Treatment of P190 Bcr/Abl lymphoblastic leukemia cells with inhibitors of the serine/threonine kinase CK2.
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Animals↗
Biomedical subjects
L A Pinna
Publications and source records attributed to L A Pinna.
Analysis of a sub-proteome which co-purifies with and is phosphorylated by the Golgi casein kinase.
In an attempt to gain information about the identity of the Golgi apparatus casein kinase(s) (G-CK), responsible for the phosphorylation of caseins in lactating mammary gland, the proteins present in fractions enriched in G-CK activity eluted from DEAE-Sepharose and heparin-Sepharose columns were resolved by two-dimensional electrophoresis and analyzed by mass spectrometry. This led to the identification of 47 proteins altogether, none of which is a bona fide protein kinase. At least 9 of the identified proteins however, are readily phosphorylated by co-purifying G-CK activity, and 7 are physically associated with it to give supramolecular complex(es) of about 500 kDa as judged from Superdex S200 gel fitration and glycerol gradient ultracentrifugation experiments. In contrast, the apparent molecular weight of G-CK estimated from an in gel activity assay after SDSPAGE and renaturation is about 41 kDa. Many of the proteins phosphorylated by and/or associated with G-CK belong to the category of chaperonines, including HSP90, GRP-94 and -78, and various isoforms of protein disulfide isomerases, suggesting a global role of this kinase in the modulation of protein folding.
Amino Acid Sequence↗
Protein kinase CK2: a newcomer in the 'druggable kinome'.
The acronym CK2 (derived from the misnomer 'casein kinase' 2) denotes one of the most pleiotropic members of the eukaryotic protein kinase superfamily, characterized by an acidic consensus sequence in which a carboxylic acid (or pre-phosphorylated) side chain at position n+3 relative to the target serine/threonine residue plays a crucial role. The latest repertoire of CK2 substrates includes approx. 300 proteins, but the analysis of available phosphopeptide databases from different sources suggests that CK2 alone may be responsible for the generation of a much larger proportion (10-20%) of the eukaryotic phosphoproteome. Although for the time being CK2 is not included among protein kinases whose inhibitors are in clinical practice or in advanced clinical trials, evidence is accumulating that elevated CK2 constitutive activity co-operates to induce a number of pathological conditions, including cancer, infectious diseases, neurodegeneration and cardiovascular pathologies. The development and usage of cell-permeant, selective inhibitors discloses a scenario whereby CK2 plays a global anti-apoptotic role, which under special circumstances may lead to untimely and pathogenic cell survival.
Adenosine Triphosphate↗
Protein kinase CK2 phosphorylates and upregulates Akt/PKB.
Treatment of Jurkat cells with specific inhibitors of protein kinase CK2 induces apoptosis. Here we provide evidence that the anti-apoptotic effect of CK2 can be at least partially mediated by upregulation of the Akt/PKB pathway. Such a conclusion is based on the following observations: (1) inhibition of CK2 by cell treatment with two structurally unrelated CK2 inhibitors induces downregulation of Akt/PKB, as judged from decreased phosphorylation of its physiological targets, and immunoprecipitate kinase assay; (2) similar results are observed upon reduction of CK2 catalytic subunit by the RNA-interference technique; (3) Akt/PKB Ser129 is phosphorylated by CK2 in vitro and in vivo; (4) such a phosphorylation of activated Akt/PKB correlates with a further increase in catalytic activity. These data disclose an unanticipated mechanism by which constitutive phosphorylation by CK2 may be required for maximal activation of Akt/PKB.
Amino Acid Sequence↗
Expression of the Stp1 LMW-PTP and inhibition of protein CK2 display a cooperative effect on immunophilin Fpr3 tyrosine phosphorylation and Saccharomyces cerevisiae growth.
Although the yeast genome does not encode bona fide protein tyrosine kinases, tyrosine-phosphorylated proteins are numerous, suggesting that besides dual-specificity kinases, some Ser/Thr kinases are also committed to tyrosine phosphorylation in Saccharomyces cerevisiae. Here we show that blockage of the highly pleiotropic Ser/Thr kinase CK2 with a specific inhibitor synergizes with the overexpression of Stp1 low-molecular-weight protein tyrosine phosphatase (PTP) in inducing a severe growth-defective phenotype, consistent with a prominent role for CK2 in tyrosine phosphorylation in yeast. We also present in vivo evidence that immunophilin Fpr3, the only tyrosine-phosphorylated CK2 substrate recognized so far, interacts with and is dephosphorylated by Spt1. These data disclose a functional correlation between CK2 and LMW-PTPs, and suggest that reversible phosphorylation of Fpr3 plays a role in the regulation of growth rate and budding in S. cerevisiae.
