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PubMed2007

Mitosis-specific MPM-2 phosphorylation of DNA topoisomerase IIalpha is regulated directly by protein phosphatase 2A.

Recent results suggest a role for topoIIalpha (topoisomerase IIalpha) in the fine-tuning of mitotic entry. Mitotic entry is accompanied by the formation of specific phosphoepitopes such as MPM-2 (mitotic protein monoclonal 2) that are believed to control mitotic processes. Surprisingly, the MPM-2 kinase of topoIIalpha was identified as protein kinase CK2, otherwise known as a constitutive interphase kinase. This suggested the existence of alternative pathways for the creation of mitotic phosphoepitopes, different from the classical pathway where the substrate is phosphorylated by a mitotic kinase. In the present paper, we report that topoIIalpha is co-localized with both CK2 and PP2A (protein phosphatase 2A) during interphase. Simultaneous incubation of purified topoIIalpha with CK2 and PP2A had minimal influence on the total phosphorylation levels of topoIIalpha, but resulted in complete disappearance of the MPM-2 phosphoepitope owing to opposite sequence preferences of CK2 and PP2A. Accordingly, short-term exposure of interphase cells to okadaic acid, a selective PP2A inhibitor, was accompanied by the specific appearance of the MPM-2 phosphoepitope on topoIIalpha. During early mitosis, PP2A was translocated from the nucleus, while CK2 remained in the nucleus until pro-metaphase thus permitting the formation of the MPM-2 phosphoepitope. These results underline the importance of protein phosphatases as an alternative way of creating cell-cycle-specific phosphoepitopes.

Antigens, Neoplasm↗
PubMed1995

Direct identification of a polyamine binding domain on the regulatory subunit of the protein kinase casein kinase 2 by photoaffinity labeling.

Phosphorylation of many protein substrates by the protein kinase casein kinase 2 (CK2) is stimulated severalfold in the presence of polyamines such as spermine. Previous experiments have shown that CK2 is a polyamine binding protein and that the regulatory beta subunit is required for this binding activity. To delineate the spermine binding site of CK2, we have applied a photoaffinity labeling method using a tritiated photoactivable analog of spermine, [3H]sperminediazonium. The photoaffinity labeled beta subunit was cleaved with cyanogen bromide, and two labeled peptides were separated by high performance liquid chromatography. The major one was the peptide T72EQAAEM78 and the minor one was a 22-amino acid peptide comprising residues Ile98 to Met119. Thr72 and His108 were identified as the labeled amino acids of the Thr72-Met78 and Ile98-Met119 peptides, respectively. In the same manner, we succeeded in determining the residue Leu220 as an alpha subunit residue covalently bound to the probe. The photoaffinity labeling method described here enabled the first elucidation, by direct microsequencing, of a polyamine binding site on CK2 for which we propose a provisional structural model. These observations suggest a possible mechanism for CK2 activation by polyamines at the molecular level.

Affinity Labels↗
PubMed1998

Protein kinase CK2alpha' is induced by serum as a delayed early gene and cooperates with Ha-ras in fibroblast transformation.

Protein kinase CK2 is an ubiquitous and pleiotropic Ser/Thr protein kinase composed of two catalytic (alpha and/or alpha') and two noncatalytic (beta) subunits forming a heterotetrameric holoenzyme involved in cell growth and differentiation. Here we report the identification, cloning, and oncogenic activity of the murine CK2alpha' subunit. Serum treatment of quiescent mouse fibroblasts induces CK2alpha' mRNA expression, which peaks at 4 h. The kinetics of CK2alpha' expression correlate with increased kinase activity toward a specific CK2 holoenzyme peptide substrate. The ectopic expression of CK2alpha' (or CK2alpha) cooperates with Ha-ras in foci formation of rat primary embryo fibroblasts. Moreover, we observed that BALB/c 3T3 fibroblasts transformed with Ha-ras and CK2alpha' show a faster growth rate than cells transformed with Ha-ras alone. In these cells the higher growth rate correlates with an increase in calmodulin phosphorylation, a protein substrate specifically affected by isolated CK2 catalytic subunits but not by CK2 holoenzyme, suggesting that unbalanced expression of a CK2 catalytic subunit synergizes with Ha-ras in cell transformation.

3T3 Cells↗
PubMed1999

Tyrosine versus serine/threonine phosphorylation by protein kinase casein kinase-2. A study with peptide substrates derived from immunophilin Fpr3.

