Tyr-716 in the Platelet-Derived Growth Factor 3-Receptor Kinase Insert Is

Tyr-716 in the Platelet-Derived Growth Factor 3-Receptor Kinase Insert Is Involved in GRB2 Binding and Ras Activation ANN-KRISTIN ARVIDSSON,l* EVA RUPP,' EEWA NANBERG,2 JULIAN DOWNWARD,3 LARS RONNSTRAND,1 STEFAN WENNSTROM,1 JOSEPH SCHLESSINGER,4 CARL-HENRIK HELDIN,1 AND LENA CLAESSON-WELSH1 Ludwig Institute for Cancer Research, Biomedical Center, 5-751 24 Uppsala, 1 and Department of Pathology, University Hospital, S-751 85 Uppsala,2 Sweden; Signal Transduction Laboratory, Imperial Cancer Research Fund, London WC2A 3PX, United Kingdom3; and Department of Pharmacology, New York University Medical Center, New York New York 100164 Received 8 April 1994/Returned for modification 19 May 1994/Accepted 19 July 1994

Ligand stimulation of the platelet-derived growth factor (PDGF) P-receptor leads to activation of its intrinsic tyrosine kinase and autophosphorylation of the intracellular part of the receptor. The autophosphorylated tyrosine residues mediate interactions with downstream signal transduction molecules and thereby initiate different signalling pathways. A pathway leading to activation of the GTP-binding protein Ras involves the adaptor molecule GRB2. Here we show that Tyr-716, a novel autophosphorylation site in the PDGF ,8-receptor kinase insert, mediates direct binding of GRB2 in vitro and in vivo. In a panel of mutant PDGF ,-receptors, in which Tyr-716 and the previously known autophosphorylation sites were individually mutated, only PDGFRjY716F failed to bind GRB2. Furthermore, a synthetic phosphorylated peptide containing Tyr-716 bound GRB2, and this peptide specifically interrupted the interaction between GRB2 and the wild-type receptor. In addition, the Y716(P) peptide significantly decreased the amount of GTP bound to Ras in response to PDGF in permeabilized fibroblasts as well as in porcine aortic endothelial cells expressing transfected PDGF ,8-receptors. The mutant PDGFRjIY716F still mediated activation of mitogen-activated protein kinases and an increased DNA synthesis in response to PDGF, indicating that multiple signal transduction pathways transduce mitogenic signals from the activated PDGF j-receptor.

