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.

5-HT(3) receptors demonstrate significant structural and functional homology to other members of the Cys-loop ligand-gated ion channel superfamily. The extracellular domains of these receptors share similar sequence homology (approximately 20%) with Limnaea acetylcholine binding protein, for which an x-ray crystal structure is available. We used this structure as a template for computer-based homology modeling of the 5-HT(3) receptor extracellular domain. AutoDock software was used to dock 5-HT into the putative 5-HT(3) receptor ligand-binding site, resulting in seven alternative energetically favorable models. Residues located no more than 5 A from the docked 5-HT were identified for each model; of these, 12 were found to be common to all seven models with five others present in only certain models. Some docking models reflected the cation-pi interaction previously demonstrated for W183, and data from these and other studies were used to define our preferred models.

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.

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We review here signalling complexes that we have defined using X-ray analysis in our laboratory. They include growth factors and their receptors: nerve growth factor (NGF) and its hetero-hexameric 7S NGF storage complex, hepatocyte growth factor/scatter factor (HGF/SF) NK1 dimers and fibroblast growth factor (FGF1) in complex with its receptor (FGFR2) ectodomain and heparin. We also review our recent structural studies on intracellular signalling complexes, focusing on phosducin transducin GPry, CK2 protein kinase and its complexes, and the cyclin D-dependent kinase, Cdk6, bound to the cell cycle inhibitor p19INK4d. Comparing the structures of these complexes with others we show that the surface area buried in signalling interactions does not always give a good indication of the strength of the interactions. We show that conformational changes are often important in complexes with intermediate buried surface areas of 1500 to 2000 A2, such as Cdk6INK4 interactions. Some interactions involve recognition of continuous epitopes, where there is no necessity for a tertiary structure and very often the binding conformation is induced during the process of interaction, for example phosducin binding to the betagamma subunits (Gtbetagamma) of the heterotrimeric G protein transducin.