title: Identification of the autophosphorylation sites and characterization of their effects in the protein kinase DYRK1A.
abstract: Protein kinases of the DYRK ('dual-specificity tyrosine-regulated kinase') family are characterized by a conserved Tyr-Xaa-Tyr motif (Tyr-319-Tyr-321) in a position exactly corresponding to the activation motif of the mitogen-activated protein kinase (MAP kinase) family (Thr-Xaa-Tyr). In a molecular model of the catalytic domain of DYRK1A, the orientation of phosphorylated Tyr-321 is strikingly similar to that of Tyr-185 in the known structure of the activated MAP kinase, extracellular-signal-regulated kinase 2. Consistent with our model, substitution of Tyr-321 but not of Tyr-319 by phenylalanine markedly reduced the enzymic activity of recombinant DYRK1A expressed in either Escherichia coli or mammalian cells. Direct identification of phosphorylated residues by tandem MS confirmed that Tyr-321, but not Tyr-319, was phosphorylated. When expressed in COS-7 cells, DYRK1A was found to be fully phosphorylated on Tyr-321. A catalytically inactive mutant of DYRK1A contained no detectable phosphotyrosine, indicating that Tyr-321 is autophosphorylated by DYRK1A. MS identified Tyr-111 and Ser-97 as additional autophosphorylation sites in the non-catalytic N-terminal domain of bacterially expressed DYRK1A. Enzymic activity was not affected in the DYRK1A-Y111F mutant. The present experimental data and the molecular model indicate that the activity of DYRK1A is dependent on the autophosphorylation of a conserved tyrosine residue in the activation loop.
authors.affiliation: Institut für Pharmakologie und Toxikologie, Medizinische Fakultät der RWTH Aachen, Wendlingweg 2, D-52057 Aachen, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Himpel
authors.forename: P
authors.initials: P
authors.lastname: Panzer
authors.forename: K
authors.initials: K
authors.lastname: Eirmbter
authors.forename: H
authors.initials: H
authors.lastname: Czajkowska
authors.forename: M
authors.initials: M
authors.lastname: Sayed
authors.forename: L C
authors.initials: LC
authors.lastname: Packman
authors.forename: T
authors.initials: T
authors.lastname: Blundell
authors.forename: H
authors.initials: H
authors.lastname: Kentrup
authors.forename: J
authors.initials: J
authors.lastname: Grötzinger
authors.forename: H G
authors.initials: HG
authors.lastname: Joost
authors.forename: W
authors.initials: W
authors.lastname: Becker
mesh_terms: D000818:Animals; D002460:Cell Line; D017930:Genes, Reporter; D013058:Mass Spectrometry; D008958:Models, Molecular; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism*; D017354:Point Mutation; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D000097603:Dyrk Kinases
chemical_list: D019000:Phosphotyrosine; D017346:Protein Serine-Threonine Kinases; D011505:Protein-Tyrosine Kinases; D011993:Recombinant Fusion Proteins; D000097603:Dyrk Kinases