Platelet-derived growth factor (PDGF) is a connective tissue in the PDGF P-receptor (reviewed in reference 9). The cell mitogen, consisting of dimers of disulfide-bonded A and B specificity of these interactions is determined by the abilities of polypeptide chains, which combine to form the three isoforms, different SH2 domains to recognize different sequences sur- PDGF-AA, -AB, and -BB (for a review, see reference 21). Two rounding the phosphorylated tyrosine residues in the receptor types of PDGF tyrosine kinase receptors have been identified (16, 49, 50). These interactions initiate different signal trans- and cloned. The ox-receptor binds both A and B chains with duction pathways that ultimately result in cell division and high affinity, whereas the p-receptor binds only the B chain. other biological responses. Signal transduction through tyrosine kinase receptors fol- A principal signal transduction pathway by which tyrosine lows a general scheme whereby ligand binding induces dimer- kinases stimulate cell growth and differentiation involves the ization or oligomerization of receptor molecules, activation of activation of Ras guanine nucleotide-binding proteins (30). the receptor tyrosine kinase, and autophosphorylation in trans Ras activity is regulated by the opposing actions of GTPase- on multiple tyrosine residues in the intracellular region (re- activating proteins (GAPs) and guanine nucleotide exchange viewed in reference 44). Thereby, binding sites are created for factors (5). The notion that Ras plays a critical role in the intracellular signal transduction molecules, containing one or transmission of mitogenic signals from receptor tyrosine ki- two copies of Src homology 2 (SH2) domains. There are two nases was originally suggested by the growth-inhibitory effect principal groups of signal transduction molecules: those which of microinjecting neutralizing Ras antibodies into mammalian are equipped with a catalytic domain, and those which lack fibroblasts (34, 48). More recent data from genetic studies of such domains but which serve as adaptors and associate with Caenorhabditis elegans and Drosophila melanogaster revealed catalytically active molecules (50). For the PDGF P-receptor, that the adaptor molecule GRB2/SEM5 is a key molecule in considerable information has accumulated regarding the posi- the signal transduction pathway that links receptor tyrosine tions of autophosphorylation sites as well as which signal kinases to Ras activation (12, 36, 46). GRB2 has also been transduction molecules interact with individual sites. Thus, shown to cooperate with Ras, since microinjection of GRB2 members of the Src family, i.e., phospholipase C--y, the Ras together with H-Ras protein into quiescent rat embryo fibro- GTPase-activating protein (GAP), phosphatidylinositol 3'-ki- blasts resulted in DNA synthesis, whereas the injection of nase (P13-kinase), the phosphotyrosine phosphatase PTP1D/ either alone had no effect (28). A number of reports and the molecules Shc and have been shown protein Syp, adaptor Nck, have recently shown that GRB2 forms a complex with a to bind to regions involving different autophosphorylation sites guanine nucleotide exchange factor for Ras, designated Sos. The GRB2-Sos complex associates with activated receptor tyrosine kinases, and the exchange factor is thereby brought * Corresponding author. Mailing address: Ludwig Institute for into contact with its target, Ras, at the plasma membrane (8, Cancer Research, Biomedical Center, Box 595, S-751 24 Uppsala, 15, 18, 26, 38). Sweden. Phone: 46 18 55 16 88. Fax: 46 18 50 68 67. Work from several laboratories has shown that GRB2 binds 6715 6716 ARVIDSSON ET AL. MOL. CELL. BIOL. either directly to activated tyrosine kinase receptors, e.g., the Peroxidase-conjugated sheep anti-mouse immunoglobulins epidermal growth factor (EGF) receptor (8, 28), or indirectly, were from Amersham. The mouse phosphotyrosine monoclo- via Shc (39, 47) or PTPlD/Syp (27). Here we identify Tyr-716 nal antibody PY20 was purchased from Transduction Labora- in the PDGF n-receptor kinase insert as a novel autophosphor- tories. The Ras monoclonal antibody Y13-259 was purchased ylation site and show that phosphorylated Tyr-716 binds GRB2 from Oncogene Science. Antiserum HL-2 (53) recognizes directly. We also demonstrate that a phosphorylated peptide amino acid residues 701 to 732 in the PDGF 3-receptor and encompassing Tyr-716 is able to specifically decrease the was a kind gift from J. van Zoelen, Nijmegen, The Nether- amount of GTP bound to Ras in response to PDGF in lands. permeabilized PDGF 3-receptor-expressing cells. 125I-PDGF-BB binding experiment, [3H]thymidine incorporation assay, and actin reorganization. Procedures for labeling MATERIALS AND METHODS PDGF-BB with 1251, radioreceptor assay, measuring of DNA synthesis, and actin reorganization were carried out as previ- Site-directed mutagenesis. Site-directed mutagenesis was ously described (3). performed with the Altered Sites Mutagenesis system (Pro- In vitro kinase assay. The cells were serum starved over- mega Corp.). A cDNA encompassing the entire coding region night in Ham's F12 medium. PDGF-BB was added to a final of the human PDGF ,-receptor (10) was subcloned into the concentration of 100 ng/ml, and the cells were incubated at 4°C pAlter-1 vector, using the 5' EcoRI site and the HindIII site at for 1 h and at 37°C for 8 min. The cells were lysed and nucleotide 3629 in the PDGF 3-receptor cDNA. Point muta- centrifuged, and the supernatants were incubated with a GAP tions which change tyrosine residues (single mutations at antiserum or with monoclonal antibody PDGFR-B2 for 2 h at Tyr-716, Tyr-763, Tyr-771, and a double mutation at Tyr-775 4°C. Protein A-Sepharose CL-4B (Pharmacia) was used to and Tyr-778, according to the numbering in reference 10) to precipitate the immune complexes. Kinase assays were per- phenylalanine residues were then introduced into the insert, formed on the immunoprecipitated samples as described pre- using the following oligonucleotides: 5'-GC GCG GAG CTC sodium dodecyl TFTC AGC AAT GC -3' (PDGFRPY716F), 5'-GAC GTC viously (3), and the samples were analyzed by AAA TTT1 GCA GAC ATC G -3' (PDGFR,BY763F), 5'-CC sulfate (SDS)-gel electrophoresis. The gels were treated with 1 TCC AAC TTC ATG GCC CC -3' (PDGFRIY771F), and M KOH for 1 h at 55°C to reduce levels of serine-bound 5'-GCC CCT TTC GAT AAC TTC GTT CCC -3' phosphate and subjected to autoradiography. (PDGFRPY775/778F). All mutations were confirmed by se- P13-kinase assay. Cells, kept in Ham's F12 medium supple- quencing. The mutated cDNAs were excised from the pAlter-1 mented with 1% fetal calf serum overnight, were stimulated vector and cloned into the eukaryotic expression vector with PDGF-BB as described above, rinsed with cold 20 mM pcDNA1/Neo (Invitrogen). Mutagenesis and constructions to N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES; create PDGFRPY740F, PDGFR3Y751F, and PDGFRIY740/ pH 7.5)-150 mM NaCl-0.1 mM Na3VO4, and then lysed in this 751F have been described earlier (54). buffer supplemented with 10% glycerol, 1% Nonidet P-40 Cell culture and transfection. Porcine aortic endothelial (Sigma), 5 mM EDTA, 1% Trasylol, and 1 mM phenylmeth- (PAE) cells (31), which lack endogenous PDGF cx- and ylsulfonyl fluoride (PMSF) on ice for 10 min. Clarified cell 3-receptors, were cultured in Ham's F12 medium (Biochrom) lysates were immunoprecipitated with antiserum PDGFR-3. supplemented with 10% fetal calf serum (FCS; GIBCO), 100 The immune complexes, immobilized on Sepharose, were then U of penicillin per ml, 100 ,ug of streptomycin per ml, and 2 subjected to P13-kinase assay essentially as described by Fukui mM L-glutamine. Transfection of PAE cells was performed by and Hanafusa (17). Briefly, the immobilized immune com- electroporation as described earlier (55). Selection of trans- plexes were washed three times with phosphate-buffered saline fected cells was initiated after 48 h by adding Geneticin (G418 (PBS) containing 1% Nonidet P-40, once with PBS, once with sulfate; GIBCO) at 0.4 mg/ml in the culture medium. Several 0.1 M Tris-HCl (pH 7.5)-0.5 M LiCl, once with distilled water, clones were expanded, and after analysis of PDGF 3-receptor and once with 20 mM Tris-HCl (pH 7.5)-100 mM NaCl-1 mM expression by immunoprecipitation, two clonal cell lines for EDTA on ice. The beads were suspended in 50 pl of 20 mM each of the receptor mutants were chosen and further ana- Tris-HCl (pH 7.5)-100 mM NaCl-0.5 mM EGTA-0.2 mg of lyzed. The cell lines with wild-type and mutated receptors phosphatidylinositol (sonicated for 15 min at 4°C; Sigma) per bound PDGF-BB with similar affinities and expressed the ml and preincubated at room temperature for 10 min. Then 10 following number of binding sites per cell, determined as ,uCi of [_y-32P]ATP and MgCl2 (final concentration, 10 mM) described previously (32): 4.8 x 10 (wild type), 3.2 x 104 were added, and the samples were further incubated for 10 (PDGFRPY716F), 2.8 x 104 (PDGFRIY763F), 1.8 x 104 min. Reactions were stopped by addition of chloroform- (PDGFRPY771F), and 2.4 x 104 (PDGFRIY775/778F). The methanol-11.6 M HCl (50:100:1), phospholipids were ex- mutant PDGF 3-receptors Y740F, Y751F, and Y740/751F tracted with chloroform, and the organic phase washed with have previously been described (54) and express 4.6 x 104, 2.8 methanol-1 M HCl (1:1). Reaction products were concen- x 10, and 5.6 x 104 binding sites per cell, respectively. The trated in vacuo, dissolved in chloroform, spotted on Silica Gel mutant PDGFR,BY1009F (40) has 3.2 x 104 binding sites per 60 plates (Merck) impregnated with 1% potassium oxalate, cell. All mutant receptors were stably expressed in PAE cells. and resolved by chromatography in chloroform-methanol- Antisera. For immunoprecipitation, we used the mouse 28% ammonia-water (43:38:5:7) for 45 min. Phosphorylated monoclonal antibody PDGFR-B2 (41) and the rabbit anti- products were detected by autoradiography on Fuji RX films. serum PDGFR-3 (11), both specifically reactive with the Peptide synthesis and purification. Peptides were synthe- human PDGF ,B-receptor. The rabbit antiserum against human sized by Fmoc (9-fluorenylmethoxycarbonyl) chemistry and GAP (PW 6.1) has been described elsewhere (4). The rabbit phosphorylated by use of the global phosphorylation method antisera raised against murine GRB2/SEM-5 (alternatively, (2, 24), using di-t-butyl-N,N-diisopropylphosphoramidite, as the rabbit GRB2 antiserum 327 was used), murine SH-PTP2/ previously described (33). All peptides had free carboxy ter- Syp, and a synthetic peptide corresponding to residues 333 to minals and were purified by reverse-phase high-pressure liquid 367 of the rat ERK1/mitogen-activated protein (MAP) kinase chromatography. The collected fractions were analyzed by were purchased from Upstate Biotechnology Incorporated. plasma desorption mass spectrometry using a Bio Ion 20 VOL. 14, 1994 GRB2 BINDS DIRECTLY TO THE PDGF 3-RECEPTOR 6717

