Cloning, expression and properties of the α' subunit of case in kinase 2 from zebrafish (Danio rerio)

Abstract

The protein kinase casein kinase 2 (CK2) is ubiquitous in eukaryotic cells and is apparently involved in the control of cell division. The holoenzyme is a tetramer composed of two catalytic subunits (α and/or α') and regulatory subunits β2). The α and α' subunits are encoded by different genes but are very similar in amino acid sequence, except that α' is normally considerably shorter. There have been extensive biochemical studies with recombinant α and β subunits of many species, but only one previous description of the activity of an isolated recombinant α' subunit from human CK2 (Bodenbach, L., Fauss, J., Robitzki, A., Krehan, A., Lorenz, P., Lozeman, F.J. and Pyerin, W. (1994) Recombinant human casein kinase II. A study with the complete set of subunits (α, α', and β), site-directed autophosphorylation mutants and a bicistronically expressed holoenzyme, Eur. J. Biochem. 220, 263 -273). In the present work, the isolation and bacterial expression of a cDNA coding for the α' subunit of zebrafish (Danio rerio) is reported. The clone covers the complete coding region that generates a protein of 348 amino acids that is 86% identical to the α' subunits of human and chicken, and 82% identical to the sequenced portion of the CK2α subunit of zebrafish. The recombinant α' subunit has apparent K(m) values for ATP (6 μM), GTP (20 μM), case in (2.0 mg/ml) and the model peptide RRRDDDSEDD (0.3 mM) which are very similar to those of the recombinant α subunit of Xenopus laevis. The α' subunit k(cat) was 7.2 min-1 which is again similar to that of Xenopus laevis α subunit (7.5 min-1). The α' subunit also behaved similarly to CK2α with regard to optimal concentrations for Mg+2 or Mn+2 and to the inhibition by heparin and the poly(Glu80Tyr20) Peptide. However α' kinase activity was less sensitive to poly(U) inhibition than α, it was more heat stable than α, and α' was slightly more sensitive to KCl inhibition than α. The difference in salt sensitivity, however, was enhanced by the presence of the regulatory β subunit which shifted the optimal salt concentration of the phosphorylating activity. The α'2β2 holoenzyme was inhibited by KCl concentrations above 100 mM, while the α2β2 enzyme was stimulated by KCl concentrations up to 150 mM and required 180 mM for inhibition. Another important difference between α and α' is seen in the degree of the stimulation of casein phosphorylation activity in the presence of the regulatory β subunit. When assayed at 100 mM KCl stoichiometric amounts of CK2β produced maximal stimulation of both α' (D. rerio) and α (X. laevis), however the activity levels with α' were stimulated 20-fold by β while the addition of β stimulated α (X. laevis) only 7-8-fold.