The Tyrosine-phosphorylated Hepatocyte Growth Factor/Scatter Factor Receptor Associates with Phosphatidylinositol 3-Kinase”

The receptor for hepatocyte growth factor, also known as scatter factor (HGF/SF), has recently been identified as the 190-kDa heterodimeric tyrosine ki- nase encoded by the MET proto-oncogene ( ~ 1 9 0 ~ 9 . The signaling pathway@) triggered by HGF/SF are unknown. In A549 cells, a lung epithelial cell line, nanomolar concentrations of HGF/SF induced tyrosine phosphorylation of the p190Mm receptor. The auto-phosphorylated receptor coprecipitated with phospha- tidylinositol3-kinase (PI 3-kinase) activity. In GTL16 cells, a cell line derived from a gastric carcinoma, the p190Mm receptor, overexpressed and constitutively phosphorylated on tyrosine, coprecipitated with PI 3- kinase activity and with the 85-kDa PI 3-kinase subunit. In these cells activation of protein kinase C or the increase of intracellular [Ca”’] inhibits tyrosine phos- phorylation of the p190Mm receptor as well as the association with both PI 3-kinase activity and the 85- kDa subunit of the enzyme. In an in vitro assay, tyrosine phosphorylation of the immobilized p190Mm receptor was required for binding of PI 3-kinase from cell lysates. These

biological effects of hepatocyte growth factor/scatter factor (HGF/SF)' are triggered by binding to the same receptor, the protein encoded by the MET proto-oncogene (7). The Met protein had already been identified as the receptor for hepatocyte growth factor (8,9).
The MET-encoded receptor is a 190-kDa heterodimeric transmembrane protein (p190MET) made of a 50-kDa a subunit disulfide-linked to a 145-kDa p subunit (~145') (10, 11). The a subunit and the NH-terminal portion of ~1 4 5~ are exposed at the cell surface (12). The carboxyl-terminal portion is cytoplasmic and includes a tyrosine kinase domain (13-15), and phosphorylation sites are involved in the regulation of its activity (16). The kinase activity is positively regulated by autophosphorylation on tyrosine (17), and it is negatively regulated by protein kinase C activation (18) or a transient increase of intracellular Ca2+ concentration (19). Stimulation of ~1 4 5~ subunit tyrosine phosphorylation after exposure to HGF/SF was observed both in intact cells (7-9) and in vitro with partially purified Met protein (9).
The signaling pathway(s) triggered by HGF/SF have not yet been investigated. Similarly, the substrates of the p190MET receptor kinase are unknown. In this work we show that the tyrosine-phosphorylated form of p190MET receptor associates with phosphatidylinositol 3-kinase, a key enzyme involved in the transduction of signals generated by tyrosine kinase receptors (20, 21).

EXPERIMENTAL PROCEDURES
Reagents, Cell Lines, and Antibodies-All reagents, unless specified, were purchased from Sigma. Protein A covalently coupled to Sepharose was purchased from Pharmacia LKB Biotechnology Inc. Reagents for SDS-PAGE were from Bio-Rad. I4C-Methylated molecular mass standards were from Amersham Corp.
A549 lung carcinoma cells were obtained from American Type Cell Catalogue. GTL16 is a clonal cell line derived from a poorly differentiated gastric carcinoma (12). Cells were cultured in RPMI 1640 medium (Seromed) containing 10% fetal calf serum (Seromed) and maintained at 37 'C in a humidified atmosphere with 5% CO,.
HGF/SF was purified from the supernatant of MRC5 human fibroblasts by heparin-Sepharose column chromatography (Pharmacia) eluted by a linear gradient of 0.5-1.8 M NaCl as described (5).
