Association of CDC37-nanoKAZ with the immunoprecipitated GFP-DYRK1B constructs (WT, H90P, R102C) or GFP-DYRK4 is shown relative to wild type GFP-DYRK1B (means and SD, n?=?4)
Association of CDC37-nanoKAZ with the immunoprecipitated GFP-DYRK1B constructs (WT, H90P, R102C) or GFP-DYRK4 is shown relative to wild type GFP-DYRK1B (means and SD, n?=?4). as compared to crazy type DYRK1B. PF-543 Citrate These results support the hypothesis the mutations in the DH package interfere with the maturation of DYRK1B by tyrosine autophosphorylation and compromise the PF-543 Citrate conformational stability of the catalytic website, which renders the kinase susceptible to misfolding. Intro DYRK1B (dual-specificity tyrosine(Y)-phosphorylation controlled kinase 1B) is definitely a member of the DYRK family of protein kinases that attain full catalytic activity by co-translational autophosphorylation on a conserved tyrosine residue in the activation loop of the catalytic website1C3. DYRK1B is definitely overexpressed in certain cancers and has been characterized like a regulator of cell differentiation and cell cycle progression4C6. Two missense mutations of the gene, H90P and R102C, were recently found to co-segregate having a rare autosomal-dominant form of metabolic syndrome called AOMS3 (abdominal obesity-metabolic syndrome 3, OMIM access #615812). Affected individuals develop early-onset coronary artery disease, hypertension, PF-543 Citrate central obesity, and diabetes7. This important discovery demonstrates a single cause can trigger the full phenotype of metabolic syndrome. Based on the results of cell-based assays, the mutant alleles were proposed to encode gain-of-function variants of DYRK1B7. Overexpression of DYRK1B-H90P or DYRK1B-R102C in HepG2 hepatoma cells resulted in a higher induction of the key gluconeogenic enzyme, glucose-6-phosphatase (G6Pase), than overexpression of crazy type DYRK1B. Furthermore, the R102C mutation potentiated the effect of DYRK1B within the adipogenic differentiation of 3T3-L1 preadipocytes7. How the mutations alter the molecular function of DYRK1B in these assays remains unknown. In contrast to the gain-of-function effects in these cellular assays, DYRK1B-R102C was found to have reduced catalytic activity in completely co-segregate having a familial form of metabolic syndrome raises the query how these mutations alter the properties of DYRK1B to give rise to this complex phenotype. The present study is designed to elucidate the effects of the H90P and R102C mutations on a molecular and cellular level. Our results suggest that both mutations compromise the maturation by tyrosine autophosphorylation of DYRK1B, which leads to conformational instability and improved misfolding of the mutant proteins. Results Structural model of DYRK1B Sequence analysis revealed the residues affected by the pathogenic mutations belong to a conserved sequence motif designated DYRK homology package (DH package) in the N-terminal website of DYRK1B (Fig.?1A)1. To day no experimental structure is available of DYRK1B. However, the high sequence conservation of the DYRK1A and DYRK1B isoforms allowed us to generate a homology model using available Rabbit Polyclonal to eIF2B DYRK1A crystal constructions16, 17. In the crystal structure of DYRK1A, the DH package participates in considerable relationships with the catalytic website17. Comparisons of the generated model and the DYRK1A structure revealed that many of the DH package relationships are conserved between DYRK1A and DYRK1B, including the hydrogen relationship network involving the central Y147, E153 and R231 (Y99, E105, R183 in DYRK1B) (Fig.?1B). However, a hydrophobic cluster (Y136, Y145) that fixes the N-terminal end of the C helix in the active kinase conformation of DYRK1A is probably less efficiently stabilizing DYRK1B due to loss of hydrophobic and aromatic relationships (L88 and H97). Importantly, the mutated amino PF-543 Citrate acids (H90 and R102) are exposed to the surface of the protein and don’t make direct contacts with the catalytic website. The missense mutations did not result in obvious structural perturbations of the active conformation of DYRK1B. It appears possible the mutation of H90 to P may indirectly impact the backbone conformation of L88 and therefore indirectly disturb the placing of the C helix. It should be noted that our model does not include sequences N-terminal of the DH package and does not allow definitive conclusions concerning the effects of the missense mutations within the function of the kinase. Open in a separate window Number 1 Location of the mutated amino acids in the N-terminal website of DYRK1B. (A).