TABLE 1. Phosphorylated peptides used (PAE), rinsed twice with an ice-cold solution containing 20 Peptide Sequencea mM Tris-HCl (pH 7.5) and 150 mM NaCl (Tris-buffered saline), and lysed in a solution containing 20 mM Tris-HCl (pH Y579(P) ..... V-S-S-D-G-H-E-Y(P)-I-Y-V-R-P-M-Q-L-P-Y 7.5) 150 mM NaCl, 1% Triton X-100, 10% glycerol, 1 mM Y581(P) ..... V-S-S-D-G-H-E-Y-I-Y(P)-V-R-P-M-Q-L-P-Y dithiothreitol, 1% Trasylol, 1 mM PMSF, and 100 p.M Y716(P) ..... L-Q-H-H-S-D-K-R-R-P-P-S-A-E-L-Y(P)-S-N-A-L-P-V-G Na3VO4. GST-GRB2 fusion protein (10 p.g/ml) was incubated Y740(P) ..... G-E-S-D-G-G-Y(P)-M-D-M-S-K-D-E-S Y751(P) ..... M-S-K-D-E-S-V-D-Y(P)-V-P-M-L-D-M-K-G with the different PAE cell lysates at 4°C for 2 h. The Y771(P) .... A-D-I-E-S-S-N-Y(P)-M-A-P-Y-D-N-Y-V-P-S GST-GRB2 fusion protein-PDGF ,B-receptor complex was Y775(P) .... I-E-S-S-N-Y-M-A-P-Y(P)-D-N-Y-V-P-S-A-P-E-R precipitated from lysates by using 90 plI (50%, vol/vol) of Y1009(P) .... L-D-T-S-S-V-L-Y(P)-T-A-V-Q-P-N-E-G glutathione-Sepharose 4B at 4°C for 1 h. The precipitates were Y1021(P) ..... P-N-E-G-D-N-D-Y(P)-I-I-P-L-P-D-P-K washed five times with lysis buffer and once with H20. Bound a Y(P), phosphorylated tyrosine. The corresponding unphosphorylated pep- proteins were released by boiling in SDS sample buffer for 5 tides (designated Y579, Y581, Y716, Y740, Y751, Y771, Y775, Y1009, and min and analyzed by SDS-gel electrophoresis and immuno- Y1021) were also synthesized. blotting. The NIH 3T3 cell lysates were used for immunoprecipitation with the GRB2 antiserum. Peptide inhibition studies were done in an identical manner with the exception that lysates were preincubated with 25 p.M (NIH 3T3) or 100 p.M instrument (Applied BioSystems). The sequences of the phos- (PAE) of different phosphorylated or unphosphorylated pep- phorylated peptides are given in Table 1. tides for 1 h at 4°C prior to the addition of GRB2 antiserum Preparation of GST-GRB2 fusion proteins. A glutathione (NIH 3T3) or GST-GRB2 fusion protein (PAE). The amount S-transferase (GST) fusion protein which contains the SH2 of coprecipitated PDGF 13-receptor (NIH 3T3) was quanti- domain of GRB2 (27) was used. Bacterial cultures of 500 ml tated with an enhanced laser densitometer and compared with were grown for 2.5 h at 30°C and were induced with 0.1 mM the amount of coprecipitated PDGF (3-receptor incubated with isopropylthiogalactopyranoside (IPTG) for 5 h. Cells were unphosphorylated peptides. The amount of bound PDGF pelleted and resuspended in 10 ml of lysis buffer (PBS, 1% ,3-receptor (PAE) was quantitated as described above and Triton X-100, 2 mM EDTA, 0.2 mM PMSF, 0.1% mercapto- compared with the amount of PDGF 13-receptor bound to ethanol). Resuspended cells were sonicated for 10 min on ice wheat germ agglutinin (WGA)-agarose (EC Diagnostics AB, and cleared of debris by centrifugation for 10 min at 10,000 x Uppsala, Sweden), analyzed in parallel with the same washing g. Cleared lysates were incubated with 2.5 ml of 50% (vol/vol) conditions. glutathione-Sepharose 4B (Pharmacia) for 30 min at 4°C. The Identification of phosphorylation sites by Edman degrada- Sepharose was then washed four times with cold lysis buffer, tion. To label the autophosphorylation sites in the PDGF and fusion proteins were eluted with two 2-ml washes with 3-receptor, PAE cells expressing wild-type ,B-receptors were freshly made elution buffer (50 mM Tris-HCl [pH 8.0], 20 mM rinsed twice with phosphate-free Ham's F12, incubated for 2.5 glutathione). h at 37°C in Ham's F12 medium supplemented with 20 mM Binding of fusion proteins to immobilized phosphopeptides. HEPES (pH 7.4), 0.1% dialyzed FCS, and 1 mCi of 32Pi Purified GST-GRB2 fusion protein was incubated for 2 h with (Amersham) per ml, and subsequently stimulated with 100 ,ul of 50% (vol/vol) AH-Sepharose 4B (Pharmacia) to PDGF-BB (100 ng/ml) for 60 min at 4°C. Cells were rinsed which the different synthetic peptides had been coupled indi- with PBS and lysed in a buffer containing 20 mM HEPES (pH vidually. Beads were washed 10 times with lysis buffer (20 mM 7.5), 150 mM NaCl, 1% Nonidet P-40, 10% glycerol, 1 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1% Triton X-100, 10% dithiothreitol, 100 puM Na3VO4, 1% Trasylol, and 1 mM glycerol, 1% Trasylol, 1 mM PMSF, 100 puM Na3VO4), and PMSF. The cell lysates were centrifuged and incubated with bound proteins were eluted by boiling 5 min in SDS-sample PDGFR-3 and protein A-Sepharose CL-4B. The immobilized buffer. The proteins were separated by SDS-gel electrophore- immunoprecipitates were washed three times with lysis buffer sis, using a gel consisting of 7% polyacrylamide, and then and eluted with SDS sample buffer. Samples were subjected to electrophoretically transferred to nitrocellulose membranes SDS-gel electrophoresis in a gradient gel consisting of 5 to (Amersham), which were blocked with 3% bovine serum 10% polyacrylamide; the material was then electrotransferred albumin in PBS and 0.2% Tween 20 (PBS-T) overnight at 4°C. to nitrocellulose membranes. After exposure to film, the filter Membranes were incubated with a GRB2 antiserum at a piece corresponding to the PDGF receptor was cut out and 1:1,000 dilution in PBS-T for 1 h at room temperature. After incubated with 0.5% polyvinylpyrrolidone-40-0.6% acetic acid incubation, membranes were rinsed twice, washed three times for 30 min at 37°C essentially as described by Aebersold et al. for 10 min each time in PBS-T, and then further incubated in (1). The filter piece was washed three times with water and the presence of peroxidase-conjugated sheep anti-mouse im- incubated for 12 h at 37°C with 200 pL. of 50 mM ammonium munoglobulin at a 1:1,000 dilution in PBS-T for 1 h at room bicarbonate containing 1 p.g of modified sequencing-grade temperature. The membranes were again rinsed twice, washed trypsin (Promega). The digest was lyophilized, oxidized with extensively for 90 min in PBS-T, and developed with chemilu- performic acid, lyophilized again, and then resuspended in 50 minescence reagents (ECL kit; Amersham). To quantitate the p.1 of 50 mM ammonium bicarbonate and digested with trypsin binding observed in the Western blot (immunoblot), we used for another 12 h at 37°C. an enhanced laser densitometer (LKB Ultroscan XL). For immunoprecipitation of tryptic peptides, 50 p.l of car- Detection of GRB2-PDGF 18 receptor complex formation by rier-bound a2-macroglobulin (Boehringer Mannheim) was immunoblotting and peptide inhibition. Confluent monolayers added to the tryptic digest, and incubation was continued for of NIH 3T3 cells or of PAE cells expressing wild-type or 30 min at 4°C. The beads were spun down, and the protease- different mutant receptors were grown in 15-cm-diameter petri free supernatant was transferred to a new tube. After being dishes (Falcon). After the cells were starved overnight in washed once with 50 mM ammonium bicarbonate, superna- culture medium without serum, PDGF-BB was added to a final tants were combined and 20 p.l of antiserum HL-2 was added. concentration of 100 ng/ml; cells were then incubated at 37°C After 2 h of incubation at 4°C, 50 p.1 of a 1:1 slurry of protein for 5 min (NIH 3T3) or at 4°C for 1 h and at 37°C for 8 min A-Sepharose CL-4B was added and incubation was continued 6718 ARVIDSSON ET AL. MOL. CELL. BIOL. at 4°C for 30 min. The immunoprecipitate was washed two extracellular TM intracellular times with 0.05% Triton X-100-50 mM ammonium bicarbonate, and the immunoprecipitated peptides were eluted with JM TK1 KI TK2 CT 70% formic acid. Lyophilized peptides were subjected to ILLIjZe~ ZZJ PDGFR3 phosphoamino acid analysis as described by Boyle et al. (7). / \ For peptide sequencing, lyophilized peptides were coupled to /\ a Sequelon-AA membrane (Milligen/Bioresearch, Milford, Mass.) according to the manufacturer's instructions, and Ed- Y71 6F man degradation was performed in an Applied Biosystems V PDGFRPY71 6F gas-phase sequencer modified for solid-phase sequencing. In- Y740F dividual fractions were spotted onto a thin-layer chromatogra- PDGFR3 Y740F phy (TLC) cellulose plate (Merck), dried, and exposed by Y751 F using a PhosphorImager. Radioactivity in the individual fractions was quantified with the Image Quant program. PDGFR Y751F Analysis of Ras-bound GDP and GTP. Human foreskin Y740/751 F fibroblasts (AG1523) and PAE cells were cultured in Eagle's PDGFRP Y740/751 F minimal essential medium supplemented with 10% fetal bo- Y763F vine serum, 100 U of penicillin per ml, and 50 ,ug of strepto- PDGFRO Y763F mycin per ml. The cells were serum starved in MCDB 104 containing 0.5 mM CaCl2 for 48 h prior to the experiment. Y771F Permeabilization with streptolysin 0, immunoprecipitation of PDGFRP Y771 F Ras proteins, and determination of the amount of associated Y775/778F labeled GTP and GDP were performed essentially as previ- PDGFRO Y775/778F ously described The cells were with 0.5 (14, 35). permeabilized FIG. 1. Schematic illustration of wild-type and mutated PDGF (AG1523) or 0.2 (PAE) U of streptolysin 0 per ml for 5 min ,B-receptors. The different regions in the intracellular domains are at 23°C (AG1523) or 37°C (PAE), and peptides were added at indicated. TM, transmembrane domain; JM, juxtamembrane domain; 20 ,uM. After 2 min of incubation, 20 ng of PDGF-BB or TK1, tyrosine kinase domain 1; KI, kinase insert; TK2, tyrosine kinase vehicle per ml and 10 ,uCi of [ot-32P]GTP per ml were added. domain 2; CT, C-terminal tail. Arrows indicate the positions of The cells were transferred to 4°C, lysed, and immunoprecipi- tyrosine residues in the kinase insert that were replaced with phenyl- tated with antibody Y13-259. The Ras-bound guanosine nucle- alanine residues. otides were eluted from the immunoprecipitates and applied onto polyethyleneimide-cellulose TLC plates. The TLC plates were exposed to X-Omat film (Kodak), and the spots corre- 110 kDa, most probably representing the regulatory subunit sponding to GTP and GDP were identified. Those areas were (p85) and the catalytic subunit (p110) of P13-kinase, were excised, and the amount of radioactivity was determined by found to associate with all mutant receptors except PDGFR scintillation technique. ,BY740/751F. In vitro P13-kinase assays were also performed to determine the abilities of the different mutant receptors to RESULTS bind and activate P13-kinase. Consistent with previous findings (16, 22, 54), cells expressing PDGFR1Y740/751F failed to Expression and characterization of mutant PDGF ,I-recep- display any ligand-stimulatable P13-kinase activity (Fig. 2B), tors with tyrosine residues in the kinase insert replaced with whereas similar amounts of phosphorylated PI were formed in phenylalanine residues. The kinase insert of the PDGF ,B-re- all the other mutants. ceptor has been shown to play an important role in signal GRB2-SH2 domain binds to a peptide containing phosphor- transduction. Binding sites for several signal transduction ylated Tyr-716. Different SH2 domains bind in a highly specific molecules, i.e., p120 GAP, P13-kinase, and Nck, are present in manner to phosphotyrosine residues and flanking amino acid this domain. In this work, we have analyzed the characteristics residues. In particular, the three amino acid residues C termi- of cell lines expressing mutated PDGF 13-receptors in which nal of the phosphotyrosine residue appear to be directly each tyrosine residue within the kinase insert was changed, involved in binding (47, 49, 50). Using a peptide library for individually or in combination, to phenylalanine residues (Fig. binding of GRB2 SH2, Songyang et al. (49) identified the motif 1). The mutant PDGF 1-receptors Y716F, Y740F, Y751F, Y(P)-L/V-N-g/p as important for GRB2 binding. The strongest Y740/751F, Y763F, Y771F, and Y775/778F were generated by selection was for N in position +2 in relation to the phospho- site-directed mutagenesis and stably introduced into PAE cells. tyrosine. Phosphotyrosine 1068 in the EGF receptor is sur- Fourteen different clones with similar numbers of receptors rounded by Y(P)-I-N-Q and has been shown to constitute a and similar rates of receptor synthesis were chosen for further binding site for GRB2 (8). In the PDGF 13-receptor intracel- characterization. lular domain, two tyrosine residues are flanked by N in position To ensure that the mutated PDGF 3-receptors could func- +2, Tyr-716 (Y-S-N-A) and Tyr-775 (Y-D-N-Y). To analyze tion as ligand-stimulatable tyrosine kinases, in vitro kinase whether the GRB2-SH2 domain has the capacity to bind to assays were performed as previously described (3). As shown in these sites as well as to other known tyrosine phosphorylation Fig. 2A, all of the different mutant receptors became autophos- sites in the 1-receptor, we incubated GST-GRB2 SH2 fusion phorylated in response to PDGF. The requirement for binding protein with immobilized phosphorylated peptides from differ- of p120 GAP was investigated by immunoprecipitating the ent regions of the receptor (see Materials and Methods), and different PDGF 13-receptor mutants with an anti-GAP anti- the amount of GST-GRB2 fusion protein bound to the beads serum followed by an in vitro kinase assay. As seen in Fig. 2A, was determined by immunoblotting. We found that peptides only the mutant PDGFR1Y771F was unable to associate with Y716(P) and Y740(P) bound the GST-GRB2 fusion protein, p120 GAP, in agreement with previous reports (16, 22). while peptides Y751(P), Y775(P), or Y1009(P) did not bind Components with molecular masses of approximately 85 and (Fig. 3). The corresponding nonphosphorylated versions of the VOL. 14, 1994 GRB2 BINDS DIRECTLY TO THE PDGF (-RECEPTOR 6719

