title: Determination of the transphosphorylation sites of Jak2 kinase.
abstract: Janus kinases are the key enzymes involved in the initial transmission of signals in response to type I and II cytokines. Activation of the signal begins with the transphosphorylation of Jak kinases. Substrates that give rise to downstream events are recruited to the receptor complex in part by interactions with phosphorylated tyrosines. The identity of many of the phosphotyrosines responsible for recruitment has been elucidated as being receptor-based tyrosines. The ability of Jaks to recruit substrates through their own phosphotyrosines has been demonstrated for tyrosines in the kinase activation loop. Recent studies demonstrate that other tyrosines have implications in regulatory roles of Jak kinase activity. In this study, baculovirus-produced Jak2 was utilized to demonstrate that transphosphorylation of Jak kinases occurs on multiple residues throughout the protein. We demonstrate that among the tyrosines phosphorylated, those in the kinase domain occur as expected, but many other sites are also phosphorylated. The tyrosines conserved in the Jak family are the object of this study, although many of them are phosphorylated, many are not. This result suggests that conservation of tyrosines is perhaps as important in maintaining structure of the Jak family. Additionally, non-Jak family conserved tyrosines are phosphorylated suggesting that the individual Jaks ability to phosphorylated specific tyrosines may influence signals emitting from activated Jaks.
authors.affiliation: Department of Immunology, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan. tmatsuda@pham.hokudai.ac.jp
authors.forename: Tadashi
authors.initials: T
authors.lastname: Matsuda
authors.forename: Jian
authors.initials: J
authors.lastname: Feng
authors.forename: Bruce A
authors.initials: BA
authors.lastname: Witthuhn
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Sekine
authors.forename: James N
authors.initials: JN
authors.lastname: Ihle
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer / Q000379:methods; D017353:Gene Deletion; D047468:Immunoprecipitation; D053614:Janus Kinase 2; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010748:Phosphopeptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera / Q000166:cytology; D014357:Trypsin / Q000378:metabolism; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism
chemical_list: D010446:Peptide Fragments; D010748:Phosphopeptides; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D053614:Janus Kinase 2; D014357:Trypsin