Phosphotyrosine antibodies were raised as previously described (22). Monoclonal antibodies against the anti-extracellular domain of the Met protein were obtained as described elsewhere (48). Polyclonal antibodies against the 85-kDa subunit of PI 3-kinase were raised in chickens immunized with a synthetic peptide as described elsewhere.* Preparationof CeULysates, Immunoprecipitation, and Western Blotting-Subconfluent A549 cells were starved for 2 days in serum-free medium, stimulated by HGF/SF as indicated, and then lysed in buffer A (25 mM Hepes buffer, pH 8, 10% glycerol, 1% Nonidet P-40, 150 mM NaC1, 5 mM EDTA, 2 mM EGTA, 0.5 mM dithiothreitol, 0.2 mM The abbreviations used are: HGF/SF, hepatocyte growth factor/ scatter factor; PI 3-kinase, phosphatidylinositol 3-kinase; PI 4-kinase, 22087 phenylmethylsulfonyl fluoride, 1 pg/ml leupeptin, 1 pg/ml aprotinin, and 1 pg/ml soybean trypsin inhihitors) supplemented with 0.5 mM sodium vanadate, 0.1 mM ammonium molyhdate, 10 p~ phenylarsine oxide, and 1 mM ZnCI,. Suhconfluent monolayers of GTLl6 cells were serum-starved for 2 days and, unless otherwise indicated, lysed in buffer A containing 0.1 mM vanadate. Lysates were spun a t 15,000 X ,q for 15 min, and the supernatants were immunoprecipitated after 2 h of incuhation with monoclonal anti-Met antibodies cross-linked to rabbit anti-mouse Ig-Protein A-Sepharose. The immunoprecipitates were washed as previously descrihed (23). Western blotting from both whole cell lysate or immunoprecipitates was performed as described before (17,19).
PI 3-Kinose Assay-PI &kinase assay was performed directly on the heads as descrihed by Auger et al. (23). Adenosine (0.2 mM) was added to the reaction mixture t,o inhibit residual PI(4) kinase activity (33). For definitive identification, PIP, PIP,, and PIPa separated by T L C were eluted, deacylated, and the respective glycerophosphoinositol phosphate derivatives separated on HPLC hy a Partisil Sax column (Whatman) as descrihed (2.7, 24).
Bindin,q of P I 3-Kinme to pI9@"~"" in Vitro-Confluent GTLl6 cells were lysed in buffer A, and p190METwas purified by immunoprecipitation as descrihed above. The Protein A-Sepharose-immobilized p19OME'' was dephosphorylated hy alkaline phosphatase (Promega) treatment for 20 min a t 15 "C. The immunocomplexes were washed as in the immunoprecipitation protocol. When indicated samples were rephosphorylated by incubation for 15 min at room temperature with 0.1 mM ATP, 10 mM MnCI2. The immunocomplexes were extensively washed as ahove. A549 cells were made quiescent by 48h serum starvation and lysed with buffer A supplemented with 0.5 mM sodium vanadate, 0.1 mM ammonium molyhdate, and 1 mM ZnCI2. The lysates were then incuhated for 1 h a t 4 "C with the immobilized pl9OMkT. The complexes were washed as descrihed in the immunoprecipitation protocol and prohed as indicated.

HGFISF Induces Tyrosine Phosphorylation of p 1 9 p E T Receptor and Association with PI 3-Kincrse in A549 Cells-
Stimulation of A549 cells for 15 min with 50 ng/ml purified HGF/SF induced tyrosine phosphorylation of the p190' "' " receptor. This was observed by probing the anti-Met immunoprecipitates with anti-phosphotyrosine antibodies. The ~1 4 5~ subunit was heavily phosphorylated on tyrosine after 15 min of stimulation with HGF/SF (Fig. 1, A and I?). Notably, two additional proteins of molecular mass close to 85 and 60 kDa were also co-precipitated by anti-MET antibodies and were found to contain phosphotyrosine. 85 kDa is the molecular mass of the putative regulatory subunit of PI 3-kinase (26)(27)(28)37), which was found to be phosphorylated on tyrosine when present in a complex with several tyrosinephosphorylated proteins (reviewed in 20). PI 3-kinase activity, assayed in the presence of adenosine to inhibit residual nonspecifically bound PI 4-kinase (33), was present in the immunocomplexes from HGF/SF-stimulated cells. Fig. 1C shows the TLC separation of the D-3-phosphorylated inositol lipids generated in vitro by incubation of the anti-Met immunocomplex in the presence of sonicated lipid substrates and [ y -: ' T ] ATP. The identities of PIP, PIPz, and PIPn generated in vitro were confirmed by HPLC analysis of the deacylated derivatives to be PI(3)P, PI(3,4)Pz, and PI(3,4,5)Pn (not shown). No PI 3-kinase activity was detectable in immunocomplexes precipitated from unstimulated cells.

