title: Sequential cis/trans autophosphorylation in TrkB tyrosine kinase.
abstract: TrkB, a member of the tyrosine kinase family of growth factor receptors, is activated by binding of brain-derived neurotrophic factor or neurotrophin 4/5. The intracellular kinase domain of TrkB (ICD-TrkB) was prepared by an insect cell expression system and characterized to identify the mechanism of autophosphorylation. The time course of autophosphorylation, which shows a biphasic progression with a slow nonlinear phase followed by a fast linear phase, indicates the existence of autophosphorylation-induced activation in ICD-TrkB. This is also supported by the finding that phosphorylated ICD-TrkB shows significantly higher activity than control naive ICD-TrkB. Interestingly, the autophosphorylation rate in the linear phase clearly depends on the ICD-TrkB concentration, whereas the rate of initial autophosphorylation is independent of the concentration of ICD-TrkB in the reaction mixture. This observation suggests a two-step autophosphorylation, first an intramolecular activating step and then an intermolecular step. This mechanism is confirmed by the result that only the later phase of autophos-phorylation is inhibited by addition of glycerol which interferes with intermolecular interactions. Therefore, we propose the mechanism of ICD-TrkB autophosphorylation as a sequential cis/trans phosphorylation.
authors.affiliation: Bio-Organic Research Department, Ciba-Geigy (Japan) Limited, Takarazuka.
authors.forename: Y
authors.initials: Y
authors.lastname: Iwasaki
authors.forename: H
authors.initials: H
authors.lastname: Nishiyama
authors.forename: K
authors.initials: K
authors.lastname: Suzuki
authors.forename: S
authors.initials: S
authors.lastname: Koizumi
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D004789:Enzyme Activation; D005822:Genetic Vectors / Q000378:metabolism; D007424:Intracellular Fluid / Q000201:enzymology / Q000378:metabolism; D051379:Mice; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D020794:Receptor Protein-Tyrosine Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020813:Receptor, trkB; D017475:Receptors, Nerve Growth Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism
chemical_list: D017475:Receptors, Nerve Growth Factor; D011994:Recombinant Proteins; D011505:Protein-Tyrosine Kinases; D020794:Receptor Protein-Tyrosine Kinases; D020813:Receptor, trkB