title: Repair by Src kinase of function-impaired RET with multiple endocrine neoplasia type 2A mutation with substitutions of tyrosines in the COOH-terminal kinase domain for phenylalanine.
abstract: An oncogenic mutant of c-RET as a receptor-type tyrosine kinase, termed RET-MEN2A, displays both cell-transforming activity in vivo and strong catalytic activity in vitro. In this study, we compared the activities of mutant RET-MEN2A with substitutions of each of nine tyrosines for phenylalanine (Y1062F, Y1015F, Y981F, Y952F, Y928F, Y905F, Y900F, Y864F, and Y826F), which had been transfected into NIH 3T3 cells. In RET-MEN2A with the Y905F mutation, the cell-transforming activity was drastically reduced with a great reduction in the in vitro catalytic activity. Unexpectedly, we found that in vitro kinase activity was severely impaired in RET-MEN2A with Y981F, Y952F, or Y928F mutation, which displayed near-normal cell-transforming activity and only a partially impaired tyrosine phosphorylation level in vivo. Phosphoamino acid analysis actually demonstrated some increase in phosphotyrosine in the Y905F mutant but no or barely detectable increase in the Y981F, Y952F, or Y928F mutant after incubation for in vitro kinase assay. This suggested a crucial role of the Y981/Y952/Y928-linked structural integrity of the COOH end of the catalytic domain of RET in starting Y905 autophosphorylation. Interestingly, the apparent defect in intrinsic kinase activity in vitro in the Y981F, Y952F, or Y928F mutant, but not the reduction in activity in the Y905F mutant, could be partially repaired or restored by c-Src or, more extensively, by v-Src, which promoted Y905 phosphorylation in trans. A complex was shown to be formed between v-Src and RET-MEN2A through association of both with a cholesterol-rich membrane microdomain known as "a raft," possibly for efficient contact of submembranous domains of Src and RET to promote phosphorylation of Y905 of the latter. Finally, endogenous c-Src was shown to promote Y905 phosphorylation of the Y981F mutant in vivo. These results reveal a novel Src kinase-mediated repair mechanism of otherwise function-impaired mutant RET kinases.
authors.affiliation: Department of Immunology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, Aichi 466-8550, Japan.
authors.forename: Masashi
authors.initials: M
authors.lastname: Kato
authors.forename: Kozue
authors.initials: K
authors.lastname: Takeda
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Kawamoto
authors.forename: Toshihide
authors.initials: T
authors.lastname: Iwashita
authors.forename: Anwarul A
authors.initials: AA
authors.lastname: Akhand
authors.forename: Takeshi
authors.initials: T
authors.lastname: Senga
authors.forename: Masahiko
authors.initials: M
authors.lastname: Yamamoto
authors.forename: Gen
authors.initials: G
authors.lastname: Sobue
authors.forename: Michinari
authors.initials: M
authors.lastname: Hamaguchi
authors.forename: Masahide
authors.initials: M
authors.lastname: Takahashi
authors.forename: Izumi
authors.initials: I
authors.lastname: Nakashima
mesh_terms: D016475:3T3 Cells; D019943:Amino Acid Substitution; D000818:Animals; D002384:Catalysis; D002471:Cell Transformation, Neoplastic; D029721:Drosophila Proteins; D006801:Humans; D051379:Mice; D018813:Multiple Endocrine Neoplasia Type 2a / Q000235:genetics / Q000378:metabolism*; D016296:Mutagenesis; D010649:Phenylalanine / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D051096:Proto-Oncogene Proteins c-ret; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism; D019061:src-Family Kinases / Q000378:metabolism*
chemical_list: D029721:Drosophila Proteins; D011518:Proto-Oncogene Proteins; D019000:Phosphotyrosine; D014443:Tyrosine; D010649:Phenylalanine; D051096:Proto-Oncogene Proteins c-ret; D020794:Receptor Protein-Tyrosine Kinases; C416649:Ret protein, Drosophila; C494999:Ret protein, mouse; D019061:src-Family Kinases