Cloning, expression and properties of the alpha' subunit of casein kinase 2 from zebrafish (Danio rerio).
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 (alpha and/or alpha') and regulatory subunits (beta 2). The alpha and alpha' subunits are encoded by different genes but are very similar in amino acid sequence, except that alpha' is normally considerably shorter. There have been extensive biochemical studies with recombinant alpha and beta subunits of many species, but only one previous description of the activity of an isolated recombinant alpha' subunit from human CK2 (Bodenbach, L., Fauss, J., Robitzki, A., Krehan, A., Lorenz, P., Lozeman, F. J. & Pyerin, W. (1994) Recombinant human casein kinase II. A study with the complete set of subunits (alpha, alpha', and beta), 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 alpha' 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 alpha' subunits of human and chicken, and 82% identical to the sequenced portion of the CK2 alpha subunit of zebrafish. The recombinant alpha' subunit has apparent K(m) values for ATP (6 microM), GTP (20 microM), casein (2.0 mg/ml) and the model peptide RRRDDDSEDD (0.3 mM) which are very similar to those of the recombinant alpha subunit of Xenopus laevis. The alpha' subunit kcat was 7.2 min-1 which is again similar to that of Xenopus laevis alpha subunit (7.5 min-1). The alpha' subunit also behaved similarly to CK2 alpha with regard to optimal concentrations for Mg+2 or Mn+2 and to the inhibition by heparin and the poly(Glu80Tyr20) peptide. However alpha' kinase activity was less sensitive to poly(U) inhibition than alpha, it was more heat stable than alpha, and alpha' was slightly more sensitive to KCl inhibition than alpha. The difference in salt sensitivity, however, was enhanced by the presence of the regulatory beta subunit which shifted the optimal salt concentration of the phosphorylating activity. The alpha' 2 beta 2 holoenzyme was inhibited by KCl concentrations above 100 mM, while the alpha 2 beta 2 enzyme was stimulated by KCl concentrations up to 150 mM and required 180 mM for inhibition. Another important difference between alpha and alpha' is seen in the degree of the stimulation of casein phosphorylation activity in the presence of the regulatory beta subunit. When assayed at 100 mM KCl stoichiometric amounts of CK2 beta produced maximal stimulation of both alpha' (D. rerio) and alpha (X. laevis), however the activity levels with alpha' were stimulated 20-fold by beta while the addition of beta stimulated alpha (X. laevis) only 7-8-fold.
Departamento de Bioquímica, Facultad de Medicina Universidad de Chile, Santiago, Chile.
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Antonelli
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Daniotti
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Rojo
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Allende
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Allende
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002147:Calmodulin / Q000378:metabolism; D047390:Casein Kinase II; D003001:Cloning, Molecular; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011072:Poly U / Q000494:pharmacology; D011189:Potassium Chloride / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D014982:Xenopus laevis; D015027:Zebrafish
D002147:Calmodulin; D011994:Recombinant Proteins; D011072:Poly U; D011189:Potassium Chloride; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases