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 (Delta2-6, Delta2-12, Delta2-18, Delta2-24, Delta2-30) expected to affect these interactions are more or less detrimental to catalytic activity of the a-subunit of human CK2, the deleted mutants Delta2-24 and Delta2-30 being nearly inactive under normal assay conditions. Kinetic analyses showed that impaired catalytic activity of mutants Delta2-12, Delta2-18, Delta2-24, 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 Delta2-12, Delta2-18, Y26F, E180A, and Y182F to wild type level and also conferred catalytic activity to the intrinsically inactive mutants, Delta2-24 and Delta2-30. 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.

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

GHISELLINI, PAOLA;
2002

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 (Delta2-6, Delta2-12, Delta2-18, Delta2-24, Delta2-30) expected to affect these interactions are more or less detrimental to catalytic activity of the a-subunit of human CK2, the deleted mutants Delta2-24 and Delta2-30 being nearly inactive under normal assay conditions. Kinetic analyses showed that impaired catalytic activity of mutants Delta2-12, Delta2-18, Delta2-24, 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 Delta2-12, Delta2-18, Y26F, E180A, and Y182F to wild type level and also conferred catalytic activity to the intrinsically inactive mutants, Delta2-24 and Delta2-30. 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.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11567/602742
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