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Tyr-716 in the Platelet-Derived Growth Factor 3-Receptor Kinase Insert Is MOLECULAR AND CELLULAR BIOLOGY, OCt. 1994, p. 6715-6726 Vol. 14, No. 10 0270-7306/94/$04.00+0 Copyright © 1994, American Society for Microbiology 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 autophosphor- ylated 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 incorpo- ration 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).
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