TY - JOUR
T1 - Identification of RET Autophosphorylation Sites by Mass Spectrometry
AU - Kawamoto, Yoshiyuki
AU - Takeda, Kozue
AU - Okuno, Yusuke
AU - Yamakawa, Yoshinori
AU - Ito, Yasutomo
AU - Taguchi, Ryo
AU - Kato, Masashi
AU - Suzuki, Haruhiko
AU - Takahashi, Masahide
AU - Nakashima, Izumi
PY - 2004/4/2
Y1 - 2004/4/2
N2 - 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 [32P]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 Tyr806, Tyr809, Tyr900, Tyr905, Tyr981, Tyr1062, Tyr1090, 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 Tyr900 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 Tyr806, Tyr809, and Tyr900 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.
AB - 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 [32P]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 Tyr806, Tyr809, Tyr900, Tyr905, Tyr981, Tyr1062, Tyr1090, 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 Tyr900 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 Tyr806, Tyr809, and Tyr900 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.
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U2 - 10.1074/jbc.M312600200
DO - 10.1074/jbc.M312600200
M3 - Article
C2 - 14711813
AN - SCOPUS:11144355646
SN - 0021-9258
VL - 279
SP - 14213
EP - 14224
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 14
ER -