Casein Kinase II↗
The carboxy-terminal domain of Grp94 binds to protein kinase CK2 alpha but not to CK2 holoenzyme.
Surface plasmon resonance analysis shows that the carboxy-terminal domain of Grp94 (Grp94-CT, residues 518-803) physically interacts with the catalytic subunit of protein kinase CK2 (CK2 alpha) under non-stressed conditions. A K(D) of 4 x 10(-7) was determined for this binding. Heparin competed with Grp94-CT for binding to CK2 alpha. CK2 beta also inhibited the binding of Grp94-CT to CK2 alpha, and CK2 holoenzyme reconstituted in vitro was unable to bind Grp94-CT. The use of CK2 alpha mutants made it possible to map the Grp94-CT binding site to the four lysine stretch (residues 74-77) present in helix C of CK2 alpha. Grp94-CT stimulated the activity of CK2 alpha wild-type but was ineffective on the CK2 alpha K74-77A mutant.
Amino Acid Sequence↗
Autocatalytic tyrosine-phosphorylation of protein kinase CK2 alpha and alpha' subunits: implication of Tyr182.
CK2 is a pleiotropic and constitutively active serine/threonine protein kinase composed of two catalytic (alpha and/or alpha') and two regulatory beta-subunits, whose mechanism of modulation is still obscure. Here we show that CK2 alpha/alpha' subunits undergo intermolecular (trans) tyrosine-autophosphorylation, which is dependent on intrinsic catalytic activity and is suppressed by the individual mutation of Tyr182, a crucial residue of the activation loop, to phenylalanine. At variance with serine-autophosphorylation, tyrosine-autophosphorylation of CK2alpha is reversed by ADP and GDP and is counteracted by the beta-subunit and by a peptide reproducing the activation loop of CK2alpha/alpha' (amino acids 175-201). These results disclose new perspectives about the mode of regulation of CK2 catalytic subunits.
Adenosine Triphosphate↗
Possible implication of the Golgi apparatus casein kinase in the phosphorylation of vesicle docking protein p115 Ser-940: a study with peptide substrates.
Phosphorylation of human vescicle docking protein p115 at Ser-942 (homologous to Ser-940 in rat p115) promotes its dissociation from the Golgi membrane. Here we show that a peptide encompassing the 934--950 sequence of p115 is unaffected or poorly phosphorylated by a variety of Ser/Thr protein kinases with the notable exception of the Golgi apparatus casein kinase (G-CK) which phosphorylates it with an efficiency comparable to that of its optimal peptide substrates. In contrast phosphorylation of the p115 peptide by protein kinase CK2 is negligible compared to that of the specific peptide substrates of this kinase. Phosphorylation by G-CK is abolished if a conserved cluster of acidic residues at position between n + 4 and n + 9 (EDDDDE) is replaced by a neutral stretch (GAGAGA). These data strongly support the view that G-CK but not the other two classes of ubiquitous "casein kinases" (CK1 and CK2) is the natural phosphorylating agent of p115.
Amino Acid Sequence↗
Selectivity of 4,5,6,7-tetrabromobenzotriazole, an ATP site-directed inhibitor of protein kinase CK2 ('casein kinase-2').
The specificity of 4,5,6,7-tetrabromo-2-azabenzimidazole (TBB), an ATP/GTP competitive inhibitor of protein kinase casein kinase-2 (CK2), has been examined against a panel of 33 protein kinases, either Ser/Thr- or Tyr-specific. In the presence of 10 microM TBB (and 100 microM ATP) only CK2 was drastically inhibited (>85%) whereas three kinases (phosphorylase kinase, glycogen synthase kinase 3 beta and cyclin-dependent kinase 2/cyclin A) underwent moderate inhibition, with IC(50) values one--two orders of magnitude higher than CK2 (IC(50)=0.9 microM). TBB also inhibits endogenous CK2 in cultured Jurkat cells. A CK2 mutant in which Val66 has been replaced by alanine is much less susceptible to inhibition by TBB as well as by another ATP competitive inhibitor, emodin. These data show that TBB is a quite selective inhibitor of CK2, that can be used in cell-based assays.
Adenosine Triphosphate↗
Antennapedia/HS1 chimeric phosphotyrosyl peptide: conformational properties, binding capability to c-Fgr SH2 domain and cell permeability.