t peptides failed to bind GST-GRB2 SH2. To explore the A possibility that an additional molecule was involved in the N- N- (2 N-N binding of GRB2 to the PDGF (3-receptor, we added cell lysate n a-o c c from unstimulated PDGF (-receptor expressing cells to a aGAP B2 aGAP B2 aGAP B2 aGAP B2 -GAP B2 -GAP B2 parallel series of incubations of fusion protein and Sepharose- PDGF-BB + + coupled peptides. The addition of cell lysate did not enhance or specifically change the efficiency of binding to any of the PDGFR tested peptides (Fig. 3). We also tested phosphopeptides corresponding to other known autophosphorylation sites, Y579(P), Y581(P), Y771(P), and Y1021(P), of which none was found to bind GST-GRB2 fusion protein (data not shown). pl 20 GAP Tyr-716 is phosphorylated in vivo in response to PDGF. The results from the experiment with immobilized peptides suggested that Tyr-740 and Tyr-716 may be involved in binding of GRB2. Tyr-740 has been shown to be phosphorylated in vivo (16, 22). To determine whether Y-716 is phosphorylated in vivo, "Pi-labeled cells expressing wild-type or PDGFR,BY716F receptors were stimulated with PDGF-BB; the (3-receptor was then immunoprecipitated, isolated by SDS-gel electrophoresis, and transferred to a nylon membrane. After tryptic digestion of

