Identification of the autophosphorylation sites and characterization of their effects in the protein kinase DYRK1A

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 MA-P kinase, extracellular-signal-regulated kinase 2. Consis tent with our model, substitution of Tyr-321 but not of Tyr-319 by phenylal anine markedly reduced the enzymic activity of recombinant DYRK1A expressed in either Escherichia coli or mammalian cells. Direct identification of ph osphorylated 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 phospho tyro sine, indicating that Tyr-321 is autop hosphorylated by DYRK1A. MS identified Tyr-111 and Ser-97 as additional aut ophosphorylation sites in the non-catalytic N-terminal domain of bacteriall y expressed DYRK1A. Enzymic activity was not affected in the DYRK1A-Y111F m utant. The present experimental data and the molecular model indicate that the activity of DYRK1A is dependent on the autophosphorylation of a conserv ed tyrosine residue in the activation loop.