Reconstitution of normal and hyperactivated forms of casein kinase-2 by variably mutated beta-subunits.
Twenty-one mutants of the noncatalytic beta-subunit of human casein kinase-2 have been created, expressed in Escherichia coli, and purified to homogeneity. They are either modified at the autophosphorylation site (mutants beta delta 1-4 and beta A 5,6) or bear variable deletions in their C-terminal part (mutants beta delta 209-215, beta delta 194-215, beta delta 181-215, beta delta 171-215, beta delta 150-215) or have undergone Ala substitutions for the acidic and basic residues which are concentrated in the sequences 55-70 and 171-180, respectively. All these mutants have been examined for their ability to functionally replace the wild type beta-subunit. All substitutions and the deletions delta 1-4, delta 194-215, and delta 209-215 are compatible with effective binding of the catalytic alpha-subunit, as judged by sucrose density gradient analysis, stimulation of catalytic activity, and protection against thermal denaturation. Deletions delta 171-215 and delta 150-215, however, give rise to truncated molecules which are unable to associate with the alpha-subunit. The intermediate deletion delta 181-215 is still compatible with association, albeit the reconstituted holoenzyme exhibits an altered sedimentation coefficient. The holoenzymes reconstituted with substituted mutants beta A 55,57, beta A55-57, and, to a lesser extent, beta A 59-61, beta A63,64, and beta A5,6 display a basal activity which is higher (up to 4-fold) than that of the wild type holoenzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
Boldyreff
B
B
Institut für Humangenetik, Universität des Saarlandes, Homburg, Germany.
Meggio
F
F
Pinna
L A
LA
Issinger
O G
OG
D000595:Amino Acid Sequence; D047388:Casein Kinases; D002384:Catalysis; D003001:Cloning, Molecular; D004789:Enzyme Activation / Q000187:drug effects; D014644:Genetic Variation; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011494:Protein Kinases / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D012965:Sodium Chloride / Q000494:pharmacology
D012965:Sodium Chloride; D006493:Heparin; D011494:Protein Kinases; D047388:Casein Kinases