RESUMO
The Ser/Thr kinase CK2, a member of the superfamily of eukaryotic protein kinases, has an acidophilic substrate profile with the substrate recognition sequence S/T-D/E-X-D/E, and it is inhibited by polyanionic substances like heparin. The latter, a highly sulphated glucosamino glycan composed mainly of repeating 2-O-sulpho-α-l-idopyranuronic acid/N,O6-disulpho-α-d-glucosamine disaccharide units, is the longest known substrate-competitive CK2 inhibitor. The structural basis of CK2's preference for anionic substrates and substrate-competitive inhibitors is only vaguely known which limits the value of the substrate-binding region for the structure-based development of CK2 bisubstrate inhibitors. Here, a tetragonal and a monoclinic co-crystal structure of CK2α, the catalytic subunit of CK2, with a decameric heparin fragment are described. In the tetragonal structure, the heparin molecule binds to the polybasic stretch at the beginning of CK2α's helix αC, whereas in the monoclinic structure it occupies the central substrate-recognition region around the P+1 loop. Together, the structures rationalize the inhibitory efficacy of heparin fragments as a function of chain length. The monoclinic CK2α/heparin structure, in which the heparin fragment is particularly well defined, is the first CK2 structure with an anionic inhibitor of considerable size at the central part of the substrate-recognition site. The bound heparin fragment is so close to the binding site of ATP-competitive inhibitors that it can guide the design of linkers and pave the way to efficient CK2 bisubstrate inhibitors in the future.
Assuntos
Caseína Quinase II/antagonistas & inibidores , Desenho de Fármacos , Heparina/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Caseína Quinase II/isolamento & purificação , Caseína Quinase II/metabolismo , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Heparina/química , Humanos , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteínas Quinases/química , Relação Estrutura-AtividadeRESUMO
Resistance to Inhibitors of Cholinesterase-8 (Ric-8) proteins are molecular chaperones that fold heterotrimeric G protein α subunits shortly after biosynthesis. Ric-8 proteins also act as test tube guanine nucleotide exchange factors (GEF) that promote Gα subunit GDP for GTP exchange. The GEF and chaperoning activities of Ric-8A are regulated by phosphorylation of five serine and threonine residues within protein kinase CK2 consensus sites. The traditional way that Ric-8A proteins have been purified is from Spodoptera frugiperda (Sf9) or Trichoplusia ni (Tni) insect cells. Endogenous insect cell kinases do phosphorylate the critical regulatory sites of recombinant Ric-8A reasonably well, but there is batch-to-batch variability among recombinant Ric-8A preparations. Additionally, insect cell-production of some Ric-8 proteins with phosphosite alanine substitution mutations is proscribed as there seems to be interdependency of multi-site phosphorylation for functional protein production. Here, we present a method to produce wild type and phosphosite mutant Ric-8A proteins that are fully occupied with bound phosphate at each of the regulatory positions. Ric-8A proteins were expressed and purified from E. coli. Purified Ric-8A was phosphorylated in vitro with protein kinase CK2 and then re-isolated to remove kinase. The phosphorylated Ric-8A proteins were â¼99% pure and the completeness of phosphorylation was verified by chromatography, phos-tag SDS-PAGE mobility shifts, immunoblotting using phospho-site specific antibodies, and mass spectrometry analysis. E. coli-produced Ric-8A that was phosphorylated using this method promoted a faster rate of Gα subunit guanine nucleotide exchange than Ric-8A that was variably phosphorylated during production in insect cells.
Assuntos
Caseína Quinase II , Fatores de Troca do Nucleotídeo Guanina , Fosfoproteínas , Caseína Quinase II/química , Caseína Quinase II/genética , Caseína Quinase II/isolamento & purificação , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/isolamento & purificação , Humanos , Fosfoproteínas/química , Fosfoproteínas/genética , Fosfoproteínas/isolamento & purificação , Fosforilação , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
Emodin (1,3,8-trihydroxy-6-methyl-anthraquinone) is a natural secondary plant product, originally isolated from the rhizomes of Rheum palmatum. Many reports show its diuretic, vasorelaxant, antibacterial, antiviral, anti-ulcerogenic, immunosuppressive, hepatoprotective, anti-inflammatory and anticancer potential. Emodin is a pleiotropic molecule capable of interacting with several major molecular targets, e.g. NF-κB, AKT/mTOR and STAT3. The compound can also act as an inhibitor of some protein kinases, with special affinity to protein kinase CK2. The aim of the presented report was to evaluate antifungal properties of emodin and its activity towards CK2 isolated from Candida cells. Our studies revealed that the compound suppressed growth of the cells of reference strains as well as clinical Candida strains, with minimal inhibitory concentration and minimal fungicidal concentration values between 12.5 and 200 µg/mL. Moreover, at a low concentration, the compound was able to effectively stop hyphal formation, thus showing a distinct antivirulent potential. Interestingly, we showed that emodin added to Candida culture inhibited the phosphorylation of many cellular proteins, presumably owing to the inhibition of protein kinase CK2. Notably, the enzyme isolated from the Candida cells was susceptible to emodin with IC50 of 2.8 µg/mL. Indeed, our computational modelling revealed that emodin was able to occupy the ATP-binding pocket of CK2. Copyright © 2017 John Wiley & Sons, Ltd.