With the aim of interfering with the signaling pathways mediated by the SH2 domains of Src-like tyrosine kinases, we synthesized a tyrosyl-phospho decapeptide, corresponding to the sequence 392-401 of HS1 protein, which inhibits the secondary phosphorylation of HS1 protein catalyzed by the Src-like kinases c-Fgr or Lyn. This phospho-peptide was modified to enter cells by coupling to the third helix of Antennapedia homeodomain, which is able to translocate across cell membranes. Here we present CD and fluorescence studies on the conformational behavior in membrane-mimicking environments and on lipid interactions of Antennapedia fragment and its chimeric phosphorylated and unphosphorylated derivatives. These studies evidenced that electrostatic rather than amphiphilic interactions determine the peptide adsorption on lipids. Experiments performed with recombinant protein containing the SH2 domain of c-Fgr fused with GST and with isolated erythrocyte membranes demonstrated that the presence of the N-terminal Antennapedia fragment only slightly affects the binding of the phospho-HS1 peptide to the SH2 domain. In fact, it has been shown that in isolated erythrocyte membranes, both phospho-HS1 peptide and its chimeric derivative greatly affect either the SH2-mediated recruitment of the c-Fgr to the transmembrane protein band 3 and the following phosphorylation of the protein catalyzed by the Src-like kinase c-Fgr. The ability of the chimeric phospho-peptide to enter cells has been demonstrated by confocal microscopy analysis.
Adaptor Proteins, Signal Transducing↗
Expression of the small tyrosine phosphatase (Stp1) in Saccharomyces cerevisiae: a study on protein tyrosine phosphorylation.
Small tyrosine phoshatase 1 (Stp1) is a Schizosaccharomyces pombe low-molecular-mass phosphotyrosine-phosphatase 50% identical to Saccharomyces cerevisiae Ltp1. In order to investigate the role of Stp1 in yeast, a mutant was generated having the characteristic of a dominant negative molecule. Changes in protein tyrosine phosphorylation in S. cerevisiae proteome in response to Stp1 or its dominant negative mutant expression were analyzed by high-resolution two-dimensional (2-D) electrophoresis. The most remarkable result is the modification by phosphorylation on tyrosine of several proteins involved in carbohydrate metabolism. Twelve proteins were identified on the basis of their positions in the anti-phosphotyrosine immunoblot of the 2-D electrophoresis. Ten of these present tyrosyl residues that are within the consensus sequence for protein kinase CK2 (casein kinase-2). These data open the possibility for the identification of Stp1 substrates in yeast and provide hints about the nature of tyrosine phosphorylating agents in yeast and in other organisms where bona fide tyrosine kinases are lacking.
Acrylic Resins↗
Structural features underlying selective inhibition of protein kinase CK2 by ATP site-directed tetrabromo-2-benzotriazole.
Two novel crystal structures of Zea mays protein kinase CK2alpha catalytic subunit, one in complex with the specific inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB) and another in the apo-form, were solved at 2.2 A resolution. These structures were compared with those of the enzyme in presence of ATP and GTP (the natural cosubstrates) and the inhibitor emodin. Interaction of TBB with the active site of CK2alpha is mainly due to van der Waals contacts, with the ligand fitting almost perfectly the cavity. One nitrogen of the five-membered ring interacts with two charged residues, Glu 81 and Lys 68, in the depth of the cavity, through two water molecules. These are buried in the active site and are also generally found in the structures of CK2alpha enzyme analyzed so far, with the exception of the complex with emodin. In the N-terminal lobe, the position of helix alphaC is particularly well preserved in all the structures examined; the Gly-rich loop is displaced from the intermediate position it has in the apo-form and in the presence of the natural cosubstrates (ATP/GTP) to either an upper (with TBB) or a lower position (with emodin). The selectivity of TBB for CK2 appears to be mainly dictated by the reduced size of the active site which in most other protein kinases is too large for making stable interactions with this inhibitor.
Adenosine Triphosphate↗
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.
Alanine↗
HIV-1 Rev transactivator: a beta-subunit directed substrate and effector of protein kinase CK2.
The phosphorylation of HIV-1 Rev by protein kinase CK2 is strictly dependent on the regulatory beta subunit of the kinase and is deeply affected by conformational changes of the substrate outside the phosphorylation site. Here we show that Rev modulates a variety of CK2 properties, including autophosphorylation, catalytic activity toward calmodulin, and susceptibility to polycationic effectors, whose common denominator is the involvement of the beta subunit. Rev's two major CK2 sites are located at its N-terminus, immediately adjacent to a helix-loop-helix motif. By comparing the behaviour of full-size Rev with that of synthetic peptides reproducing, with suitable modifications, its N-terminal 26 amino acids including the phosphoacceptor site (Ser 5, Ser 8) and amphipathic helix-1, it appears that the functional interaction of the N-terminal portion of Rev with the N-terminal domain of the beta subunit must rely on both electrostatic and hydrophobic interactions. The former mainly involve Rev's arginine-rich domain (residues 35-50) in helix-2, while the latter are mostly mediated by residues 12-24 of helix-1. These data disclose the possibility that, besides displaying protective, regulatory and targeting properties with respect to the catalytic subunit, the CK2 beta subunit also plays a role as a docking site for a subset of CK2 substrates.