N- N- rs s~~~~~~~~r the material on the membrane, the tryptic fragments were B cc 0 m - N- N- N N- N- immunoprecipitated by antiserum HL-2 and subjected to >- N- r- >- 2- a= Ca phosphoamino acid analysis and Edman degradation. Phos- cc v c. Cv c phoamino acid analysis of the immunoprecipitates revealed

PDGF-BB + - + - + - + - + + that Tyr was the only amino acid phosphorylated both in wild-type and mutant receptors under the conditions used. PIP 990 099 Since antiserum HL-2 recognizes amino acid residues 701 to 732 in the human (3-receptor, tryptic fragments comprising amino acids 698 to 707 or 698 to 708 (without tyrosine residues) and 708 to 758 or 709 to 758 (containing Tyr-716, Tyr-741, and Tyr-751) can potentially be recognized. Edman Ori * eO@e* * . degradation of the material obtained from the wild-type receptor revealed most radioactivity in cycle 8, which is the expected FIG. 2. Characterization of PDGF (3-receptor mutants in which position of phosphorylated Tyr-716, in peptide 709-758 (Fig. tyrosine residues in the kinase insert have been replaced with phenyl- in alanine residues. (A) PAE cells expressing wild-type or mutant PDGF 4). Tyr-740 and Tyr-751, which are phosphorylated vivo, (-receptors were stimulated with PDGF-BB, lysed, and immunopre- would appear in cycles 32 and 45, respectively. Because of the cipitated with a GAP antiserum (aGAP) or the monoclonal antibody loss of material with increasing cycle number, they could not be PDGFR-B2 (B2). The precipitated material was analyzed by an in detected. Edman degradation of material obtained from the vitro kinase assay followed by SDS-gel electrophoresis and autoradiog- PDGFR,BY716F mutant-expressing cells did not show any raphy. The positions of PDGFR( and p120 GAP are indicated. (B) clear peak of radioactivity in fraction 8 (Fig. 4). Taken TLC of PI3-kinase reaction products from unstimulated or ligand- together, these data strongly suggest that Tyr-716 is used as an stimulated cells. Cells expressing either wild-type or mutant PDGF autophosphorylation site in the PDGF (-receptor in vivo. (-receptors were incubated without (-) or with (+) PDGF-BB. The The PDGF P-receptor mutant Y716F fails to associate with cell lysates were then immunoprecipitated with antiserum PDGFR-3. whether Immune complexes were subjected to a P13-kinase assay, and the GRB2 upon ligand binding. To investigate phosphor- PI3-kinase reaction products were analyzed by TLC and autoradiog- ylated Tyr-716 or Tyr-740 in the intact PDGF (-receptor is raphy. The positions of phosphatidylinositol phosphate (PIP) and the critical for interaction with GRB2, we compared the abilities of origin (Ori) are indicated. wild-type and mutant PDGF (3-receptors to associate with the GST-GRB2 fusion protein. PAE cells expressing the wild-type receptor or different PDGF (3-receptor mutants were stimu-

R ;a. .1. M 1- m M. F. ,, ,, 10 CD cl- - ;C- r) C-D CD VI CD 0 z :_- z ", r- C) 0 :,- E: 2 2 - r, -4 14 >. >1 >. >. >1 >1 >, >. >1 N- N- N >1 >1 >1

I 4bI4I~~~~ N .9N6

no addi-ior + cell lysate FIG. 3. Association of the GRB2 SH2 domain to phosphotyrosine-containing peptides. GST-GRB2 SH2, in the absence or presence of PDGF (3-receptor cell lysate, was incubated with a series of unphosphorylated or phosphorylated peptides coupled to AH-Sepharose 4B. Material bound to the Sepharose beads was separated by SDS-gel electrophoresis, transferred onto a nitrocellulose membrane, and immunoblotted with an anti-GRB2 antiserum, using the ECL detection system. 6720 ARVIDSSON ET AL. MOL. CELL. BIOL.

PA A:

8 OPT 'C". . 0E 'Zvpy

0=n 4_

2 _

0 _. Eq -MU cycle #: 1 2 3 4 5 6 7 8 9 10 11 12 13 14 tryptic fragments: R R P P S A E L Y S N A L P V 716 K R R P P S A E L Y S N A L P 716 K H T F L Q H H S D K FIG. 4. Edman degradation of in vivo-autophosphorylated PDGF 1-receptor. PAE cells expressing the wild-type PDGF 3-receptor or the PDGFR,BY716F mutant were labeled with 32Pi, stimulated with PDGF-BB, lysed, and immunoprecipitated with antiserum PDGFR-3. The immunoprecipitates were separated by SDS-gel electrophoresis and transferred onto a nitrocellulose filter, and the band corresponding to the phosphorylated receptor was cut out and digested with trypsin. The tryptic fragments of the phosphorylated wild-type ,B-receptors (solid bars) and of the PDGFR13Y716F mutant (hatched bars) were immunoprecipitated with antiserum HL-2 (recognizing amino acid residues 701 to 732). Radioactivity in individual cycles following Edman degradation of the 32P-labeled tryptic peptides immunoprecipitated with HL-2 is shown. The amino acid sequences of the potentially immunoprecipitated tryptic fragments are shown below the cycle numbers. The inset shows phosphoamino acid analysis (PAA) of HL-2-immunoprecipitated tryptic peptides. pS, phosphoserine; pT, phosphothreonine; pY, phosphotyrosine.