Assuntos
Antifúngicos/farmacologia , Biofilmes/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Caseína Quinase II/antagonistas & inibidores , Emodina/farmacologia , Hifas/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Candida albicans/crescimento & desenvolvimento , Caseína Quinase II/isolamento & purificação , Hifas/crescimento & desenvolvimento , Testes de Sensibilidade Microbiana , Microscopia de FluorescênciaRESUMO
CK2 is a ubiquitous protein kinase involved in many cell functions. During the last years it became an interesting target in cancer research. A series of flavonoid compounds was tested as inhibitors of protein kinase CK2. Several substances were found to be highly active against both catalytic subunits with IC50 values below 1 µM in case of CK2α'. The most promising inhibitor we identified is chrysoeriol with IC50 values of 250 and 34 nM for CK2α and CK2α', respectively.
Assuntos
Caseína Quinase II/antagonistas & inibidores , Flavonas/isolamento & purificação , Flavonas/farmacologia , Sequência de Aminoácidos , Caseína Quinase II/química , Caseína Quinase II/isolamento & purificação , Caseína Quinase II/farmacologia , Domínio Catalítico , Flavonas/química , Humanos , Estrutura MolecularRESUMO
Ethanol, the major fermentation product of Saccharomyces cerevisiae, has long been known as an inducer of heat shock response, but the underlying mechanisms by which ethanol activates heat shock transcription factor (HSF) are not well understood. We demonstrate that CK2-dependent phosphorylation on S608 is an ethanol stress-specific repression mechanism of Hsf1, which does not affect the basal or heat-induced activity of Hsf1. This repression is relieved by dephosphorylation by Ppt1 which directly interacts with Hsf1 via its tetratricopeptide repeat (TPR) domain. In response to ethanol stress, PPT1 deletion and CK2 overexpression exert synergistic inhibitory effects on Hsf1 activation, whereas Hsf1(S608A) mutant shows enhanced activation. Therefore, regulation of the Hsf1 S608 phosphorylation status by reciprocal actions of CK2 and Ppt1 might play an important role to determine Hsf1 sensitivity towards ethanol stress.
Assuntos
Caseína Quinase II/metabolismo , Proteínas de Ligação a DNA/metabolismo , Etanol/metabolismo , Proteínas de Choque Térmico/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Western Blotting , Caseína Quinase II/genética , Caseína Quinase II/isolamento & purificação , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/genética , Fosforilação , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Estresse Fisiológico , Fatores de Transcrição/química , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
Spt2/Sin1 is a DNA binding protein with HMG-like domains. It plays a role in chromatin modulations associated with transcription elongation in Saccharomyces cerevisiae. Spt2 maintains the nucleosome level in coding regions and is important for the inhibition of spurious transcription in yeast. In this work, we undertook a biochemical approach to identify Spt2-interacting partners. Interestingly, casein kinase 2 (CK2) interacts with Spt2 and phosphorylates it in vitro as well as in vivo on two small regions, region I (RI) (amino acids 226 to 230) and RII (amino acids 277 to 281), located in its essential C-terminal domain. Mutation of the phosphorylation sites in RI and RII to acidic residues, thereby mimicking CK2 phosphorylation, leads to the inhibition of Spt2 function in the repression of spurious transcription and to a loss of its recruitment to coding regions. Inversely, depleting cells of CK2 activity leads to an increased Spt2 association with genes. We further show that Spt2 physically interacts with the essential histone chaperone Spt6 and that this association is inhibited in vitro and in vivo by CK2-dependent phosphorylation. Taken together, our data suggest that CK2 regulates the function of Spt2 by modulating its interaction with chromatin and the histone chaperone Spt6.
