Search

The PI3K/Akt pathway is interconnected to protein kinase CK2, which directly phosphorylates Akt1 at S129. We have previously found that, in HK-2 renal cells, downregulation of the CK2 regulatory subunit β (shCK2β cells) reduces S129 Akt phosphorylation. Here, we investigated in more details how the different CK2 isoforms impact on Akt and other signaling pathways. We found that all CK2 isoforms phosphorylate S129 in vitro, independently of CK2β. However, in HK-2 cells the dependence on CK2β was confirmed by rescue experiments (CK2β re-expression in shCK2β HK-2 cells), suggesting the presence of additional components that drive Akt recognition by CK2 in cells. We also found that CK2β downregulation altered the phosphorylation ratio between the two canonical Akt activation sites (pT308 strongly reduced, pS473 slightly increased) in HK-2 cells. Similar results were found in other cell lines where CK2β was stably knocked out by CRISPR-Cas9 technology. The phosphorylation of rpS6 S235/S236, a downstream effector of Akt, was strongly reduced in shCK2β HK-2 cells, while the phosphorylation of two Akt direct targets, PRAS40 T246 and GSK3β S9, was increased. Differently to what observed in response to CK2β down-regulation, the chemical inhibition of CK2 activity by cell treatment with the specific inhibitor CX-4945 reduced both the Akt canonical sites, pT308 and pS473. In CX-4945-treated cells, the changes in rpS6 pS235/S236 and GSK3β pS9 mirrored those induced by CK2β knock-down (reduction and slight increase, respectively); on the contrary, the effect on PRAS40 pT246 phosphorylation was sharply different, being strongly reduced by CK2 inhibition; this suggests that this Akt target might be dependent on Akt pS473 status in HK-2 cells. Since PI3K/Akt and ERK1/2/p90rsk pathways are known to be interconnected and both modulated by CK2, with GSK3β pS9 representing a convergent point, we investigated if ERK1/2/p90rsk signaling was affected by CK2β knock-down and CX-4945 treatment in HK-2 cells. We found that p90rsk was insensitive to any kind of CK2 targeting; therefore, the observation that, similarly, GSK3β pS9 was not reduced by CK2 blockade suggests that GSK3β phosphorylation is mainly under the control of p90rsk in these cells. However, we found that the PI3K inhibitor LY294002 reduced GSK3β pS9, and concomitantly decreased Snail1 levels (a GSK3β target and Epithelial-to-Mesenchymal transition marker). The effects of LY294002 were observed also in CK2β-downregulated cells, suggesting that reducing GSK3β pS9 could be a strategy to control Snail1 levels in any situation where CK2β is defective, as possibly occurring in cancer cells. [ABSTRACT FROM AUTHOR]

The cystic fibrosis transmembrane conductance regulator (CFTR) harbors, close to Phe-508, whose deletion is the commonest cause of cystic fibrosis, a conserved potential CK2 phospho-acceptor site (Ser511), which however is not susceptible to phosphorylation by CK2. To shed light on this apparent paradox, a series of systematically substituted peptides encompassing Ser511 were assayed for their ability to be phosphorylated. The main outcomes of our study are the following: (a) Tyr512 plays a prominent role as a negative determinant as its replacement by Ala restores Ser511 phosphorylation by CK2; (b) an even more pronounced phosphorylation of Ser511 is promoted if Tyr512 is replaced by phospho-tyrosine instead of alanine; (c) Tyr512 and, to a lesser extent, Tyr515 are readily phosphorylated by Lyn, a protein tyrosine kinase of the Src family, in a manner which is enhanced by the concomitant Phe508 deletion. Collectively taken, our data, in conjunction with the notion that Tyr515 is phosphorylated in vivo, disclose the possibility that CFTR Ser511 can be phosphorylated by the combined action of tyrosine kinases and CK2 and disclose a new mechanism of hierarchical phosphorylation where the role of the priming kinase is that of removing negative determinant(s). [ABSTRACT FROM AUTHOR]

