Recombinant human casein kinase II.

Human casein kinase II (CKII) is a ubiquitous and multipotential Ser/Thr kinase involved in Ihe regulation of cell growth and differentiation. Biochemically, two characteristics are particularly notable; first, the tetrameric composition of two catalytic subunits (α and/or α′) and two regulatory subunits (β); second, the autophosphorylation of the holoenzyme at the N-terminus of CKIIβ, suspected to be involved in tuning of the kinase activity. Whether CKIIα and CKIIα′ reconstitute comparably with CKIIβ to form holoenzyme is unclear. For a systematic investigation, the complete set of recombinant CKII subunits and of autophosphorylation mutants of CKIIβ were expressed in Escherichia coli and comparative reconstitutions carried out. At 1:1 molar ratio, CKIIβ stimulated both catalytic subunits roughly fivefold with phosvitin as a substrate. The level of activity reached with both of the reconstituted CKII isoforms was of the same order of magnitude as that of holoenzyme isolated from human placenta. It was also similar to a recombinant α₂β₂ holoenzyme whose expression had been attained in E. coli with a bicistronic construct containing the coding regions of CKIIβ and CKIIα in a tandem arrangement. Both Ser2 and Ser3 were identified as the autophosphorylation sites; replacement of one of these with Ala by oligonucleotide-mediated site-directed mutagenesis influenced only the extent of CKIIβ autophospborylation, replacement of both resulted in a loss of autophosphorylation. Despite these differences, the stimulatory effect of all the CKIIβ mutants was comparable both to each other and to that of wild-type CKIIα′. This was also obtained when substrates other than phosvitin were employed such as tubulin, or upstream-binding factor (UBF). However, the degree of stimulation was substrate specific and ranged from 2--5-fold with no major differences between CKIIα and CKIIα′ stimulation. Calmodulin phosphorylation by both CKIIα and CKIIα′ was decreased similarly by CKIIβ and the CKIIβ mutants. Proteins such as cAMP-responsive-element-binding protein (CREB), HPV16 E7 or Jun were not phosphorylated by either catalytic subunit but became substrates of both in the presence of CKIIβ or CKIIβ mutants. The data suggest that CKIIα and CKIIα′ form similar CKII holoenzymes and that the tuning of holoenzyme activity is independent of the autopbosphorylation status of CKIIβ. [ABSTRACT FROM AUTHOR]