Assuntos
Caseína Quinase II/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Transcrição Gênica , Caseína Quinase II/química , Caseína Quinase II/isolamento & purificação , Domínio Catalítico , Cromatina/genética , Cromatina/metabolismo , Cromatografia de Afinidade , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/isolamento & purificação , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/isolamento & purificação , Fases de Leitura Aberta , Fosforilação , Regiões Promotoras Genéticas , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Processamento de Proteína Pós-Traducional , Subunidades Proteicas/isolamento & purificação , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Serina/metabolismo , Tirosina/metabolismoRESUMO
Alternative splicing of pre-mRNA is a highly regulated process that allows cells to change their genetic informational output. These changes are mediated by protein factors that directly bind specific pre-mRNA sequences. Although much is known about how these splicing factors regulate pre-mRNA splicing events, comparatively little is known about the regulation of the splicing factors themselves. Here, we show that the Drosophila splicing factor P element Somatic Inhibitor (PSI) is phosphorylated at at least two different sites by at minimum two different kinases, casein kinase II (CK II) and tousled-like kinase (tlk). These phosphorylation events may be important for regulating protein-protein interactions involving PSI. Additionally, we show that PSI interacts with several proteins in Drosophila S2 tissue culture cells, the majority of which are splicing factors.
Assuntos
Caseína Quinase II/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Splicing de RNA/genética , Proteínas de Ligação a RNA/metabolismo , Animais , Caseína Quinase II/isolamento & purificação , Proteínas de Drosophila/química , Drosophila melanogaster/citologia , Espectrometria de Massas , Mutação/genética , Proteínas Nucleares/química , Fosforilação , Mapeamento de Interação de Proteínas , Proteínas de Ligação a RNA/químicaRESUMO
The Mediterranean fruit fly Ceratitis capitata is an insect capable of wreaking extensive damage to a wide range of fruit crops. Protein kinase CK2 is a ubiquitous Ser/Thr kinase that is highly conserved among eukaryotes; it is a heterotetramer composed of two catalytic (α) and a dimer of regulatory (ß) subunits. We present here the construction of the cDNA molecules of the CK2α and CK2ß subunits from the medfly C. capitata by the 5'/3' RACE and RT-PCR methods, respectively. CcCK2α catalytic subunit presents the characteristic and conserved features of a typical protein kinase, similar to the regulatory CcCK2ß subunit, that also possess the conserved features of regulatory CK2ß subunits, as revealed by comparison of their predicted amino acid sequences with other eukaryotic species. The recombinant CcCK2α and CcCK2ß proteins were purified by affinity chromatography to homogeneity, after overexpression in Escherichia coli. CcCK2α is capable to utilize GTP and its activity and is inhibited by polyanions and stimulated by polycations in phosphorylation assays, using purified acidic ribosomal protein P1 as a substrate.
Assuntos
Caseína Quinase II/genética , Caseína Quinase II/isolamento & purificação , Ceratitis capitata/enzimologia , Ceratitis capitata/genética , Subunidades Proteicas/genética , Subunidades Proteicas/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação , Sequência de Aminoácidos , Animais , Caseína Quinase II/química , Domínio Catalítico , Cromatografia de Afinidade , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Escherichia coli/metabolismo , Proteínas Imobilizadas/metabolismo , Dados de Sequência Molecular , Subunidades Proteicas/química , Proteínas Recombinantes/genética , Alinhamento de SequênciaRESUMO
Protein kinase CK2 is a ubiquitous, highly pleiotropic, and constitutively active phosphotransferase that phosphorylates mainly serine and threonine residues. CK2 has been studied and characterized in many organisms, from yeast to mammals. The holoenzyme is generally composed of two catalytic (α and/or α') and two regulatory (ß) subunits, forming a differently assembled tetramer. The free and catalytically active α/α' subunits can be present in cells under some circumstances. We present here the isolation of a putative catalytic CK2α subunit and holoenzyme from gills of the mussel Mytilus galloprovincialis capable of phosphorylating the purified recombinant ribosomal protein rMgP1. For further analysis of M. galloprovincialis protein kinase CK2, the cDNA molecules of CK2α and CK2ß subunits were constructed and cloned into expression vectors, and the recombinant proteins were purified after expression in Escherichia coli. The recombinant MgCK2ß subunit and MgP1 were phosphorylated by the purified recombinant MgCK2α subunit. The mussel enzyme presented features typical for CK2: affinity for GTP, inhibition by both heparin and ATP competitive inhibitors (TBBt, TBBz), and sensitivity towards NaCl. Predicted amino acid sequence comparison showed that the M. galloprovincialis MgCK2α and MgCK2ß subunits have similar features to their mammalian orthologs.