Abstract: The processivity factor of human cytomegalovirus DNA polymerase, phosphoprotein ppUL44, is essential for viral replication. During viral infection ppUL44 is phosphorylated by the viral kinase pUL97, but neither the target residues on ppUL44 nor the effect of phosphorylation on ppUL44''s activity are known. We report here that ppUL44 is phosphorylated when transiently expressed in mammalian cells and coimmunoprecipitates with cellular kinases. Of three potential phosphorylation sites (S413, S415, S418) located upstream of ppUL44''s nuclear localization signal (NLS) and one (T427) within the NLS itself, protein kinase CK2 (CK2) specifically phosphorylates S413, to trigger a cascade of phosphorylation of S418 and S415 by CK1 and CK2, respectively. Negative charge at the CK2/CK1 target serine residues facilitates optimal nuclear accumulation of ppUL44, whereas negative charge on T427, a potential cyclin-dependent 1 phosphorylation site, strongly decreases nuclear accumulation. Thus, nuclear transport of ppUL44 is finely tuned during viral infection through complex phosphorylation events. [Copyright &y& Elsevier]

Examines the protein kinase CK2 cooperative modulation by separate domains of regulatory beta-subunit. Properties of protein kinase CK2; Effects of a beta-subunit truncated form on protein functions; Account on synthetic peptide interaction with CDK2 PSTAIRE motif; Importance of a peptide reproducing the beta181-203 residue sequence.

Explores the factors determining the site recognition and phosphorylation by rat liver casein kinase-2 (CK-2) with a set of 14 related hexapeptidases each including a single phosphorylatable amino acid and five acidic plus neutral residues. Features of the peptides; Testing of substrates and/or competitive inhibitors of CK-2 and their kinetic and inhibition constants; Affinity for the protein-binding site.

Protein kinase CK2 is a ubiquitous pleiotropic serine/threonine protein kinase whose holoenzyme is comprised of two catalytic (α and/or α') and two non-catalytic, β-subunits. The β-subunit possesses antagonist functions that can be physically dissected by generating synthetic fragments encompassing its N-terminal and C-terminal domains. Here we show that by mutating basic residues in the 74-77 and in the 191- 198 regions of the α-subunit, the negative regulation by the β-subunit and by its N-terminal synthetic fragment CK2β-(41-77), which is observable using calmodulin as a substrate for phosphorylation, is drastically reduced. In contrast, the positive regulation by a C-terminal CK2β-(155-215)-peptide is unaffected or even increased. Moreover, the basal activity of α mutants K74-77A, K79R80K83A. and R191R195K198A toward specific peptide substrates is stimulated by the β-subunit many fold more than rival of α wild type, while extrastimulation by β mutant D55L56E57A, observable with α wild type, is abolished with these mutants. These data support the conclusion that down regulation by the acidic residues clustered in the N-terminal moiety of β is mediated by basic residues in the 74-83 and in the 191-198, sequences of the α-subunit. These are also implicated in substrate recognition consistent with the concept that the N-terminal acidic region of the β subunit operates as a pseudosubstrate. In contrast, another CK2α mutant, V66A, is more sensitive to inhibition by either β-subunit or its N-terminal. CK2β(1 - 77)-peptide, while its stimulation by the C-terminal peptide, C K 2β-(155- 2l5 ), is comparable to that of a wild type. These observations suggest an indirect rote of Val66, in conferring to the α-subunit a conformation less sensitive to down regulation by β-subunit. [ABSTRACT FROM AUTHOR]

Recombinant isolated β-subunit of protein kinase CK2 is readily phosphorylated by p34cdc2/cyclin B kinase at Ser209 with favourable kinetic constants (K1 = 1.7 μM, Vmax = 20 nmol · min-1 · mg -1). Two synthetic peptides reproducing the 170–215 and the 206–215 C-terminal fragments of the β-subunit are also phosphorylated though with tenfold higher Km values (19.5 and 28.0 μM, respectively). In contrast, both the β-subunit associated with the α-subunit to give the heterotetrameric holoenzyme and the native CK2 are not appreciably phosphorylated by p34cdc2. These data suggest that the Ser209 β-subunit phosphorylation observed in intact cells occurs prior to β-subunit incorporation into the holoenzyme. The isolated CK2 α-subunit is not phosphorylated to any appreciable extent by p34cdc2 kinase. Its catalytic activity is nevertheless increased up to fivefold upon incubation with pcdc2/cyclin B kinase complex. Such a stimulation of activity is comparable to that induced by the β-subunit and it is paralleled by a 40% decrease of p34cdc2/cyclin B catalytic activity. Similar to β-subunit, p34cdc2/cyclin B also protects the α-subunit against thermal inactivation. CK2 holoenzyme is also stimulated by p34cdc2/cyclin B, albeit less dramatically than the isolated α-subunit. Such an effect is also evident with CK2 holoenzyme reconstituted with a mutated β-subunit lacking the p34cdc2 phosphorylation site and it is not accompanied by any appreciable phosphorylation of either the β or the α-subunit. These data indicate that in vitro CK2 α-subunit interacts with and is activated by p34cdc2/cyclin B kinase by a mechanism that does not imply the phosphorylation of CK2. [ABSTRACT FROM AUTHOR]