Protein kinase casein kinase-2 (CK2) is a spontaneously active, ubiquitous, and pleiotropic enzyme that phosphorylates seryl/threonyl residues specified by multiple negatively charged side chains, the one at position n + 3 being of crucial importance (minimum consensus S/T-x-x-E/D/S(P)/T(P). Recently CK2 has been reported to catalyze phosphorylation of the yeast nucleolar immunophilin Fpr3 at a tyrosyl residue (Tyr(184)) fulfilling the consensus sequence of Ser/Thr substrates (Wilson, L.K., Dhillon, N., Thorner, J., and Martin, G.S. (1997) J. Biol. Chem. 272, 12961-12967). Here we show that, by contrast to other tyrosyl peptides fulfilling the consensus sequence for CK2, a peptide reproducing the sequence around Fpr3 Tyr(184) (DEDADIY(184)DEEDYDL) is phosphorylated by CK2, albeit with much higher K(m) (384 versus 4. 3 microM) and lower V(max) (8.4 versus 1,132 nmol.min(-1).mg(-1)) than its derivative with Tyr(184) replaced by serine. The replacement of Asp at position n + 1 with alanine and, to a lesser extent, of Ile at n - 1 with Asp are especially detrimental to tyrosine phosphorylation as compared with serine phosphorylation, which is actually stimulated by the Ile to Asp modification. In contrast the replacement of Glu at n + 3 with alanine almost suppresses serine phosphorylation but not tyrosine phosphorylation. It can be concluded that CK2 is capable to phosphorylate, under special circumstances, tyrosyl residues, which are specified by structural features partially different from those that optimize Ser/Thr phosphorylation.

Amino Acid Sequence↗
PubMed2005

Cooperative phosphorylation of the tumor suppressor phosphatase and tensin homologue (PTEN) by casein kinases and glycogen synthase kinase 3beta.

The phosphatase and tensin homologue (PTEN) tumor suppressor is a phosphatidylinositol D3-phosphatase that counteracts the effects of phosphatidylinositol 3-kinase and negatively regulates cell growth and survival. PTEN is itself regulated by phosphorylation on multiple serine and threonine residues in its C terminus. Previous work has implicated casein kinase 2 (CK2) as the kinase responsible for this phosphorylation. Here we showed that CK2 does not phosphorylate all sites in PTEN and that glycogen synthase kinase 3beta (GSK3beta) also participates in PTEN phosphorylation. Although CK2 mainly phosphorylated PTEN at Ser-370 and Ser-385, GSK3beta phosphorylated Ser-362 and Thr-366. More importantly, prior phosphorylation of PTEN at Ser-370 by CK2 strongly increased its phosphorylation at Thr-366 by GSK3beta, suggesting that the two may synergize. Using RNA interference, we showed that GSK3 phosphorylates PTEN in intact cells. Finally, PTEN phosphorylation was affected by insulin-like growth factor in intact cells. We concluded that multiple kinases, including CK2 and GSK3beta, participate in PTEN phosphorylation and that GSK3beta may provide feedback regulation of PTEN.

Amino Acid Sequence↗
PubMed2006

v-Src-dependent down-regulation of the Ste20-like kinase SLK by casein kinase II.

We have previously shown that the Ste20-like kinase SLK is a microtubule-associated protein inducing actin stress fiber disassembly. Here, we show that v-Src expression can down-regulate SLK activity. This down-regulation is independent of focal adhesion kinase but requires v-Src kinase activity and membrane translocation. SLK down-regulation by v-Src is indirect and is accompanied by SLK hyperphosphorylation on serine residues. Deletion analysis revealed that casein kinase II (CK2) sites at position 347/348 are critical for v-Src-dependent modulation of SLK activity. Further studies show that CK2 can directly phosphorylate SLK at these positions and that inhibition of CK2 in v-Src-transformed cells results in normal kinase activity. Finally, CK2 and SLK can be co-localized in fibroblasts spreading on fibronectin-coated substrates, suggesting a mechanism whereby SLK may be regulated at sites of actin remodeling, such as membrane lamellipodia and ruffles, through CK2.

Animals↗
PubMed1997

Casein kinase 2 associates with and phosphorylates dishevelled.

The dishevelled (dsh) gene of Drosophila melanogaster encodes a phosphoprotein whose phosphorylation state is elevated by Wingless stimulation, suggesting that the phosphorylation of Dsh and the kinase(s) responsible for this phosphorylation are integral parts of the Wg signaling pathway. We found that immunoprecipitated Dsh protein from embryos and from cells in tissue culture is associated with a kinase activity that phosphorylates Dsh in vitro. Purification and peptide sequencing of a 38 kDa protein co-purifying with this kinase activity showed it to be identical to Drosophila Casein Kinase 2 (CK2). Tryptic phosphopeptide mapping indicates that identical peptides are phosphorylated by CK2 in vitro and in vivo, suggesting that CK2 is at least one of the kinases that phosphorylates Dsh. Overexpression of Dfz2, a Wingless receptor, also stimulated phosphorylation of Dsh, Dsh-associated kinase activity, and association of CK2 with Dsh, thus suggesting a role for CK2 in the transduction of the Wg signal.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Role of G protein and protein kinase signalling in influenza virus budding in MDCK cells.