Assuntos
Caseína Quinase II/genética , Caseína Quinase II/isolamento & purificação , DNA Complementar/biossíntese , Mytilus/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Primers do DNA/genética , Brânquias/enzimologia , Dados de Sequência Molecular , Fosfoproteínas/metabolismo , Fosforilação , Subunidades Proteicas/genética , Subunidades Proteicas/isolamento & purificação , Proteínas Ribossômicas/metabolismo , Análise de Sequência de DNA , Homologia de SequênciaRESUMO
The checkpoint clamp Rad9-Hus1-Rad1 (9-1-1) is loaded by the Rad17-RFC complex onto chromatin after DNA damage and plays a key role in the ATR-dependent checkpoint activation. Here, we demonstrate that in vitro casein kinase 2 (CK2) specifically interacts with human 9-1-1 and phosphorylates serines 341 and 387 (Ser-341 and Ser-387) in the C-terminal tail of Rad9. Interestingly, phosphorylated Ser-387 has previously been reported to be required for interacting with a checkpoint mediator TopBP1. Indeed, 9-1-1 purified from Escherichia coli and phosphorylated in vitro by CK2 physically interacts with TopBP1. Further analyses showed that phosphorylation at both serine residues occurs in vivo and is required for the efficient interaction with TopBP1 in vitro. Furthermore, when over-expressed in HeLa cells, a mutant Rad9 harboring phospho-deficient substitutions at both Ser-341 and Ser-387 residues causes hypersensitivity to UV and methyl methane sulfonate (MMS). Our observations suggest that CK2 plays a crucial role in the ATR-dependent checkpoint pathway through its ability to phosphorylate Ser-341 and Ser-387 of the Rad9 subunit of the 9-1-1 complex.
Assuntos
Proteínas de Transporte/metabolismo , Caseína Quinase II/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Exonucleases/metabolismo , Proteínas Nucleares/metabolismo , Caseína Quinase II/isolamento & purificação , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/isolamento & purificação , Células Cultivadas , Células HeLa , Humanos , Mutação , Fosforilação , Serina/metabolismoRESUMO
A previously described wheat germ protein kinase (Yan, T. F., and Tao, M. (1982) J. Biol. Chem. 257, 7037-7043) was identified unambiguously as CK2 using mass spectrometry. CK2 is a ubiquitous eukaryotic protein kinase that phosphorylates a wide range of substrates. In previous studies, this wheat germ kinase was shown to phosphorylate eIF2alpha, eIF3c, and three large subunit (60 S) ribosomal proteins (Browning, K. S., Yan, T. F., Lauer, S. J., Aquino, L. A., Tao, M., and Ravel, J. M. (1985) Plant Physiol. 77, 370-373). To further characterize the role of CK2 in the regulation of translation initiation, Arabidopsis thaliana catalytic (alpha1 and alpha2) and regulatory (beta1, beta2, beta3, and beta4) subunits of CK2 were cloned and expressed in Escherichia coli. Recombinant A. thaliana CK2beta subunits spontaneously dimerize and assemble into holoenzymes in the presence of either CK2alpha1 or CK2alpha2 and exhibit autophosphorylation. The purified CK2 subunits were used to characterize the properties of the individual subunits and their ability to phosphorylate various plant protein substrates. CK2 was shown to phosphorylate eIF2alpha, eIF2beta, eIF3c, eIF4B, eIF5, and histone deacetylase 2B but did not phosphorylate eIF1, eIF1A, eIF4A, eIF4E, eIF4G, eIFiso4E, or eIFiso4G. Differential phosphorylation was exhibited by CK2 in the presence of various regulatory beta-subunits. Analysis of A. thaliana mutants either lacking or overexpressing CK2 subunits showed that the amount of eIF2beta protein present in extracts was affected, which suggests that CK2 phosphorylation may play a role in eIF2beta stability. These results provide evidence for a potential mechanism through which the expression and/or subcellular distribution of CK2 beta-subunits could participate in the regulation of the initiation of translation and other physiological processes in plants.
