RECOMBINANT HUMAN CASEIN KINASE-II - A STUDY WITH THE COMPLETE SET OFSUBUNITS (ALPHA,ALPHA' AND BETA), SITE-DIRECTED AUTOPHOSPHORYLATION MUTANTS AND A BICISTRONICALLY EXPRESSED HOLOENZYME

Human casein kinase II (CKII) is a ubiquitous and multipotential Ser/T hr kinase involved in the regulation of cell growth and differentiatio n. Biochemically, two characteristics are particularly notable; first, the tetrameric composition of two catalytic subunits (alpha and/or al pha') and two regulatory subunits (beta); second, the autophosphorylat ion of the holoenzyme at the N-terminus of CKII beta, suspected to be involved in tuning of the kinase activity. Whether CKII alpha and CKII alpha' reconstitute comparably with CKII beta to form holoenzyme is u nclear. For a systematic investigation, the complete set of recombinan t CKII subunits and of autophosphorylation mutants of CKII beta were e xpressed in Escherichia coli and comparative reconstitutions carried o ut. At 1:1 molar ratio, CKII beta stimulated both catalytic subunits r oughly fivefold with phosvitin as a substrate. The level of activity r eached with both of the reconstituted CKII isoforms was of the same or der of magnitude as that of holoenzyme isolated from human placenta. I t was also similar to a recombinant alpha(2) beta(2) holoenzyme whose expression had been attained in E. coli with a bicistronic construct c ontaining the coding regions of CKII beta and CKII alpha in a tandem a rrangement. Both Ser2 and Ser3 were identified as the autophosphorylat ion sites; replacement of one of these with Ala by oligonucleotide-med iated site-directed mutagenesis influenced only the extent of CKII bet a autophosphorylation, replacement of both resulted in a loss of autop hosphorylation. Despite these differences, the stimulatory effect of a ll the CKII beta mutants was comparable both to each other and to that of wild-type CTII beta. This was also obtained when substrates other than phosvitin were employed such as tubulin, or upstream-binding fact or (UBF). However, the degree of stimulation was substrate specific an d ranged from 2-5-fold with no major differences between CKII alpha an d CKII alpha' stimulation. Calmodulin phosphorylation by both CKII alp ha and CKII alpha' was decreased similarly by CKII beta and the CKII b eta mutants. Proteins such as cAMP-responsive-element-binding protein (CREB), HPV16 E7 or Jun were not phosphorylated by either catalytic su bunit but became substrates of both in the presence of CKII beta or CK II beta mutants. The data suggest that CKII alpha and CKII alpha' form similar CKII holoenzymes and that the tuning of holoenzyme activity i s independent of the autophosphorylation status of CKII beta.