EFFICIENT AUTOPHOSPHORYLATION AND PHOSPHORYLATION OF THE BETA-SUBUNITBY CASEIN KINASE-2 REQUIRE THE INTEGRITY OF AN ACIDIC CLUSTER 50 RESIDUES DOWNSTREAM FROM THE PHOSPHOACCEPTOR SITE

Various beta-mutants were investigated either as subunits or as substr ates for casein kinase 2 (CK-2), in the absence or presence of polylys ine. A total of 21 beta-mutants were characterized for their susceptib ility to au- tophosphorylation, by combining them in equimolar amounts with the recombinant cy-subunit. Six mutants, i.e. beta A(5,6), beta A(59-61,63,64), beta A(55,57), beta A(55-57), beta Delta 171-215, and beta Delta 150-215 exhibited a >70% reduction in autophosphorylation. This strongly suggests that in addition to amino acid residues 5,6, di stant amino acid residues within the sequence 55-64 are also involved in the process of autophosphorylation, possibly by means of a loop for mation. The results obtained with the COOH-terminal-deleted mutants su pport the view that reconstitution of a functional holoenzyme must occ ur to allow efficient autophosphorylation. Polylysine prevents the aut ophosphorylation of beta(wt) (86% inhibition) inducing a parallel incr ease of the alpha-subunit autophosphorylation. The autophosphorylation of all mutants, with the exception of beta A(55-57) and beta A(59-61, 63,64), is also inhibited by polylysine (>64%). The alpha-subunit auto phosphorylation is increased with all mutants reconstituting a tetrame ric holoenzyme. Only with the three largest COOH-terminal deletion mut ants beta Delta 150-215, beta Delta 171-215, and beta Delta 181-215 is no significant alpha-subunit autophosphorylation observed. The phosph orylation of the beta-subunit mutants added in large molar excess to C K-2 holoenzyme (either native or recombinant) is also severly impaired by Ala for Glu/Asp substitutions at position 5,6 and in the 55-64 reg ion and by the deletion of the COOH-terminal segments 150-215 and 171- 215. Such a phosphorylation is inhibited by polylysine, with the excep tion of mutants beta Delta 171-215 and beta Delta 150-215, whose phosp horylation is conversely stimulated by polylysine. The decreased phosp horylation efficiency of those mutants that are poor substrates is inv ariably accounted for by lower V-max values, whereas the affinity for CK-2 is actually increased (K-m values lower than that of beta(wt)).