Assuntos
Arabidopsis/enzimologia , Caseína Quinase II/metabolismo , Fatores de Iniciação de Peptídeos/metabolismo , Arabidopsis/genética , Biocatálise , Caseína Quinase II/química , Caseína Quinase II/isolamento & purificação , Cromatografia em Gel , Biologia Computacional , Eletroforese em Gel de Poliacrilamida , Holoenzimas/metabolismo , Fosforilação , Plantas Geneticamente Modificadas , Proteínas Quinases/metabolismo , Estabilidade Proteica , Estrutura Quaternária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Triticum/enzimologiaRESUMO
Altogether 2 holoenzymes and 4 catalytic CK2 constructs were expressed and characterized i.e. CK2alpha(2)1-335 beta2; CK2alpha'-derived holoenzyme; CK2alpha1-335; MBP-CK2alpha'; His-tagged CK2alpha and His-tagged CK2alpha'. The two His-tagged catalytic subunits were expressed in insect cells, all others in Escherichia coli. IC50 studies involving the established CK2 inhibitors DMAT, TBBt, TBBz, apigenin and emodin were carried out and the Ki values calculated. Although the differences in the Ki values found were modest, there was a general tendency showing that the CK2 holoenzymes were more sensitive towards the inhibitors than the free catalytic subunits. Thermal inactivation experiments involving the individual catalytic subunits showed an almost complete loss of activity after only 2 min at 45 degrees C. In the case of the two holoenzymes, the CK2alpha'-derived holoenzyme lost ca. 90% of its activity after 14 min, whereas CK2alpha2(1-335) beta2 only showed a loss of ca. 40% by this time of incubation. Gel filtration analyses were performed at high (500 mM) and low (150 mM) monovalent salt concentrations in the absence or presence of ATP. At 500 mM NaCl the CK2alpha'-derived holoenzyme eluted at a position corresponding to a molecular mass of 105 kDa which is significantly below the elution of the CK2alpha(2)1-335 beta2 holoenzyme (145 kDa). Calmodulin was not phosphorylated by either CK2alpha2(1-335) beta2 or the CK2alpha'-derived holoenzyme. However, in the presence of polylysine only the CK2alpha(2)1-335 beta2 holoenzyme could use calmodulin as a substrate such as the catalytic subunits, in contrast to the CK2alpha'-derived holoenzyme which only phosphorylated calmodulin weakly. This attenuation may be owing to a different structural interaction between the catalytic CK2alpha' subunit and non-catalytic CK2beta subunit.
Assuntos
Caseína Quinase II/química , Caseína Quinase II/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Homologia de Sequência de Aminoácidos , Sequência de Aminoácidos , Animais , Autorradiografia , Calmodulina/metabolismo , Caseína Quinase II/antagonistas & inibidores , Caseína Quinase II/isolamento & purificação , Linhagem Celular , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática/efeitos dos fármacos , Holoenzimas/química , Holoenzimas/metabolismo , Insetos , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Estrutura Quaternária de Proteína , Alinhamento de Sequência , Cloreto de Sódio/farmacologia , TemperaturaRESUMO
Human cytomegalovirus (HCMV) UL84 is a phosphoprotein that shuttles from the nucleus to the cytoplasm and is required for oriLyt-dependent DNA replication and viral growth. UL84 was previously shown to interact with IE2 (IE86) in infected cells, and this interaction down-regulates IE2-mediated transcriptional activation in transient assays. UL84 and IE2 were also shown to cooperatively activate a promoter within HCMV oriLyt. UL84 alone can interact with an RNA stem-loop within oriLyt and is bound to this structure within the virion. In an effort to investigate the binding partners for UL84 in infected cells, we pulled down UL84 from protein lysates prepared from HCMV-infected human fibroblasts by using a UL84-specific antibody and resolved the immunoprecipitated protein complexes by two-dimensional gel electrophoresis. We subsequently identified individual proteins by matrix-assisted laser desorption ionization-tandem time of flight analysis. This analysis revealed that UL84 interacts with viral proteins UL44, pp65, and IE2. In addition, a number of cell-encoded proteins were identified, including ubiquitin-conjugating enzyme E2, casein kinase II (CKII), and the multifunctional protein p32. We also confirmed the interaction between UL84 and IE2 as well as the interaction of UL84 with importin alpha. UL44, pp65, and CKII interactions were confirmed to occur in infected and cotransfected cells by coimmunoprecipitation assays followed by Western blotting. Ubiquitination of UL84 occurred in the presence and absence of the proteasome activity inhibitor MG132 in infected cells. The identification of UL84 binding partners is a significant step toward the understanding of the function of this significant replication protein.
