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Biochemical characterization of the recombinant human Drosophila homologues Timekeeper and Andante involved in the Drosophila circadian oscillator
Authors:Tine Rasmussen  Iben H E Skjøth  Hans H Jensen  Karsten Niefind  Brigitte Boldyreff  Olaf-Georg Issinger
Institution:(1) BMB, Syddansk Universitet, Odense, Denmark;(2) Institut für Biochemie, Universität zu Köln, Germany;(3) KinaseDetect, ApS, Odense, Denmark;(4) BMB, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark
Abstract:The Drosophila clock proteins timekeeper (CK2αTik) and andante (CK2βAnd) are mutated CK2α and CK2β subunits, respectively.In order to revisit the hypothesis concerning a perturbation of the β/β and/or α/β subunit association, involving the andante mutant we have cloned, expressed and purified the recombinant andante mutant CK2βAnd and a CK2 holoenzyme composed of CK2βAnd and the wildtype CK2α subunit. Biochemical analyses using gel filtration analysis, inhibitor and heat treatment, as well as urea denaturation studies did not yield significant differences between the wildtype holoenzyme (α2β2) and a holoenzyme containing wildtype CK2α and andante CK2βAnd.The timekeeper mutant, CK2αTik has been reported to show a significant reduction in enzyme activity. In order to closely investigate the reason for this reduction in activity, we have also cloned and expressed the human homologue of Drosophila timekeeper. Using a CK2 holoenzyme containing the human timekeeper mutant and the wildtype CK2β subunit we could confirm a strongly reduced activity towards CK2 substrates, but also a significant reduction in the autophosphorylation of the CK2β in the absence of any substrate. Based on a structure-based model we postulate that the mutation M161K in Drosophila (i.e. M163K in human) is responsible for the drastic loss of activity, where the lysine residue may cause improper binding of the tri-nucleotide.
Keywords:andante  circadian rhythm  clock proteins  protein kinase CK2  timekeeper
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