Identification of Tyr-762 in the platelet-derived growth factor alpha-receptor as the binding site for Crk proteins.
Tyr-762 is an autophosphorylation site in the human platelet-derived growth factor (PDGF) alpha-receptor. In order to investigate whether phosphorylated Tyr-762 serves as a docking site for downstream signal transduction molecules, affinity purification using an immobilized synthetic peptide containing phosphorylated Tyr-762 and its surrounding amino acid residues was performed. Proteins in HeLa cell lysate of molecular sizes 27, 38 and 40 kDa bound to the phosphorylated, but not to the unphosphorylated peptide. Analyses of partial amino acid sequences of the purified proteins indicated that they were identical to CrkI, CrkII and CrkL respectively. The wild-type PDGF alpha-receptor, when expressed in porcine aortic endothelial cells, formed complexes with CrkII and CrkL upon ligand stimulation, which was specifically inhibited by a synthetic peptide containing phosphorylated Tyr-762. Replacement of Tyr-762 with a phenylalanine residue in the PDGF alpha-receptor abrogated ligand-induced binding of Crk proteins. Tyrosine phosphorylation of CrkII and CrkL increased by 1.8- and 1.3-fold, respectively, upon ligand stimulation of the wild-type alpha-receptor. In contrast, the Y762F mutant PDGF alpha-receptor failed to induce tyrosine phosphorylation of Crk proteins. CrkII and CrkL constitutively formed complex with the guanine nucleotide exchange factor C3G, in unstimulated as well as PDGF-stimulated cells. Moreover, the activated wild-type PDGF alpha-receptor but not the Y762F mutant receptor was found in a C3G immunoprecipitate, suggesting that a ternary complex between the activated PDGF alpha-receptor, Crk and C3G was formed. DNA synthesis stimulated by PDGF-BB as well as PDGF-induced MAP kinase activation was similar in cells expressing wild-type and mutant receptors. Interestingly, the activated PDGF beta-receptor was found not to bind Crk proteins. Instead, Tyr-771 of the beta-receptor, which is localized at an analogous position to Tyr-762 in the alpha-receptor, binds RasGAP. RasGAP is not bound to the alpha-receptor. Thus, this region in the kinase inserts of the two receptors may be important for the divergency in signaling from the two PDGF receptors.
Yokote
K
K
Second Department of Internal Medicine, Chiba University School of Medicine, Chiba city, Japan.
Hellman
U
U
Ekman
S
S
Saito
Y
Y
Rönnstrand
L
L
Saito
Y
Y
Heldin
C H
CH
Mori
S
S
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D001011:Aorta; D000077214:Becaplermin; D001665:Binding Sites; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D004730:Endothelium, Vascular / Q000378:metabolism; D004789:Enzyme Activation; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D010982:Platelet-Derived Growth Factor / Q000378:metabolism / Q000494:pharmacology; D017354:Point Mutation; D011494:Protein Kinases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011518:Proto-Oncogene Proteins; D050737:Proto-Oncogene Proteins c-crk; D020574:Proto-Oncogene Proteins c-sis; D020796:Receptor, Platelet-Derived Growth Factor alpha; D020797:Receptor, Platelet-Derived Growth Factor beta; D017479:Receptors, Platelet-Derived Growth Factor / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013552:Swine; D014162:Transfection; D014443:Tyrosine; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; C082869:CRKL protein; D009687:Nuclear Proteins; D010455:Peptides; D010982:Platelet-Derived Growth Factor; D011518:Proto-Oncogene Proteins; D050737:Proto-Oncogene Proteins c-crk; D020574:Proto-Oncogene Proteins c-sis; D011994:Recombinant Proteins; D000077214:Becaplermin; D014443:Tyrosine; D011494:Protein Kinases; D020796:Receptor, Platelet-Derived Growth Factor alpha; D020797:Receptor, Platelet-Derived Growth Factor beta; D017479:Receptors, Platelet-Derived Growth Factor; D017871:Calcium-Calmodulin-Dependent Protein Kinases