Assuntos
Citomegalovirus/fisiologia , Proteínas Virais/metabolismo , Caseína Quinase II/isolamento & purificação , Caseína Quinase II/metabolismo , Linhagem Celular , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Eletroforese em Gel Bidimensional , Fibroblastos/química , Fibroblastos/virologia , Humanos , Proteínas Imediatamente Precoces/isolamento & purificação , Proteínas Imediatamente Precoces/metabolismo , Fosfoproteínas/isolamento & purificação , Fosfoproteínas/metabolismo , Ligação Proteica , Proteoma/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Transativadores/isolamento & purificação , Transativadores/metabolismo , Enzimas de Conjugação de Ubiquitina/isolamento & purificação , Enzimas de Conjugação de Ubiquitina/metabolismo , Proteínas da Matriz Viral/isolamento & purificação , Proteínas da Matriz Viral/metabolismo , Proteínas Virais/isolamento & purificaçãoRESUMO
Protein kinase CK2 is a highly conserved Ser/Thr protein kinase that is ubiquitous among eucaryotic organisms and appears to play an important role in many cellular functions. This enzyme in yeast has a tetrameric structure composed of two catalytic (alpha and/or alpha') subunits and two regulatory beta and beta' subunits. Previously, we have reported isolation from yeast cells four active forms of CK2, composed of alphaalpha'betabeta', alpha2betabeta', alpha'2betabeta' and a free alpha'-catalytic subunit. Now, we report that in Saccharomyces cerevisiae CK2 holoenzyme regulatory beta subunit cannot substitute other beta' subunit and only both of them can form fully active enzymatic unit. We have examined the subunit composition of tetrameric complexes of yeast CK2 by transformation of yeast strains containing single deletion of the beta or beta' regulatory subunits with vectors carrying lacking CKB1 or CKB2 genes. CK2 holoenzyme activity was restored only in cases when both of them were present in the cell. Additional, co-immunoprecypitation experiments show that polyadenylation factor Fip1 interacts with catalytic alpha subunits of CK2 and interaction with beta subunits in the holoenzyme decreases CK2 activity towards this protein substrate. These data may help to elucidate the role of yeast protein kinase CK2beta/beta' subunits in the regulation of holoenzyme assembly and phosphotransferase activity.
Assuntos
Caseína Quinase II/metabolismo , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/enzimologia , Caseína Quinase II/isolamento & purificação , Holoenzimas/isolamento & purificação , Holoenzimas/metabolismo , Imunoprecipitação , Fosforilação , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismoRESUMO
CK2 is a ubiquitous but enigmatic kinase. The difficulty in assigning a role to CK2 centers on the fact that, to date, no biologically relevant modulator of its function has been identified. One common theme revolves around a constellation of known substrates involved in growth control, compatible with its concentration in the nucleus and nucleolus. We had previously described the identification of two catalytic subunits of CK2 in Trypanosoma brucei and characterized one of them. Here we report the characterization of the second catalytic subunit, CK2alpha', and the identification and characterization of the regulatory subunit CK2beta. All three subunits are primarily localized to the nucleolus in T. brucei. We also show that CK2beta interacts with the nucleolar protein NOG1, adding to the interaction map which previously linked CK2alpha to the nucleolar protein NOPP44/46, which in turn associates with the rRNA binding protein p37. CK2 activity has four distinctive features: near equal affinity for GTP and ATP, heparin sensitivity, and stimulation by polyamines and polybasic peptides. Sequence comparison shows that the parasite orthologues have mutations in residues previously mapped as important in specifying affinity for GTP and stimulation by both polyamines and polybasic peptides. Studies of the enzymatic activity of the T. brucei CK2s show that both the affinity for GTP and stimulation by polyamines have been lost and only the features of heparin inhibition and stimulation by polybasic peptides are conserved.