Tyrosine Phosphorylation of p190""T Receptor Regulates the Physical Association with PI 3-Kinase in Intact GTL16 Celk-
In GTL16 cells, due to the amplification of the M E T gene, the p190' "' receptor is overexpressed and constitutively phosphorylated on tyrosine (10). Moreover, we have previously shown that in these cells it is possible to negatively regulate the kinase activity of p19OM"'" by activation of protein kinase C or by increasing the intracellular Ca2+ concentration (18, 19). We therefore took advantage of this model system t o further investigate if tyrosine phosphorylation of p190ME" regulates its association with PI 3-kinase. Subconfluent cultures of GTL16 cells were starved for 3 days in serum-free medium and treated either with TPA (160 nM for 1 h ) or with the Ca2+ ionophore A23187 (10 pM for 5 min). p190""7' tyrosine phosphorylation was assessed by probing the blotted whole cell lysates with anti-phosphotyrosine and with anti- Met antibodies (Fig. 2). The p190MET receptor was then immunoprecipitated from control and TPA or A23187 Ca2+ ionophore-treated cells; immunoprecipitates were blotted and probed with either anti-phosphotyrosine or anti-Met antibodies. The amount of p190ME7' tyrosine phosphorylated in the immunoprecipitates from untreated cells was severalfold higher (Fig. 3, A and B ) . The PI 3-kinase activity, measured as D-3-phosphoinositides generated in vitro, co-precipitated with tyrosine phosphorylated p190MET from control cells but could not be detected in the immunocomplexes from TPA and Ca2+ ionophore-treated cells (Fig. 30).
To demonstrate the physical association of the 85-kDa subunit of PI 3-kinase with tyrosine-phosphorylated p190MET, immunoprecipitates from GTL16 cells, either control or treated with TPA or the Ca2+ ionophore, were blotted and probed with chicken antibodies directed against the 85-kDa putative regulatory subunit of PI 3-kinase (p85). The 85-kDa subunit of PI 3-kinase was indeed found to associate only with the tyrosine-phosphorylated form of p190"'ET (Fig. 3C).
Tyrosine Phosphorylation of p l 9 p E T Receptor Is Required for Binding of PI 3-Kinase in Vitro-In order to formally prove that p190MET tyrosine phosphorylation is a switch for association of PI 3-kinase, we developed an in vitro association assay. Sepharose-immobilized p190MET receptors were dephosphorylated by alkaline phosphatase treatment and extensively washed to remove any associated PI 3-kinase activity. In half of the samples the receptors were re-phosphorylated on tyrosine as described under "Experimental Procedures." The unphosphorylated and the re-phosphorylated immobilized receptors were incubated for 1 h at 4 "C with a lysate prepared from A549 cells or with the lysis buffer alone. The  4. In vitro association of PI 3-kinase requires tyrosine phosphorylation of the HGF/SF receptor ~1 4 5~ subunit.
p190M"' HGF/SF receptor was immunopurified, immobilized to protein-A Sepharose beads, and dephosphorylated. Part of the samples were left as controls (lanes 1 and 2); part were re-phosphorylated on tyrosine in uitro (lanes 3 and 4 ) . A lysate of A549 cell was incubated cell lysates did not contain any anti-phosphotyrosine immunoprecipitable PI 3-kinase activity, suggesting that the enzyme neither is phosphorylated on tyrosine nor is associated with tyrosine-phosphorylated proteins (data not shown). However, PI 3-kinase activity from the cell lysate did bind to the tyrosine-phosphorylated p190MET receptor. No PI 3-kinase activity associated with the unphosphorylated receptor, and no residual activity was found in samples incubated with buffer alone (Fig. 4C). The amount of p190MET bound to the beads and its state of phosphorylation were not affected by the incubation with A549 cell lysate; as shown by probing with anti-Met and anti-phosphotyrosine antibodies the immunocomplexes incubated with either cell lysates or buffer alone (Fig. 4, A and B ) . This experiment demonstrates that tyrosine phosphorylation of p190' "T is a switch for association with PI 3-kinase in vitro.

