The Full Oncogenic Activity of Ret/ptc2 Depends on Tyrosine 539, a Docking Site for Phospholipase Cy

摘要: MATERIALS AND METHODSSite-directed mutagenesis and cloning of RET/PTC2-YF mutants. The cloning of the RET/PTC2 cDNA has been previously reported (5)(EMBL data bank accession number L03357). Thc 2-kb Xba I cDNA insert was cloned into the pAlter vector (Promega). Site-directed mutagenesis was performed by using an in vitro oligonucleotide mutagenesis system (Altered Sites in vitro mutagenesis system; Promega). Oligonucleotides carrying the Tyr-539–Phe and the Tyr-505–Phe mutations were 5'-AGAGGAGAGACTTCTTGGACCTTGC-3'and5'-AGCGAGGAGATGTTCCGCCTGATGCT-3', respectively, and were synthesized with an Applied Biosystems 391 apparatus. Since the introduced mutations did not destroy or create restriction sites, mutant clones were identified by selective PCR. Taking advantage of enzymatic discrimination against elongating mismatched termini (the AT terminus is …