Assuntos
Caseína Quinase II/metabolismo , Trypanosoma brucei brucei/enzimologia , Sequência de Aminoácidos , Animais , Caseína Quinase II/química , Caseína Quinase II/genética , Caseína Quinase II/isolamento & purificação , Núcleo Celular/metabolismo , Sequência Conservada , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Isoenzimas/genética , Isoenzimas/isolamento & purificação , Isoenzimas/metabolismo , Dados de Sequência Molecular , Ligação Proteica , Interferência de RNA , Alinhamento de Sequência , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/crescimento & desenvolvimentoRESUMO
In this work, we report the cloning and characterization of the first cell surface casein kinase II (CKII) substrate (Tc-1) of Trypanosoma cruzi, the causative agent of Chagas' disease. Analysis of the gene sequence revealed a 1,653-bp open reading frame coding for 550 amino acid residues. Northern blot analysis showed a 4.5-kb transcript that is expressed in invasive trypomastigotes but not in noninvasive epimastigote forms of T. cruzi. Southern blot analysis indicates that Tc-1 is a single-copy gene. At the amino acid level, Tc-1 displayed 95% and 99% identity to two hypothetical proteins recently reported by the T. cruzi genome project. Analysis of the translated amino acid sequence indicates that the Tc-1 gene has a putative transmembrane domain with multiple cytoplasmic and extracellular CKII phosphosites. Exogenous human CKII was able to phosphorylate serine residues on both recombinant Tc-1 and Tc-1 of intact trypomastigotes. This phosphorylation was inhibited by the CKII inhibitors heparin and 4,5,6,7,-tetrabromo-2-azabenzimidazole. Immunoblots of solubilized trypomastigotes, epimastigotes, and amastigotes probed with anti-recombinant Tc-1 immunoglobulin G revealed a 62-kDa protein that is expressed only in infective trypomastigotes. Immunoprecipitation of labeled surface proteins of trypomastigotes indicated that the 62-kDa protein is a surface protein, and we found that the protein is uniformly distributed on the surface of trypomastigotes by direct immunofluorescence. Antibodies to Tc-1 effectively blocked trypomastigote invasion of host cells and consequently reduced parasite load. Preincubation of either trypomastigotes or myoblasts with CKII inhibitors blocked T. cruzi infection. Thus, for the first time, we describe a cell surface CKII substrate of a protozoan parasite that is phosphorylated by human CKII and that is involved in cellular infection.
Assuntos
Caseína Quinase II/metabolismo , Clonagem Molecular , Proteínas de Membrana/genética , Mioblastos Cardíacos/parasitologia , Proteínas de Protozoários/genética , Trypanosoma cruzi/genética , Trypanosoma cruzi/metabolismo , Sequência de Aminoácidos , Animais , Caseína Quinase II/química , Caseína Quinase II/genética , Caseína Quinase II/isolamento & purificação , Humanos , Proteínas de Membrana/isolamento & purificação , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Peso Molecular , Mioblastos Cardíacos/enzimologia , Fosforilação , Proteínas de Protozoários/isolamento & purificação , Proteínas de Protozoários/metabolismo , Ratos , Especificidade por Substrato , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/patogenicidade , Fatores de Virulência/genética , Fatores de Virulência/isolamento & purificaçãoRESUMO
Protein kinase CK2 (former name: "casein kinase 2") is a pivotal and ubiquitously expressed member of the eukaryotic protein kinase superfamily. It predominantly exists as a heterotetrameric holoenzyme composed of two catalytic subunits (CK2alpha) and two regulatory subunits (CK2beta). In higher animals two paralog catalytic chains-abbreviated CK2alpha and CK2alpha'--exist which can combine with CK2beta to three isoforms of the holoenzyme: CK2alpha(2)beta(2), CK2alpha(2)(')beta(2), and CK2alphaalpha(')beta(2). While CK2alpha and the "normal" holoenzyme CK2alpha(2)beta(2) have been extensively characterized in vitro and in vivo, little is known about the enzymological properties of CK2alpha' and the "alternative" holoenzyme CK2alpha(2)(')beta(2) and about their specific physiological roles. A major reason for this lack of knowledge is the fact that so far CK2alpha' rather than CK2alpha has caused serious stability and solubility problems during standard heterologous expression procedures. To overcome them, we developed a preparation scheme for CK2alpha(2)(')beta(2) from Homo sapiens in catalytically active form based on two critical steps: first expression of human CK2alpha' as a well soluble fusion protein with the maltose binding protein (MBP) and second proteolytic cleavage of CK2alpha'-MBP in the presence of human CK2beta so that CK2alpha' subunits are incorporated into holoenzyme complexes directly after their release from MBP. This successful strategy which may be adopted in comparably difficult cases of protein/protein complex preparation is presented here together with evidence that the CK2alpha'-based and the CK2alpha-based holoenzymes are similar concerning their catalytic activities but are significantly different with respect to some well-known CK2 properties like autophosphorylation and supra-molecular aggregation.
