IDENTIFICATION OF 2 DIFFERENT MOLECULAR-FORMS OF ARABIDOPSIS-THALIANACASEIN-KINASE-II

We have isolated and identified two different molecular forms of casei n kinase II (CKIIA and CKIIB) from Arabidopsis thaliana. The use of he terologous antibodies that react with both catalytic and regulatory su bunits in animal systems, revealed that CKIIB contains a single type o f polypeptide with a molecular mass of 43 000 Da, whereas CKIIA is com posed of two different polypeptides of 43 000 and 27 000 Da, respectiv ely. Both forms show enzymatic characteristics typical of CKII activit y, such as phosphorylation of the acidic peptide RRRDDDSDDD, use of GT P as phosphate donor, inhibition of their activity by low concentratio ns of heparin and stimulation by polylysine. K-m values for beta-casei n are 0.068 mg/ml for CKIIA and 0.175 mg/ml for CKIIB. Calmodulin, a w ell-characterized substrate for casein kinases II, is significantly be tter phosphorylated by the CKIIB form than by the CKIIA form, suggesti ng an inhibitory effect of beta-subunit on the enzymatic activity for this substrate. However, no autophosphorylation of the 27-kDa polypept ide has been detected, which can be explained by the lack of the conse rved beta-autophosphorylation site in Arabidopsis thaliana, as deduced from its cDNA sequence (Collinge and Walker, 1994, Plant Mol. Biol. 2 5, 649-658). In contrast, CKIIB form, devoid of regulatory subunit, co -purifies with a protein substrate of 15-kDa and efficiently phosphory lates it. A distinct pattern of mRNA accumulation for the catalytic an d regulatory subunits suggests a different distribution of the molecul ar isoforms in the adult plant, that may reflect a functional speciali zation. (C) 1997 Elsevier Science Ireland Ltd.