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 c ells and is apparently involved in the control of cell division. The h oloenzyme is a tetramer composed of two catalytic subunits (alpha and/ or alpha') and regulatory subunits (beta(2)) The alpha and alpha' subu nits 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 bet a subunits of many species, but only one previous description of the a ctivity of an isolated recombinant alpha' subunit from human CK2 (Bode nbach, L., Fauss, J., Robitzki, A., Krehan, A., Lorenz, P., Lozeman, F . J. & Pyerin, W. (1994) Recombinant human casein kinase II. A study w ith the complete set of subunits (alpha, alpha', and beta), site-direc ted autophosphorylation mutants and a bicistronically expressed holoen zyme, Eur. J. Biochem. 220, 263-273), In the present work, the isolati on and bacterial expression of a cDNA coding for the alpha' subunit of zebrafish (Danio rerio) is reported. The clone covers the complete co ding region that generates a protein of 348 amino acids that is 86% id entical to the alpha' subunits of human and chicken. and 82% identical to the sequenced portion of the CK2 alpha subunit of zebrafish. The r ecombinant alpha' subunit has apparent K-m values for ATP (6 mu M), GT P (20 mu M), casein (2.0 mg/ml) and the model peptide RRRDDDSEDD (0.3 mM) which are very similar to those of the recombinant alpha subunit o f Xenopus laevis. The alpha' subunit k(cat) was 7.2 min(-1) which is a gain similar to that of Xenopus laevis alpha subunit (7.5 min(-1)). Th e alpha' subunit also behaved similarly to CK2 alpha with regard to op timal concentrations for Mg+2 or Mn+2 and to the inhibition by heparin and the poly(Glu(80)Tyr(20)) 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 inhib ition than alpha. The difference in salt sensitivity, however, was enh anced 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 stimulted by KCl concentrati ons up to 150 mM and required 180 mM for inhibition. Another important difference between alpha and alpha' is seen in the degree of the stim ulation of casein phosphorylation activity in the presence of the regu latory beta subunit. When assayed at 100 mM KCl stoichiometric amounts of CK2 beta produced maximal stimulation of both alpha' (D. rerio) an d alpha (X. laevis), however the activity levels with alpha' were stim ulated 20-fold by beta while the addition of beta stimulated alpha (X. laevis) only 7-8-fold.