Assuntos
Caseína Quinase II/isolamento & purificação , Proteínas de Transporte , Caseína Quinase II/genética , Holoenzimas/genética , Holoenzimas/isolamento & purificação , Humanos , Isoenzimas/genética , Isoenzimas/isolamento & purificação , Proteínas Ligantes de Maltose , Fosforilação , Ligação Proteica , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificaçãoRESUMO
CK2 is a pleiotropic constitutively active serine/threonine protein kinase composed of two catalytic alpha- and two regulatory beta-subunits, whose regulation is still not well understood. It seems to play an essential role in regulation of many cellular processes. Four active forms of CK2, composed of alphaalpha'betabeta', alpha(2)betabeta', alpha'(2)betabeta', and a free alpha'-subunit were isolated from wild-type yeast and strains containing a single deletion of the catalytic subunit. Each species exhibits properties typical for CK2, but they differ in substrate specificity and sensitivity to inhibitors. This suggests that each CK2 isomer may regulate different process or may differ in the way of its regulation.
Assuntos
Caseína Quinase II/metabolismo , Isoenzimas/metabolismo , Saccharomyces cerevisiae/enzimologia , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Caseína Quinase II/genética , Caseína Quinase II/isolamento & purificação , Catálise , Isoenzimas/isolamento & purificação , Cinética , Subunidades Proteicas/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Proteínas de Saccharomyces cerevisiae/metabolismo , Deleção de SequênciaRESUMO
By means of Mono P column chromatography, an effective phosphate acceptor (EPA) of casein kinase 2 (CK2) was purified from the Bowman-Birk-type proteinase inhibitor (BBI) fraction of soybean seeds. The most acidic EPA (aEPA, pI=approx. 3.7) was heavily phosphorylated when incubated with CK2 and 5 microM [gamma-(32)P]ATP in the presence of poly-Arg (a CK2 activator) in vitro. However, aEPA was slightly phosphorylated by casein kinase 1 (CK1) as effective as C-kinase and not at all by A-kinase in vitro. The 13 N-terminal amino acid residues (SDHSSSDDESSKP) of aEPA were 100% homologous to the corresponding sequence of soybean BBI-type proteinase inhibitor CII (SBI CII). Polyamine at 3 mM stimulated 4.6-fold the CK2-mediated phosphorylation of aEPA, and this phosphorylation was sensitive to quercetin (ID(50)=approx. 0.1 microM) in vitro. Furthermore, two basic proteins [Mr=29,000 (p29) and 17,000 (p17)] copurified with BBI were identified as proteolytic cleavage products of basic 7S globulin and functioned as potent CK2 activators in vitro. aEPA fully phosphorylated by CK2 in the presence of poly-Arg or basic proteins formed a complex with trypsin, whereas unphosphorylated aEPA was digested by trypsin in vitro. These results suggest that (i) aEPA (a BBI isoform) may coexist with two basic proteins (p29 and p17) generated from basic 7S globulin; and (ii) the physiological interaction between aEPA and its binding trypsin-like proteinases may be regulated through specific phosphorylation of aEPA by CK2 activated with the two basic proteins in legume seeds.
Assuntos
Caseína Quinase II/metabolismo , Glycine max/química , Sementes/química , Inibidor da Tripsina de Soja de Bowman-Birk/metabolismo , Sequência de Aminoácidos , Caseína Quinase II/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Ativação Enzimática , Concentração de Íons de Hidrogênio , Cinética , Fosforilação , Quercetina/metabolismo , Espermina/metabolismo , Tripsina/metabolismo , Inibidor da Tripsina de Soja de Bowman-Birk/isolamento & purificaçãoRESUMO
Posttranslational modification of circadian clock proteins by phosphorylation is an essential regulatory process in the control of eukaryotic circadian clocks. In the Neurospora circadian clock, the key clock protein FREQUENCY (FRQ) is progressively phosphorylated. The phosphorylation of FRQ is regulated by both kinases and phosphatases, and the phosphorylation is important for regulating FRQ stability and its function in the circadian negative feedback loop. The degradation of FRQ is mediated by the ubiquitin/proteasome pathway. This article discusses posttranslational regulations of the Neurospora clock and describes the methods used in the studies of FRQ phosphorylation, FRQ kinases and phosphatases, and FRQ degradation.