Generation of mutants of CK2alpha which are dependent on the beta-subunit for catalytic activity.
To shed light on the structural features underlying high constitutive activity of protein kinase CK2 a number of mutants of the human CK2alpha-subunit altered in the interactions between the N-terminal segment and the activation loop have been generated and shown to be defective in catalytic activity. In particular the truncated mutant delta2-12 displays under standard conditions an almost complete loss of catalytic activity accounted for by a dramatic rise in its Km forATP (from 10 to 206 microM) and a reduced Kcat. Such a drop in efficiency is paralleled by conformational disorganization, as judged from Superdex 75 gel filtration profile. Both catalytic properties and gel filtration behaviour similar to those of wild type CK2alpha were restored upon association with the regulatory beta-subunit, suggesting that constitutive activity is conferred to CK2alpha and to CK2 holoenzyme through different molecular mechanisms. In the holoenzyme an assumable release of tension at the backbone of Ala-193 (as seems to be indicated by a comparison of the crystal structures of maize CK2alpha alone vs. a CK2alpha-beta peptide complex) may result in the ability of the activation loop to adopt its proper conformation independently of interactions with the N-terminal segment.
Sarno
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Dipartimento di Chimica Biologica and Centro per lo Studio delle Biomembrane del CNR, Universita' di Padova, Italy.
Ghisellini
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Cesaro
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Battistutta
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Pinna
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D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D047390:Casein Kinase II; D020134:Catalytic Domain; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D017353:Gene Deletion; D006801:Humans; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000235:genetics*; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D003313:Zea mays / Q000201:enzymology
D010455:Peptides; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D000409:Alanine