title: Unique activation mechanism of protein kinase CK2. The N-terminal segment is essential for constitutive activity of the catalytic subunit but not of the holoenzyme.
abstract: CK2 is an essential, ubiquitous, and highly pleiotropic protein kinase whose catalytic subunits (alpha and alpha') and holoenzyme (composed by two catalytic and two regulatory beta-subunits) are both constitutively active, a property that is suspected to contribute to its pathogenic potential. Extensive interactions between the N-terminal segment and the activation loop are suspected to underlie the high constitutive activity of the isolated catalytic subunit. Here we show that a number of point mutations (Tyr(26) --> Phe, Glu(180) --> Ala, Tyr(182) --> Phe) and deletions (Delta 2-6, Delta 2-12, Delta 2-18, Delta 2-24, Delta 2-30) expected to affect these interactions are more or less detrimental to catalytic activity of the alpha-subunit of human CK2, the deleted mutants Delta 2-24 and Delta 2-30 being nearly inactive under normal assay conditions. Kinetic analyses showed that impaired catalytic activity of mutants Delta 2-12, Delta 2-18, Delta 2-24, and Y182F is mainly accounted for by dramatic increases in the K(m) values for ATP, whereas a drop in K(cat) with K(m) values almost unchanged was found with mutants Y26F and E180A. Holoenzyme reconstitution restored the activity of mutants Delta 2-12, Delta 2-18, Y26F, E180A, and Y182F to wild type level and also conferred catalytic activity to the intrinsically inactive mutants, Delta 2-24 and Delta 2-30. These data demonstrate that specific interactions between the N-terminal segment and the activation loop are essential to provide a fully active conformation to the catalytic subunits of CK2; they also show that these interactions become dispensable upon formation of the holoenzyme, whose constitutive activity is conferred by the beta-subunit through a different mechanism.
authors.affiliation: Department of Biological Chemistry, University of Padua, viale G. Colombo 3, 35121 Padua, Italy.
authors.forename: Stefania
authors.initials: S
authors.lastname: Sarno
authors.forename: Paola
authors.initials: P
authors.lastname: Ghisellini
authors.forename: Lorenzo A
authors.initials: LA
authors.lastname: Pinna
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D047390:Casein Kinase II; D002384:Catalysis; D020134:Catalytic Domain; D004789:Enzyme Activation; D018698:Glutamic Acid / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010649:Phenylalanine / Q000737:chemistry; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D013696:Temperature; D014443:Tyrosine / Q000737:chemistry
chemical_list: D011994:Recombinant Proteins; D018698:Glutamic Acid; D014443:Tyrosine; D010649:Phenylalanine; D000255:Adenosine Triphosphate; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D000409:Alanine