Amino Acid Motifs↗
Bovine prion protein as a modulator of protein kinase CK2.
On the basis of far-Western blot and plasmon resonance (BIAcore) experiments, we show here that recombinant bovine prion protein (bPrP) (25-242) strongly interacts with the catalytic alpha/alpha' subunits of protein kinase CK2 (also termed 'casein kinase 2'). This association leads to increased phosphotransferase activity of CK2alpha, tested on calmodulin or specific peptides as substrate. We also show that bPrP counteracts the inhibition of calmodulin phosphorylation promoted by the regulatory beta subunits of CK2. A truncated form of bPrP encompassing the C-terminal domain (residues 105-242) interacts with CK2 but does not affect its catalytic activity. The opposite is found with the N-terminal fragment of bPrP (residues 25-116), although the stimulation of catalysis is less efficient than with full-size bPrP. These results disclose the potential of the PrP to modulate the activity of CK2, a pleiotropic protein kinase that is particularly abundant in the brain.
Animals↗
Novel consensus sequence for the Golgi apparatus casein kinase, revealed using proline-rich protein-1 (PRP1)-derived peptide substrates.
Previous studies have shown that the Golgi apparatus casein kinase (G-CK) recognizes phosphoacceptor sites specified by the triplet SXE/Sp, which is found in several phosphoproteins, besides casein itself. In the present study, we report that G-CK can phosphorylate, with comparable efficiency, sequences surrounding Ser-22 of salivary proline-rich protein-1 (PRP1), which do not conform to the SXE/Sp motif. By using a series of peptide substrates derived from the PRP1 Ser-22 site, we also have shown that the optimal consensus sequence recognized by G-CK in this case was SXQXX(D/E)3, where the acidic residues at positions n+5 to n+7 and, to a lesser extent, the glutamine residue at position n+2 are the critical determinants.
Amino Acid Sequence↗
Cooperative modulation of protein kinase CK2 by separate domains of its regulatory beta-subunit.
Protein kinase CK2 ("casein kinase 2") holoenzyme is composed of two catalytic (alpha and/or alpha') and two regulatory beta-subunits. A truncated form of the beta-subunit lacking its C-terminal region (betaDelta171-215) has lost the ability to stably associate with the catalytic subunits and to display a number of properties which are mediated by structural elements still present in its sequence, notably down-regulation of catalytic activity, autophosphorylation, and responsiveness to polycationic effectors. All these functions are restored by simultaneous addition of a synthetic peptide reproducing the deleted fragment, beta170-215, which is able to associate with the catalytic subunits and to stimulate catalytic activity. This peptide includes a segment displaying significant sequence similarity with a region of cyclin A which interacts with the PSTAIRE motif of CDK2 eliciting its catalytic activity. A peptide reproducing this sequence (beta181-203), but not its derivative in which three nonpolar side chains have been replaced by polar ones, interacts with the alpha-subunit and stimulates its catalytic activity; it also partially restores the ability of truncated betaDelta171-215 to autophosphorylate. These data disclose the essential role of a structural module located between residues 181 and 203 in conferring regulatory properties to the beta-subunit of CK2.
Amino Acid Sequence↗
The replacement of ATP by the competitive inhibitor emodin induces conformational modifications in the catalytic site of protein kinase CK2.
The structure of a complex between the catalytic subunit of Zea mays CK2 and the nucleotide binding site-directed inhibitor emodin (3-methyl-1,6,8-trihydroxyanthraquinone) was solved at 2.6-A resolution. Emodin enters the nucleotide binding site of the enzyme, filling a hydrophobic pocket between the N-terminal and the C-terminal lobes, in the proximity of the site occupied by the base rings of the natural co-substrates. The interactions between the inhibitor and CK2 alpha are mainly hydrophobic. Although the C-terminal domain of the enzyme is essentially identical to the ATP-bound form, the beta-sheet in the N-terminal domain is altered by the presence of emodin. The structural data presented here highlight the flexibility of the kinase domain structure and provide information for the design of selective ATP competitive inhibitors of protein kinase CK2.
Adenosine Triphosphate↗