title: Identification of RET autophosphorylation sites by mass spectrometry.
abstract: The catalytic and signaling activities of RET, a receptor-type tyrosine kinase, are regulated by the autophosphorylation of several tyrosine residues in the cytoplasmic region of RET. Some studies have revealed a few possible autophosphorylation sites of RET by [(32)P]phosphopeptide mapping or by using specific anti-phosphotyrosine antibodies. To ultimately identify these and other autophosphorylation sites of RET, we performed mass spectrometry analysis of an originally prepared RET recombinant protein. Both the autophosphorylation and kinase activity of myelin basic protein as an external substrate of the recombinant RET protein were substantially elevated in the presence of ATP without stimulation by a glial cell line-derived neurotrophic factor, a natural ligand for RET. Mass spectrometric analysis revealed that RET Tyr(806), Tyr(809), Tyr(900), Tyr(905), Tyr(981), Tyr(1062), Tyr(1090), and Tyr(1096) were autophosphorylation sites. Levels of autophosphorylation and kinase activity of RET-MEN2A (multiple endocrine neoplasia 2A), a constitutively active form of RET with substitution of Tyr(900) by phenylalanine (Y900F), were comparable with those of original RET-MEN2A, whereas those of the mutant Y905F were greatly decreased. Interestingly, those of a double mutant, Y900F/Y905F, were completely abolished. Both the kinase activity and transforming activity were impaired in the mutants Y806F and Y809F. These results provide convincing evidence for both previously suggested and new tyrosine autophosphorylation sites of RET as well as for novel functions of Tyr(806), Tyr(809), and Tyr(900) phosphorylation in both catalytic kinase activities and cell growth. The significance of the identified autophosphorylation sites in various protein-tyrosine kinases registered in a data base is discussed in this paper.
authors.affiliation: Department of Immunology, Nagoya University Graduate School of Medicine, Showa-ku, Nagoya, Japan.
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Kawamoto
authors.forename: Kozue
authors.initials: K
authors.lastname: Takeda
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Okuno
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Yamakawa
authors.forename: Yasutomo
authors.initials: Y
authors.lastname: Ito
authors.forename: Ryo
authors.initials: R
authors.lastname: Taguchi
authors.forename: Masashi
authors.initials: M
authors.lastname: Kato
authors.forename: Haruhiko
authors.initials: H
authors.lastname: Suzuki
authors.forename: Masahide
authors.initials: M
authors.lastname: Takahashi
authors.forename: Izumi
authors.initials: I
authors.lastname: Nakashima
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D041681:NIH 3T3 Cells; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051096:Proto-Oncogene Proteins c-ret; D020794:Receptor Protein-Tyrosine Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014443:Tyrosine / Q000378:metabolism
chemical_list: D011518:Proto-Oncogene Proteins; D014443:Tyrosine; D051096:Proto-Oncogene Proteins c-ret; D020794:Receptor Protein-Tyrosine Kinases; C494999:Ret protein, mouse