DISCUSSION
In this paper we present the first evidence that tyrosine phosphorylation of the HGF/SF receptor is a switch for association of PI 3-kinase. This enzyme plays a key role in the signal transduction pathway by generating D-3-phosphoinositides (23,24), a novel class of putative intracellular second messengers (20, 26). The role in signal transduction of PI(3)P, PI(3,4)P2, and PI(3,4,5)Ps is not yet understood, although their cellular levels in vivo are elevated in cells entering the mitotic cycle or transformed by activated oncogenes (23,34-36). PI 3-kinase, recently purified to homogeneity, is a het-erodimeric protein consisting of two subunits of 110 and 85 kDa (26). The cDNA encoding the 85-kDa subunit has recently been cloned (27,28,37). The 85-kDa subunit, lacking enzymatic activity, features two SH-2 domains and directly associates with tyrosine-phosphorylated proteins (26-28, 32, 37). Moreover, upon binding to tyrosine-phosphorylated protein, the 85-kDa subunit becomes phosphorylated on tyrosine (32,38,39). This molecule, acting as a regulatory subunit for the catalytic 110-kDa subunit of PI 3-kinase may be regulated by tyrosine phosphorylation (26,27).
So far PI 3-kinase has been found to associate with and to be activated by a number of tyrosine kinase receptors, including the receptors for PDGF (33), colony-stimulating factor-1 (40), insulin (41,42), epidermal growth factor (reviewed in 20), and stem cell growth factor (44). The association is ligand-dependent and mediated by tyrosine phosphorylation of the receptor cytoplasmic domain.
It has been shown that phosphorylation of a specific tyrosine ( T Y~~~' ) in the kinase insert domain of the human p-PDGF receptor is critical for association with PI 3-kinase (45). Similarly, in vitro binding of PI 3-kinase to the mouse p-PDGF receptor is inhibited by competition with synthetic peptides containing the homologous Tyr739 in the phosphorylated state (39). This tyrosine is included in a sequence highly conserved in other receptor kinases which associate with PI 3-kinase. From these homologous sequences a putative consensus (-YXX") has been deduced (20).
We have shown that PI 3-kinase associates with the p190MET HGF/SF receptor. The association is mediated by tyrosine phosphorylation of the ~1 4 5~ subunit. The intracellular domain of ~1 4 5~ is itself a tyrosine kinase, whose activity is controlled by binding to the specific ligand. P145' also contains multiple tyrosine autophosphorylation sites (16) and a consensus sequence for binding to PI 3-kinase (-Y'313EVM-).3 It has been suggested that the recruitment of Pr3-kinase to tyrosine-phosphorylated receptors is a necessary step for activation in vivo (20). Furthermore the 85-kDa subunit of PI 3-kinase was found to be phosphorylated on tyrosine only when associated to the membrane (31). These data are compatible with the hypothesis that the 85-kDa regulatory subunit upon association to the HGF/SF-activated receptor is a target for its tyrosine kinase activity. It is conceivable that the D-3-polyphosphoinositides generated by PI 3-kinase play a role in the signaling pathway triggered by HGF/SF. This factor induces different biological responses in epithelial cells, including mitogenesis and stimulation of cell motility. Interestingly, D-3-polyphosphoinositides are generated in response to other extracellular signals inducing either cell growth (23, 40,41,42) or chemotaxis (47). P I 3-kinase may not be the only signal transducer activated by HGF/SF. Other proteins may associate with the p190MET receptor and may be the target of its tyrosine kinase activity. Consistent with this hypothesis we detected other low abundance proteins that are tyrosine-phosphorylated upon stimulation or constitutive activation of p190MET. The identification of these molecules and the analysis of their physiological significance are in progress.