming gene product. J. Biol. Chem. 258:6344 6351. Hanks, S. K., A. M. Quinn, and T. Hunter. 1988. The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science 241:42-52. Herrera, R., and 0. M. Rosen. 1986. Autophosphorylation of the insulin receptor in vitro. J. Biol. Chem. 261:11980-11985. Honegger, A. M., T. J. Dull, F. Beliot, E. Van Obberghen, D. Szapary, A. Schmidt, A. Ullrich, and J. Schlessinger. 1988. Biological activities of EGF-receptor mutants with individually altered autophosphorylation sites. EMBO J. 7:3045-3052. Honegger, A. M., T. J. Dull, D. Szapary, A. Komoriya, R. Kris, A. Ullrich, and J. Schlessinger. 1988. Kinetic parameters of the protein tyrosine kinase activity of EGF-receptor mutants with individually altered autophosphorylation sites. EMBO J. 7:3053-3060. Hunter, T. 1987. A thousand and one protein kinases. Cell 50:823-829. Hunter, T., and J. Cooper. 1985. Protein tyrosine kinases. Annu. Rev. Biochem. 54:897-930. Khazaie, K., T. J. Dull, T. Graf, J. Schlessinger, A. Ullrich, H. Beug, and B. Vennstrom. 1988. Truncation of the human EGF receptor leads to differential transforming potentials in primary avian fibroblasts and erythroblasts. EMBO J. 7:3061-3071. Klein, H. H., G. R. Friedenberg, M. Kladde, and J. M. Olefsky. 1986. Insulin activation of insulin receptor tyrosine kinase in intact rat adipocytes. J. Biol. Chem. 261:4691-4697. Kmiecik, T. E., and D. Shalloway. 1987. Activation and suppression of pp60C-src transforming ability by mutation of its primary sites of tyrosine phosphorylation. Cell 49:65-73. Laemmli, U. K. 1970. Cleavage of structural proteins during the

VOL . 1 l, 1991

38.

39. 40.

41.

42. 43.

44.

45.

46.


assembly of the head of bacteriophage T4. Nature (London) 230:680-685. Meckling-Hansen, K., R. Nelson, P. Branton, and T. Pawson. 1987. Enzymatic activation of Fujinami sarcoma virus gag-fps transforming proteins by autophosphorylation at tyrosine. EMBO J. 3:659-666. Morrison, B. D., and J. E. Pessin. 1987. Insulin stimulation of the insulin receptor kinase can occur in the complete absence of ,B subunit autophosphorylation. J. Biol. Chem. 262:2861-2868. Motoyama, T., H. Hojo, T. Suzuki, and S. Oboshi. 1984. Evaluation of the regrowth assay method as an in vitro drug sensitivity test and its application to cultured human gastric cancer cell lines. Acta Med. Biol. (Niigata University, Niigata, Japan) 27:49-52. Naldini, L., A. Stacchini, D. Cirillo, M. Aglietta, F. Gavosto, and P. M. Comoglio. 1986. Phosphotyrosine antibodies identified the p120c-ab1 tyrosine kinase and proteins phosphorylated on tyrosine in chronic myelogenous leukemia cells. Mol. Cell. Biol. 6:1803-1811. Park, M., M. Dean, C. S. Cooper, M. Schmidt, S. J. O'Brian, D. G. Blair, and G. F. Vande Woude. 1986. Mechanism of met oncogene activation. Cell 45:895-904. Park, M., M. Dean, K. Kaul, M. J. Braun, M. A. Gonda, and G. F. Vande Woude. 1987. Sequence of MET protooncogene cDNA has features characteristic of the tyrosine kinase family of growth-factor receptors. Proc. Natl. Acad. Sci. USA 84: 6379-6383. Piwnica-Worms, H., K. B. Saunders, T. M. Roberts, A. E. Smith, and S. H. Cheng. 1987. Tyrosine phosphorylation regulates the biochemical and biological properties of pp6Oc-src. Cell 49:75-82. Rosen, 0. M., R. Herrera, Y. Olowe, L. M. Petruzzelli, and M. H. Cobb. 1983. Phosphorylation activates the insulin receptor tyrosine protein kinase. Proc. Natl. Acad. Sci. USA 80: 3237-3240. Schlessinger, J. 1988. Signal transduction by allosteric receptor oligomerization. Trends Biol. Sci. 13:443-447.

1803

47. Shoelson, S. E., M. F. White, and C. R. Khan. 1988. Tryptic activation of the insulin receptor. J. Biol. Chem. 263:4852-4860. 48. Tempest, P. R., C. S. Cooper, and G. N. Major. 1986. The activated human met gene encodes a protein tyrosine kinase. FEBS Lett. 209:357-361. 49. Tempest, P. R., M. R. Stratton, and C. S. Cooper. 1988. Structure of the met protein and variation of met protein kinase activity among human tumour cell lines. Br. J. Cancer 58:3-7. 50. Tornqvist, H. E., and J. Avruch. 1988. Relationship of sitespecific 1 subunit tyrosine autophosphorylation to insulin activation of the insulin receptor (tyrosine) protein kinase activity. J. Biol. Chem. 263:4593-4601. 51. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76:4350-4353. 52. Ullrich, A., and J. Schlessinger. 1990. Signal transduction by receptors with tyrosine kinase activity. Cell 61:203-212. 53. Weber, W., P. J. Bertics, and G. N. Gill. 1984. Immunoaffinity purification of the epidermal growth factor receptor. Stoichiometry of binding and kinetics of self-phosphorylation. J. Biol. Chem. 259:14631-14636. 54. Weinmaster, G., M. J. Zoller, and T. Pawson. 1986. A lysine in the ATP binding site of pl3Ogag-fps is essential for protein tyrosine kinase activity. EMBO J. 5:69-76. 55. White, M. F., H.-U. Haring, M. Kasuga, and C. R. Khan. 1984. Kinetic properties and sites of autophosphorylation of the partially purified insulin receptor from hepatoma cells. J. Biol. Chem. 259:255-264. 56. Yarden, Y., and A. Ullrich. 1988. Growth factor receptor tyrosine kinases. Annu. Rev. Biochem. 57:443-478. 57. Yu, K.-T., and M. P. Czech. 1984. Tyrosine phosphorylation of the insulin receptor 1 subunit activates the receptor-associated tyrosine kinase activity. J. Biol. Chem. 259:5277-5286. 58. Yu, K.-T., and M. P. Czech. 1986. Tyrosine phosphorylation of insulin receptor 1B subunit activates the receptor tyrosine kinase in intact H-35 hepatoma cells. J. Biol. Chem. 261:4715-4722.