The cDNA of a novel, ubiquitously expressed protein kinase (Dyrk) was cloned from a rat brain cDNA library. The deduced amino acid sequence (763 amino acids) contains a catalytic domain that is only distantly related to that of other mammalian protein kinases. Its closest relative is the protein kinase Mnb of Drosophila, which is presumably involved in postembryonic neurogenesis (85% identical amino acids within the catalytic domain). Outside the catalytic domain, the sequence comprises several striking structural features: a bipartite nuclear translocation signal, a tyrosine-rich hydrophilic motif flanking the nuclear localization signal, a PEST region, a repeat of 13 histidines, a repeat of 17 serine/threonine residues, and an alternatively spliced insertion of nine codons. A recombinant glutathione S-transferase-Dyrk fusion protein catalyzed autophosphorylation and histone phosphorylation on tyrosine and serine/threonine residues with an apparent Km of approximately 3.4 microM. Exchange of two tyrosine residues in the "activation loop" between subdomains VII and VIII for phenylalanine almost completely suppressed the activity and tyrosine autophosphorylation of Dyrk. Tyrosine autophosphorylation was also reduced by exchange of the tyrosine (Tyr-219) in a tyrosine phosphorylation consensus motif. The data suggest that Dyrk is a dual specificity protein kinase that is regulated by tyrosine phosphorylation in the activation loop and might be a component of a signaling pathway regulating nuclear functions.

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.

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.

Background: Pseudohypoaldosteronism type 1 (PHA1) is a monogenic disease caused by mutations in the genes encoding the human mineralocorticoid receptor (MR) or the a (SCNN1A), b (SCNN1B) or g (SCNN1G) subunit of the epithelial Na C channel (ENaC). While autosomal dominant mutation of the MR cause renal PHA1, autosomal recessive mutations of the ENaC lead to systemic PHA1. In the latter, affected children suffer from neonatal onset of multi-organ salt loss and often exhibit cystic fibrosis-like pulmonary symptoms. Objective: We searched for underlying mutations in seven unrelated children with systemic PHA1, all offsprings of healthy consanguineous parents. Methods and results: Amplification of the SCNN1A gene and sequencing of all 13 coding exons unraveled mutations in all of our patients. We found five novel homozygous mutations (c.587_588insC in two patients, c.1342_1343insTACA, c.742delG, c.189COA, c.1361-2AOG) and one known mutation (c.1474COT) leading to truncation of the aENaC protein. All parents were asymptomatic heterozygous carriers of the respective mutations, confirming the autosomal recessive mode of inheritance. Five out of seven patients exhibited pulmonary symptoms in the neonatal period. Conclusion: The a subunit is essential for ENaC function and mutations truncating the pore-forming part of the protein leading to systemic PHA1. Based on current knowledge, the pulmonary phenotype cannot be satisfactorily predicted.