lated with PDGF-BB, and lysates from these cells were incu- receptors, Y771F and Y775/778F, displayed slightly reduced bated with GST-GRB2 Sepharose. Bound proteins were ana- efficiencies of association with GRB2 SH2. We conclude that lyzed by immunoblotting with phosphotyrosine antibody PY20. in PAE cells, Tyr-716 appears to be of major importance for Five percent of the total cell lysate was incubated with WGA- GRB2 binding in vitro. agarose in order to estimate the relative number of activated We next used phosphorylated peptides (see Materials and PDGF 3-receptors in each sample. As shown in Fig. 5, the Methods) corresponding to tyrosine residues 716 and 1009 of wild-type as well as the PDGF 1-receptor mutants Y740F, the PDGF ,B-receptor in blocking experiments. PAE cells Y751F, Y771F, Y775/778F, and Y1009F bound to GST- expressing the PDGF 1-receptor were stimulated with PDGF- GRB2-Sepharose with 0.3 to 2.6% efficiency compared with BB, and the cell lysate was incubated with different concentra- WGA-agarose adsorption, while the PDGFR1Y716F mutant tions of phosphorylated peptides, or the corresponding un- bound with only 0.03% efficiency. Other PDGF 1-receptor phosphorylated peptides for reference, prior to incubation mutants (Y579F, Y581F, Y763F, and Y1021F) bound to with GST-GRB2 SH2-Sepharose. Bound proteins were ana- GST-GRB2-Sepharose as efficiently as the wild-type receptor lyzed by SDS-gel electrophoresis, transferred to nitrocellulose (data not shown). Two additional tyrosine-phosphorylated membranes, and incubated with phosphotyrosine antibody proteins of 70 and 110 kDa were detected in samples from the PY20. As shown in Fig. 6A, the Y716(P) peptide significantly wild-type receptor cells as well as from the PDGFR1Y716F reduced the binding of GRB2 SH2 to the PDGF ,3-receptor, mutant cells, suggesting that GRB2 interacts directly with while the Y1009(P) peptide did not affect this binding. The these proteins. In samples from the PDGFR,BY1009F mutant Y1009(P) peptide reduced but did not completely inhibit cells, the intensity of the 70-kDa band was reduced, whereas binding of both 70- and 110-kDa components to GRB2 SH2. the PDGF 13-receptor band and the 110-kDa band remained. It To confirm the in vitro binding data, GRB2 was immuno- has previously been shown that the phosphotyrosine phos- precipitated from lysates obtained from PDGF-BB-stimulated phatase PTP1D/Syp, which associates with the PDGF 1-recep- NIH 3T3 cells incubated with 25 ,uM unphosphorylated or tor via phosphorylated Tyr-1009 (23, 25), can mediate binding phosphorylated peptide prior to the addition of antiserum. The of GRB2 in dog kidney epithelial cells (TRMP cells) (27). advantage of using NIH 3T3 cells instead of PAE cells in this Reprobing the filter in Fig. 5 with an anti-Syp antiserum experiment was that the GRB2-PDGF 13-receptor complex confirmed that the 70-kDa band indeed was immunologically appeared to be subject to a rapid turnover in the PAE cells related to Syp (data not shown). Consistent with the possibility (data not shown). The GRB2 immunoprecipitates from NIH that part of the binding of GRB2 can occur indirectly via Syp, 3T3 cells were analyzed by immunoblotting with phosphoty- the PDGFR,BY1009F mutant receptor associated slightly less rosine antibody PY20. A 190-kDa phosphoprotein correspond- with the GST-GRB2 fusion protein compared with the wild- ing to the PDGF 13-receptor was detected in GRB2 immuno- type receptor (0.3% versus 1.2%; Fig. 5). Two other mutant precipitates obtained from PDGF-BB-stimulated NIH 3T3 VOL. 14, 1994 GRB2 BINDS DIRECTLY TO THE PDGF 1-RECEPTOR 6721

PDGFR-I1 T1tirarits Y71 6F Y740F Y75 1F Y 1F 877 iSF''i'S 'FN

li 7 zr: 0 ..n 7 ~.r -1 Co m < x J:1) Affinity matrix a: z I- a: I ?: 0 3: C-1- _A l%4w[# Stw7't:~~~~~~~4-M

IN,, b. }i. i I 1 kDi

a a a LIJ Dt) k I - 0- iio 0 4,

Relative bindirng 0.03% 100% 1 .3% 1 00% 2.6% 1 00%/6 0,5%CI 1 00%Jc 0.4-: 00-c 0.3`% 1 00% 1 .2C/% 1 00%wo

FIG. 5. Association of GST-GRB2 SH2 with PDGF ,B-receptor mutants. PAE cells expressing wild-type PDGF 1-receptor or mutant receptors in which tyrosine residue 716, 740, 751, 771, 775/778, or 1009 was replaced with phenylalanine residues were stimulated with PDGF-BB, lysed, and incubated with GST-GRB2 SH2. The precipitated material was separated by SDS-gel electrophoresis, transferred onto a nitrocellulose membrane, and immunoblotted with antiserum PY20. The positions of the PDGF 1-receptor and of the 110- and 70-kDa components are indicated. For comparison, aliquots of the lysates were incubated with WGA-agarose; the extent of PDGF receptor binding to GRB2-SH2 is expressed as a percentage of the binding to WGA, which efficiently binds the receptor. cells incubated with Y716, Y1009, and Y1009(P) peptides (Fig. expressed in PAE cells. We have previously shown that PDGF 6B). In contrast, 25 ,uM Y716(P) peptide (Fig. 6B) reduced the 13-receptor mutants in which autophosphorylated residues in ability of the receptor to bind GRB2 to 12% of the binding the juxtamembrane region (Tyr-579 and Tyr-581 [33]) and in seen in the presence of unphosphorylated peptide. These data the C-terminal tail (Tyr-1009 and Tyr-1021 [40]) were mutated show that complexes containing GRB2 and the PDGF ,B-re- to phenylalanine residues are capable of mediating a mitogenic ceptor exist in the intact cell and that Tyr-716 is mediating response. The ability of PDGF-BB to stimulate DNA synthesis complex formation. of PAE cells expressing mutant PDGF 13-receptors in which Tyr-716 in the PDGF 1-receptor is involved in Ras activa- tyrosine residues in the kinase insert were mutated to phenyl- tion. PDGF stimulation increases the level of GTP bound to alanine residues was investigated by a [3H]thymidine incorpo- Ras. To evaluate the effects of various phosphopeptides on ration assay. As shown in Fig. 8, cells expressing each of the Ras activation, human foreskin fibroblasts and PAE cells mutant receptors were found to respond to stimulation by expressing wild-type PDGF 13-receptors were examined. Dif- PDGF-BB with increased incorporation of [3H]thymidine. In ferent phosphopeptides (based on the sequence around Tyr- agreement with our previous results (54), the PDGFR1Y740/ 579, Tyr-581, Tyr-716, Tyr-740, Tyr-751, Tyr-771, Tyr-775, 751F cells showed a slight decrease in incorporation of Tyr-1009, and Tyr-1021 in the PDGF 1-receptor and Tyr-1068 [3H]thymidine compared with wild-type receptor cells. in the EGF receptor) or control peptides (the corresponding unphosphorylated forms), PDGF-BB, and [a-32P]GTP were DISCUSSION added to the permeabilized cells. After 2 min, the cells were transferred to 4°C, lysed, and immunoprecipitated with the In this study, we identify Tyr-716 in the PDGF 1-receptor Ras antibody Y13-259, and the amount of labeled nucleotide kinase insert as a novel autophosphorylation site. Further- bound to Ras was determined. In human fibroblasts (Fig. 7A) more, our data indicate that GRB2 associates directly with the as well as in PAE cells (Fig. 7B), the phosphopeptide Y716(P), activated PDGF 1-receptor via Tyr-716. Finally, we present but not the corresponding unphosphorylated peptide, signifi- evidence that Tyr-716 is a dominant site for connecting the cantly reduced the increase of GTP bound to Ras. Addition of PDGF 1-receptor to the Ras pathway. phosphopeptide Y1009(P) slightly reduced the level of GTP A major mitogenic pathway employed by receptor tyrosine bound to Ras in fibroblasts, whereas the peptide corresponding kinases involves the activation of Ras. Ras is active in its to the GRB2 binding domain in the EGF receptor [Y1068(P) GTP-bound form and inactive when binding GDP. After (8)] efficiently blocked the PDGF-induced formation of Ras- activation of the PDGF 1-receptor, a rapid increase in the GTP. The extent of inhibition of GTP loading was in the same amount of GTP bound to Ras is detected (20, 42). By using order of magnitude for different concentrations of Y1068(P) microinjection of Ras monoclonal antibodies or expression of and Y716(P) (data not shown), indicating similar affinities of a dominant-negative Ras protein, the PDGF-stimulated mito- these phosphopeptides for GRB2 SH2 binding. All other genic response can be blocked (34). Recent reports have tested PDGF 13-receptor peptides lacked significant effect on shown that the adaptor molecule GRB2 functions as a direct the Ras-GTP level in PAE cells, irrespective of state of link between receptor tyrosine kinases and the Ras pathway phosphorylation. (28, 52). GRB2 binds to phosphorylated receptor tyrosine Induction of DNA synthesis by mutated PDGF P-receptors kinases via its SH2 domain, while its SH3 domains bind to the 6722 ARVIDSSON ET AL. MOL. CELL. BIOL.

