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			PubMed Journals: J Biol Chem

  Source:		PMID: 14711813


    		J Biol Chem. 2004 Apr 2;279(14):14213-24.
     		Epub 2004 Jan 6.

			Identification of RET autophosphorylation
			sites by mass spectrometry.

			Kawamoto Y(1), Takeda K, Okuno Y, Yamakawa
			Y, Ito Y, Taguchi R, Kato M, Suzuki H, Takahashi
			M, Nakashima I.

			Author Information
			(1) Department of Immunology, Nagoya University
			Graduate School of Medicine, Showa-ku, Nagoya,
			Japan.

			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.

			DOI: 10.1074/jbc.M312600200 PMID: 14711813
			[Indexed for MEDLINE]

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