EXPRESSION OF RECOMBINANT HUMAN CASEIN KINASE-II AND RECOMBINANT HEAT-SHOCK PROTEIN-90 IN ESCHERICHIA-COLI AND CHARACTERIZATION OF THEIR INTERACTIONS

To assess the interaction of human casein kinase II (CKII) with the he at shock protein 90 (HSP90) class of chaperone proteins, human CKII al pha and beta subunits and beta S2A mutant were expressed and purified separately or from a tandem coexpression construct in Escherichia coli . Recombinant human HSP90beta and recombinant yeast HSP90 as His6 cons tructs were also expressed in and purified from E. coli. The rhCKII S2 A mutant removed the regulatory beta subunit autophosphorylation site but had no effect on catalytic efficiency with peptide or protein subs trates. As a CKII substrate, recombinant hHSP90beta displayed a K(m) o f 9.8 muM and a k(cat) of 4.1 min-1 and was phosphorylated to 1.5 mol/ mol, whereas ryHSP90, lacking the known serine CKII sites of hHSP90, w as phosphorylated at a 19-fold lower k(cat/K(m) ratio to levels of 0.8 mol/mol. The endoplasmic reticulum HSP90 family member Grp94 was phos phorylated to 1.4 mol/mol but, in contrast, HSC70 and FKBP25 chaperone s were phosphorylated to <0.01 mol/mol. Neither phospho nor dephospho forms of hHSP90 showed significant activation of CKII toward the pepti de substrate RRREEETEEE in contrast to a previous report that activati on was observed at high molar ratios of chaperone to kinase.