A (43). In PAE cells, a pathway depending on the P13-kinase

-~w 0 MO binding site appears not to be dominating. 0 0 peptide Z- r- r Moreover, it was recently shown that Tyr-1009 is involved in binding PTP1D/Syp (23, 25) and that PTP1D/Syp interacts AwwmmLA.--. ..l &.-A PDGFR with the GRB2-Sos complex (27). A PDGF 3-receptor mutant VP-' lacking a number of known autophosphorylation sites (Tyr- 740, Tyr-751, Tyr-771, and Tyr-1021) but retaining Tyr-1009 showed an increase in the GTP bound Ras in response to PDGF compared with a mutant simultaneously changed at all five tyrosine residues (51). These data suggest that recruitment

110 kDa of GRB2-Sos to the PDGF 1-receptor-Syp complex may ow owt i also activate the Ras pathway. In agreement with this notion, we observed a slight decrease in the amount of GTP bound to Ras in permeabilized human fibroblasts in response to PDGF stimulation by a phosphorylated peptide encompassing Tyr-1009 (Fig. 7A). However, in permeabilized PAE cells the PDGF the phosphorylated Aft.. 70 kDa expressing wild-type 13-receptor, 4a'. MGM Tyr-1009 peptide had no effect (Fig. 7B). Moreover, the phosphorylated Tyr-1009 peptide was not able to efficiently block the in vitro or in vivo association of GRB2 SH2 to the PDGF 13-receptor in these cells (Fig. 6). This observation indicates that the contribution of different signal transduction pathways B leading to activation of Ras may differ between cell types. a_cn In addition to associating with PTP1D/Syp, GRB2 binds 0 0

N tyrosine phosphorylated Shc (39). Overexpression of Shc leads peptide >- >- > > to transformation of 3T3 cells and Ras-dependent neurite |W W #"fP|GPDGFR outgrowth of PC-12 cells (37, 39). So far, Shc has been found to associate directly with the EGF receptor and the ErbB-2 Relative binding 100% 12% 100% 70% receptor (37, 39, 45) and has recently also been found to associate with the PDGF 1-receptor (56). Several autophos- FIG. 6. Effects of receptor phosphopeptides on the complex formation of GRB2 and wild-type PDGF 1-receptor. (A) Wild-type phorylation sites (Y-579, Y-740, Y-751, and Y-771) in the PDGF 13-receptors were stimulated with PDGF-BB, lysed, and incu- PDGF 13-receptor were shown to be involved in the binding of bated with an unphosphorylated or phosphorylated peptide containing Shc, but only the PDGF 1-receptor mutant Y579F showed a Tyr-716 or Tyr-1009, as indicated, prior to addition of GST-GRB2 partially impaired binding of Shc in vivo (56). Even though all SH2. The samples were separated by SDS-gel electrophoresis, trans- Shc binding sites are present in PDGFR1Y716F, the ability of ferred onto a nitrocellulose membrane, and immunoblotted with this mutant to bind GRB2 SH2 is lost. Whereas a major role antiserum PY20. The positions of the PDGF 1-receptor and of the for Shc in mediation of PDGF-induced Ras activation there- 110- and 70-kDa components are indicated. (B) NIH 3T3 cells were stimulated with PDGF-BB, lysed, and incubated with unphosphory- fore seems unlikely, it is still possible that the low-affinity lated or phosphorylated peptides containing Tyr-716 or Tyr-1009 prior interaction of Shc to multiple sites in the receptor allows to the addition of GRB2 antiserum. The precipitated material was indirect coupling to GRB2 in the living cell. separated by SDS-gel electrophoresis, transferred onto a nitrocellulose PDGF triggers a variety of cellular responses. One rapid membrane, and immunoblotted with antiserum PY20. The coprecipi- response which occurs within minutes after stimulation is the tated PDGF 13-receptors are indicated. The extent of GRB2-PDGF reorganization of actin stress fibers and formation of mem- 13-receptor complex formation is expressed as a percentage of the brane ruffles. We have previously shown that a receptor mutant complex formation obtained with lysates incubated with unphosphor- lacking the binding site for P13-kinase, PDGFR,BY740/751F, ylated peptide. failed to mediate edge ruffle formation and circular ruffles as well as chemotaxis (54). All other PDGF 13-receptor mutants examined in this study showed edge ruffle formation to the nucleotide exchange factor Sos. Through these interactions, same extent as the wild-type receptor. The frequency of Sos is recruited to the plasma membrane and is able to induce formation of circular ruffles was decreased in several kinase Ras activation by replacing GDP with GTP. Here we show that insert mutant receptors (data not shown and Table 2). Re- a phosphorylated peptide encompassing Tyr-716 in the PDGF cently, Matuoka and coworkers (29) showed that microinjec- 1-receptor kinase insert significantly inhibits activation of Ras tion of an Ash/GRB2 antibody abolished both PDGF-stimu- by PDGF in permeabilized PAE cells as well as in fibroblasts lated mitogenesis and ruffle formation in normal rat kidney- (Fig. 7). The corresponding unphosphorylated peptide and a derived fibroblasts. Even though PDGFR13Y716F fails to series of peptides encompassing known autophosphorylation associate with GRB2, this mutant receptor, expressed in PAE sites in the PDGF 1-receptor failed to affect the level of GTP cells, has intact capacity to form ruffles and to mediate bound to Ras, irrespective of state of phosphorylation. There- chemotaxis. It will be interesting to analyze the effect of the fore, Tyr-716 is a critical structural component in PDGF- Y716F mutant receptor on motility responses after expression induced Ras activation. Other signal transduction pathways in other cell types. have previously been implicated in Ras activation. Thus, The binding site for GRB2 in the EGF receptor has been PDGF 13-receptor mutants lacking the binding site for P13- mapped to Tyr-1068, and a peptide containing phosphorylated kinase (Tyr-740 and Tyr-751) are defective in mediating Ras Tyr-1068 inhibits nucleotide exchange on Ras (8). A mutant activation, depending on the cell type used for transfection EGF receptor lacking all autophosphorylation sites is, how- A I T IT I T T

