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 heat shock protein 90 (HSP90) class of chaperone proteins, human CKII alpha and beta subunits and beta S2A mutant were expressed and purified separately or from a tandem coexpression construct in Escherichia coli. Recombinant human HSP90 beta and recombinant yeast HSP90 as His6 constructs were also expressed in and purified from E. coli. The rhCKII S2A mutant removed the regulatory beta subunit autophosphorylation site but had no effect on catalytic efficiency with peptide or protein substrates. As a CKII substrate, recombinant hHSP90 beta displayed a Km of 9.8 microM and a kcat of 4.1 min-1 and was phosphorylated to 1.5 mol/mol, whereas ryHSP90, lacking the known serine CKII sites of hHSP90, was phosphorylated at a 19-fold lower kcat/Km ratio to levels of 0.8 mol/mol. The endoplasmic reticulum HSP90 family member Grp94 was phosphorylated to 1.4 mol/mol but, in contrast, HSC70 and FKBP25 chaperones were phosphorylated to < 0.01 mol/mol. Neither phospho nor dephospho forms of hHSP90 showed significant activation of CKII toward the peptide substrate RRREEETEEE in contrast to a previous report that activation was observed at high molar ratios of chaperone to kinase.
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Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115.
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Walsh
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D000595:Amino Acid Sequence; D001483:Base Sequence; D002352:Carrier Proteins / Q000378:metabolism; D047390:Casein Kinase II; D004926:Escherichia coli / Q000235:genetics; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D022021:Tacrolimus Binding Proteins
D002352:Carrier Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; C018551:glucose-regulated proteins; C074856:FKBP3 protein, human; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D022021:Tacrolimus Binding Proteins