No abstract

CK2 is an essential, ubiquitous, and highly pleiotropic protein kinase whose catalytic subunits (␣ and ␣) and holoenzyme (composed by two catalytic and two regulatory ␤-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 3 Phe, Glu 180 3 Ala, Tyr 182 3 Phe) and deletions (⌬2-6, ⌬2-12, ⌬2-18, ⌬2-24, ⌬2-30) expected to affect these interactions are more or less detrimental to catalytic activity of the ␣-subunit of human CK2, the deleted mutants ⌬2-24 and ⌬2-30 being nearly inactive under normal assay conditions. Kinetic analyses showed that impaired catalytic activity of mutants ⌬2-12, ⌬2-18, ⌬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 ⌬2-12, ⌬2-18, Y26F, E180A, and Y182F to wild type level and also conferred catalytic activity to the intrinsically inactive mutants, ⌬2-24 and ⌬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 ␤-subunit through a different mechanism.In eukaryotic organisms nearly all aspects of cell life are controlled by reversible protein phosphorylation affecting specific seryl, threonyl, and tyrosyl residues. This reaction is catalyzed by protein kinases, which altogether make up one of the largest family of enzymes (1, 2) committed to the regulation of most cellular functions with special reference to signal transduction, metabolic pathways, gene expression, cell proliferation, and differentiation (3). Given these premises it is not surprising that protein kinases generally are tightly controlled enzymes whose activity is turned on only in response to specific stimuli, through conformational modifications induced by extracellular ligands, second messengers, intrasteric interactions, association/dissociation of regulatory subunits or domains, and phosphorylation/dephosphorylation of residues located in critical positions. Often several regulatory devices co-exist in the same kinase to ensure a tighter control over its activity, as exemplified by the cyclin-dependent kinases (CDKs) whose activation requires both the phosphorylation of a threonyl residue in the activation loop and the association of the catalytic subunit with the cognate cyclin and whose activity can be turned off by the phosphorylation of two residues in its glycine-rich loop and by the association of the active CDKcyclin complex with a variety of down-regulatory subun...

To optimize biodevice assembly and cholesterol sensing, recombinant and wild type P450scc cytochromes are extensively characterized both in solution and in thin solid films, using X-ray scattering, Brewster angle microscopy, quartz crystal nanobalance, ellipsometry, cyclic voltammetry, and circular dichroism. Efficient expression systems are implemented in microbial cells for the production of recombinant P450scc proteins, which are then purified. Modeling of the cholesterol interaction was also studied for the given application. Both types of P450 form monolayers at the air/water interface which can be transferred onto solid substrates, but only in the case of recombinant protein does the engineered monolayer turn out to be more dense and regular, and its thickness corresponds better to the native protein size. By surface pressure and surface potential measurements it is shown that at the air/water interface P450scc molecules orient themselves at the initial stage of the monolayer compression by self-assembly; increasing surface pressure yields high homogeneity, as confirmed by Brewster angle microscopy. CD measurements confirmed a significant increase in the stability of the protein secondary structure in the recombinant monolayers whose regular structure appears by X-ray measurements. The cyclic voltammetric study on LB films of cytochrome P450scc as a function of number of monolayers and of cholesterol concentration pointed to a possible electron transfer from the electrochemical process to cytochrome P450scc that in turn reacted with cholesterol, compatibly with the modeling showing that the nearest cholesterol atoms belonging to the main chain are only 4−5 Å from the heme.

The concept that the amino-terminal segment plays a role in conferring high basal activity to protein kinase CK2 K K subunit has been validated by generating two mutants (Y26F and v v2^6) which are defective both in catalytic activity and in thermal stability. The additional finding that the activity of the two mutants is fully restored upon association with the regulatory L L subunit, in conjunction with the observation that synthetic peptides reproducing the N-terminal segment (1^30) and the activation loop (175^201) of CK2K K counteract the functional effects of the C-terminal domain of the L L subunit, is consistent with a mechanism of activation of CK2 where the N-terminal domain of K K and the C-terminal domain of L L play interchangeable roles.z 1998 Federation of European Biochemical Societies.