Substitution of Tyr-321 by phenylalanine markedly reduced DYRK1A enzymic activity, demonstrating that its activity depends on autophosphorylation of this conserved tyrosine in the activation loop.
The enzymic activity of DYRK1A is dependent on the autophosphorylation of the conserved Tyr-321 residue in its activation loop.
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.
Himpel et al. (2001) studied Protein kinase DYRK1A characterization. Site-directed mutagenesis (Y321F, Y319F, Y111F) vs. Wild-type DYRK1A was evaluated on Enzymic activity and phosphorylation status. Substitution of Tyr-321 by phenylalanine markedly reduced DYRK1A enzymic activity, demonstrating that its activity depends on autophosphorylation of this conserved tyrosine in the activation loop.