The mechanism by which polybasic peptides stimulate the activity of casein kinase 2 (CK2) has been studied by comparing the effect of polylysine on the phosphorylation of a variety of protein and peptide substrates by the native CK2 holoenzyme and by its recombinant catalytic a subunit, either alone or in combination with the recombinant non-catalytic β subunit. Calmodulin is not phosphorylated by the CK2 holoenzyme, in either the native or the reconstituted form, unless polylysine is added. In the presence of polylysine, it becames a good substrate for CK2 (Km 14.2 μM, Kcat 4.6 mol · min-1 · mol CK2-1). The recombinant a subunit, however, spontaneously phosphorylates calmodulin, this phosphorylation being actually inhibited rather than stimulated by polylysine. The calmodulin tridecapeptide, RKMKDTDSEEEIR, reproducing the phosphorylation site for CK2, is spontaneously phosphorylated by either CK2 holoenzyme or the recombinant α subunit with 5.8-fold and 2.8-fold stimulation by polylysine, respectively. The recombinant β subunit of CK2 is itself a good exogenous substrate for the enzyme, its phosphorylation, however, is inhibited rather than enhanced by polylysine. On the contrary, the phosphorylation of the nonapeptide, MSSSEEVSW, reproducing the β-subunit phosphoacceptor site, is dramatically stimulated by polylysine. Using a variety of small peptide substrates, it was shown that phosphorylation rate is diversely stimulated by polylysine. The observed stimulation, moreover, is variably accounted for by changes in Vmax and/or Km, depending on the structure of the peptide substrate. Maximum stimulation with all protein/peptide substrates tested requires the presence of the β subunit, since the recombinant a subunit is much less responsive than CK2 holoenzyme, either native or reconstituted. While the phosphorylation of the peptide RRRDDDSDDD by CK2 is stimulated 2.8-fold, with 15 nM polylysine being required for half-maximal stimulation, a stimulation of only 1.9-fold, with 80 nM polylysine required for half-maximal stimulation, is attained with recombinant a subunit. The concentration of polylysine required for half-maximal stimulation is comparable to CK2 concentration and increases by increasing CK2 concentration, suggesting that polylysine primarily interacts with the enzyme, rather than with the peptide substrate. [ABSTRACT FROM AUTHOR]

Recombinant human α subunit from casein kinase-2 (CK-2) was subjected, either alone or in combination with recombinant human β subunit, to high temperature, tryptic digestion and urea treatment. In all three cases, it was shown that the presence of the β subunit could drastically reduce the loss of kinase activity, strongly suggesting a protective function for the β subunit. Assaying different peptides for specificity toward the recombinant α subunit and the recombinant reconstituted enzyme, showed that the presence of the β subunit could modify the specificity of the catalytic α subunit. Therefore, a dual function for the β subunit is proposed which confers both specificity and stability to the catalytic α subunit within the CK-2 holoenzyme complex. The peptide DLEPDEELEDNPNQSDL, reproducing the highly acidic amino acid 55 - 71 segment of the human β subunit, counteracts the stimulatory effect of the β subunit on the α subunit activity and partially substitutes the β subunit in conferring thermal stability to the α subunit. No such effect is induced by the peptide MSSSEEVSW, reproducing the N-terminal segment of the β subunit including the autophosphorylation site. It is suggested that the acidic domain of the β subunit, encompassing residues 55-71, plays a role in the interactions between the β and α subunits. [ABSTRACT FROM AUTHOR]

Abstract: CK2 is a highly conserved protein kinase controlling different cellular processes. It shows a higher activity in proliferating mammalian cells, in various types of cancer cell lines and tumors. The findings presented herein provide the first evidence of an in vivo modulation of CK2 activity, dependent on growth rate, in Saccharomyces cerevisiae. In fact, CK2 activity, assayed on nuclear extracts, is shown to increase in exponential growing batch cultures at faster growth rate, while localization of catalytic and regulatory subunits is not nutritionally modulated. Differences in intracellular CK2 activity of glucose- and ethanol-grown cells appear to depend on both increase in molecule number and k cat. Also in chemostat cultures nuclear CK2 activity is higher in faster growing cells providing the first unequivocal demonstration that growth rate itself can affect CK2 activity in a eukaryotic organism. [Copyright &y& Elsevier]