RECONSTITUTION OF ARABIDOPSIS CASEIN KINASE-II FROM RECOMBINANT SUBUNITS AND PHOSPHORYLATION OF TRANSCRIPTION FACTOR GBF1

In contrast to the well-defined tetrameric structure of animal and yea st casein kinase II (CKII), plant CKII is found in two forms: a monome ric form and an oligomeric form whose subunit composition is not well defined. The Arabidopsis homologs of the catalytic subunit alpha (CKA1 ) and the regulatory subunit beta (CKB1) of CKII were expressed in Esc herichia coli to examine their ability to form complexes, the effect o f CKB1 on the catalytic activity, and the relationship of the recombin ant enzymes to those isolated from plant material. Both subunits were found mainly in the inclusion body fraction in the bacterial expressio n strains, and they were solubilized and renatured with the recovery o f catalytic (CKA1) and stimulatory (CKB1) activities, The combination of purified CKA1 and CKB1 proteins resulted in up to 100-fold stimulat ion of casein kinase activity compared with the CKA1 activity alone, s howing that CKB1 has biochemical properties similar to those of the be ta subunit from animals, CKA1 and CKB1 spontaneously assembled into a tetrameric complex, CKA1(2)CKB1(2), which had properties very similar to those of the oligomeric CKII form isolated from broccoli, However, the properties of the catalytic subunit CKA1 alone differed from those of the broccoli monomeric form of CKII-like activity, Phosphorylation of transcription factor GBF1 with the reconstituted CKA1(2)CKB1(2) en zyme resulted in stimulation of its DNA binding activity and retardati on of the protein-DNA complex; these results are identical to those ob tained previously with isolated nuclear CKII from broccoli.