Biochemical characterization of the recombinant human Drosophila homologues Timekeeper and Andante involved in the Drosophila circadian oscillator.
The Drosophila clock proteins timekeeper (CK2a(Tik)) and andante (CK2beta(And)) are mutated CK2alpha and CK2beta subunits, respectively. In order to revisit the hypothesis concerning a perturbation of the beta/beta and/or alpha/beta subunit association, involving the andante mutant we have cloned, expressed and purified the recombinant andante mutant CK2beta(And) and a CK2 holoenzyme composed of CK2beta(And) and the wildtype CK2alpha 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 (alpha2beta2) and a holoenzyme containing wildtype CK2alpha and andante CK2beta(And). The timekeeper mutant, CK2alpha(Tik) 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 CK2beta subunit we could confirm a strongly reduced activity towards CK2 substrates, but also a significant reduction in the autophosphorylation of the CK2beta 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.
Rasmussen
Tine
T
BMB, Syddansk Universitet, Odense, Denmark
Skjøth
Iben H E
IH
Jensen
Hans H
HH
Niefind
Karsten
K
Boldyreff
Brigitte
B
Issinger
Olaf-Georg
OG
D000595:Amino Acid Sequence; D000818:Animals; D047390:Casein Kinase II / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002940:Circadian Rhythm; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000502:physiology*; D020035:Holoenzymes / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007031:Hypothalamus / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid
D029721:Drosophila Proteins; D020035:Holoenzymes; D021122:Protein Subunits; D011994:Recombinant Proteins; D047390:Casein Kinase II