la 0 I I a. Y. a VI-

I-

.~~~~ae. VI_£VI | V- V- + IL Pepde 111-- i VI V

PDGF-BB I + + + + + +

B * No peptide. no PDGF-BB *- No peped& 400

300 a.-E 0 w- a. cJO.0 CD a- 200 0-

+ 0C 0-

100

0 5 10 15 20 Time (min) FIG. 7. Effects of receptor phosphopeptides on PDGF-BB induced activation of Ras in intact cells. (A) Human fibroblasts were permeabilized with streptolysin 0 for 5 min, at which point a 20 ,M concentration of the indicated phosphorylated (hatched bars) or nonphosphorylated (shaded bars) receptor peptide was added. PDGF-BB (20 ng/ml) and [a-32P]GTP (10 p.Ci/ml) were added after 2 min of incubation, and the amount of32P radioactivity associated with Ras after 5 mm offurther incubation was determined. The amount of GTP plus GDP bound to Ras is calculated as the fold increase over the control value and represent the mean and standard error from four individual experiments. (B) [a-32P]GTP/GDP binding to Ras in streptolysin 0-permeabilized PAE cells was measured in the presence ofvarious peptides. The amount of labeled GTP plus GDP associated with Ras is indicated. Except for cells not treated with PDGF-BB, all conditions included stimulation with PDGF-BB for different time periods, as indicated. Y1068 is a tyrosine phosphopeptide based on the sequence of the EGF receptor autophosphorylation site involved in GRB2 binding. EGFR, EGF receptor. 6723 6724 ARVIDSSON ET AL. MOL. CELL. BIOL.

- 100% U

*- _80% M 6 o .0 ou Xo 60 % 0 a 5 D Eg +m 40%040 % 4-a4 '.ai 20%

-E E E--EE-E - 1J E17

0 "0 00 r- 0 0 0.W- 0 00 V 0 0 0.- 000 r-00

Y716F Y740/751 F Y763F Y771 F Y775/778F wild-type FIG. 8. Stimulation of [3H]thymidine incorporation by PDGF-BB in PAE cells expressing wild-type or mutant PDGF 1-receptors. Serum-starved cells were incubated with [3H]thymidine and 1 or 10 ng of PDGF-BB per ml or 10% FCS for 24 h. Trichloroacetic acid-precipitable radioactivity was determined and expressed as percentage of the incorporation in cells stimulated with 10% FCS.

ever, still mitogenically active, in spite of the apparent central REFERENCES role of Ras in mitogenic signal transduction (19). This finding 1. Aebersold, R. H., J. Leavitt, R. A. Saavedra, L. E. Hood, and is similar to our results for the mutant PDGFR,BY716F, which S. B. H. Kent. 1987. Internal amino acid sequence analysis of mediated increased DNA synthesis in response to PDGF and proteins separated by one- or two-dimensional gel electrophoresis thus is mitogenically active (Table 2). A common event for after in situ protease digestion on nitrocellulose. Biochemistry signaling by many mitogenic stimuli is activation of the MAP2 84:6970-6974. kinase cascade (for a review, see reference 13). Several Ras- 2. Andrews, D. M., J. Kitchin, and P. W. Seale. 1991. Solid-phase dependent and -independent pathways are probably operating synthesis of a range of O-phosphorylated peptides by post-assem- to control MAP2 kinase activation (6). In accordance, PDGFR bly phosphitylation and oxidation. Int. J. Pept. Protein Res. 38:469-475. 1Y716F and the other PDGF 1-receptor mutants tested 3. Arvidsson, A.-K., C.-H. Heldin, and L. Claesson-Welsh. 1992. (Y579, Y740F, Y751F, Y1009F, and Y1021F) induced in- Transduction of circular membrane ruffling by the platelet-derived creased phosphorylation of MAP2 kinase (data not shown). growth factor 3-receptor is dependent on its kinase insert. Cell The data presented in this report, taken together with data Growth Differ. 3:881-887. from other studies, support the notion that growth factor- 4. Basu, T. N., D. H. Gutmann, J. A. Fletcher, T. W. Glover, F. S. induced mitogenic signaling involves the activation of several Collins, and J. Downward. 1992. Aberrant regulation of ras parallel and interconnecting pathways. proteins in malignant tumour cells from type 1 neurofibromatosis patients. Nature (London) 356:713-715. ACKNOWLEDGMENTS 5. Boguski, M. S., and F. McCormick. 1993. Proteins regulating Ras and its relatives. Nature (London) 366:643-654. We thank Ulla Engstrom for peptide synthesis and Christer Wern- 6. Boudewijn, M. T. B., A. M. M. de Vries-Smits, R. H. Medema, P. C. stedt for oligomer synthesis, peptide sequencing, and analyses by van Weeren, L. G. J. Tertoolen, and J. L. Bos. 1993. Epidermal Edman degradation. growth factor induces phosphorylation of extracellular signal- This work was supported in part by the Swedish Natural Science regulated kinase 2 via multiple pathways. Mol. Cell. Biol. 13:7248- Research Council (E.N.) and Axel and Margaret Ax:son Johnsson 7256. Foundation. 7. Boyle, W. J., P. Van der Geer, and T. Hunter. 1991. Phosphopep- tide mapping and phosphoamino acid analysis by two-dimensional separation on thin-layer cellulose plates. Methods Enzymol. 201: TABLE 2. Effects of PDGF-BB on [3H]thymidine incorporation 110-149. and ruffling in different PDGF l-receptor mutants 8. Buday, L., and J. Downward. 1993. Epidermal growth factor regulates p2lras through the formation of a complex of receptor, Mutant DNA Edge Circular Reference(s) Grb2 adapter protein, and Sos nucleotide exchange factor. Cell synthesis ruffling ruffling or source 73:1-20. Y579F ++ ++ ++ 32,52 9. Claesson-Welsh, L. 1994. Signal transduction by the PDGF recep- Y581F ++ ++ ++ 32, 52 tors. Prog. Growth Factor Res. 5:37-54. Y716F ++ ++ +- This study 10. Claesson-Welsh, L., A. Eriksson, A. Moren, L. Severinsson, B. Ek, Y740/751F + - +/- 52 A. Ostman, C. Betsholtz, and C.-H. Heldin. 1988. cDNA cloning Y771F ++ ++ +/- This study and expression of a human platelet-derived growth factor (PDGF) Y775/778F + + + + +/- This study receptor specific for B-chain-containing PDGF molecules. Mol. Y1009F ++ NDa ND 39, 52 Cell. Biol. 8:3476-3486. Y1021F ++ ND ND 39, 52 11. Claesson-Welsh, L., A. Hammacher, B. Westermark, C.-H. Heldin, Y1009/1021F ++ ++ ++ 39, 52 and M. Nister. 1989. Identification and structural analysis of the A type receptor for platelet-derived growth factor. Similarities with a ND, not determined. the B type receptor. J. Biol. Chem. 264:1742-1747. VOL. 14, 1994 GRB2 BINDS DIRECTLY TO THE PDGF P-RECEPTOR 6725

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