Recently, we have shown that influenza virus budding in MDCK cells is regulated by metabolic inhibitors of ATP and ATP analogues (Hui & Nayak, Virology 290, 329-341, 2001 ). In this report, we demonstrate that G protein signalling stimulators such as sodium fluoride, aluminium fluoride, compound 48/80 and mastoparan stimulated the budding and release of influenza virus. In contrast, G protein signalling blockers such as suramin and NF023 inhibited virus budding. Furthermore, in filter-grown lysophosphatidylcholine-permeabilized virus-infected MDCK cells, membrane-impermeable GTP analogues, such as guanosine 5'-O-(3-thiotriphosphate) or 5'-guanylylimidodiphosphate caused an increase in virus budding, which could be competitively inhibited by adding an excess of GTP. These results suggest that the G protein is involved in the regulation of influenza virus budding. We also determined the role of different protein kinases in influenza virus budding. We observed that specific inhibitors or activators of protein kinase A (H-89 and 8-bromoadenosine 3',5'-cyclic monophosphate) or of protein kinase C (bisindolylmaleimide I and Ro-32-0432) or of phosphatidylinositol 3-kinase (LY294002 and wortmannin) did not affect influenza virus budding. However, the casein kinase 2 (CK2) inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole decreased virus budding. We further observed an increase in the CK2 activity during the replication cycle of influenza virus, although Western blot analysis did not reveal any increase in the amount of CK2 protein in virus-infected cells. Also, in digitonin-permeabilized MDCK cells, the introduction of CK2 substrate peptides caused a down-regulation of virus budding. These results suggest that CK2 activity also regulates influenza virus budding.

Animals↗
PubMed2001

A nuclear casein kinase 2 activity is involved in early events of transcriptional activation induced by salicylic acid in tobacco.

Salicylic acid (SA) activates immediate early transcription of genes controlled by a family of DNA promoter elements named as-1-like elements. These elements are functional in the promoter of glutathione S-transferase genes. We have previously shown that SA increases the binding of tobacco (Nicotiana tabacum cv Xanthi nc) nuclear factors to the as-1 sequence in a process mediated by protein phosphorylation. In this study we give evidence for the participation of a nuclear protein kinase CK2 (casein kinase 2) in the pathway activated by SA in tobacco. The first line of evidence comes from the evaluation of the CK2 activity in nuclear extracts prepared from tobacco plants treated with SA or water as a control. Results from these experiments indicate that SA increases the nuclear CK2 activity. The second line of evidence derives from the evaluation of the in vivo effect of 5,6-dichloro-1-(beta-D-ribofuranosyl) benzimidazole (DRB), a cell-permeable CK2 inhibitor, on the responsiveness of the as-1 sequence to SA. Results from these experiments indicate that DRB impairs the activating effect of SA on the transcription of both, the GUS reporter gene controlled by a tetramer of the as-1 element, and the endogenous gnt35 gene encoding a glutathione S-transferase, in transgenic tobacco plants. DRB also impaired the increasing effect of SA on the binding of nuclear factors to the as-1 element. Furthermore, transcription of the as-1/GUS reporter gene activated by the synthetic auxin 2,4-dichlorophenoxyacetic acid and by methyl jasmonate was also inhibited by DRB. To our knowledge, this is the first report in which activation of a CK2 enzyme by a plant hormone is reported.

Amino Acid Sequence↗
PubMed1995

A casein-kinase-2-related protein kinase is tightly associated with the large T antigen of simian virus 40.

The simian virus 40 (SV40) large T antigen is a multifunctional protein involved in SV40 cell transformation and lytic virus infection. Some of its activities are regulated by interaction with cellular proteins and/or by phosphorylation of T antigen by various protein kinases. In this study, we show that immuno-purified T antigen from SV40-transformed cells and from baculovirus-infected insect cells is tightly associated with a protein kinase that phosphorylates T antigen in vitro. In the presence of heparin or a peptide resembling a protein kinase CK2 recognition site, the phosphorylation of T antigen by the associated kinase is reduced whereas a p34cdc2-kinase-specific peptide has no influence. In addition, the T-antigen-associated protein kinase can use GTP and ATP as phosphate donors. These properties together with the observation that immunopurified T antigen can be phosphorylated by the addition of protein kinase CK2 suggest that at least one of the T-antigen-associated protein kinases is CK2 or a protein-kinase-CK2-related enzyme. The association of recombinant CK2 with T antigen was strongly confirmed by in vitro binding studies. Experiments with temperature-sensitive SV40-transformed cells provide evidence for a close correlation between cell transformation and phosphorylation of T antigen by the associated protein kinase.

Amino Acid Sequence↗
PubMed1996

Cloning, expression and properties of the alpha' subunit of casein kinase 2 from zebrafish (Danio rerio).

The protein kinase casein kinase 2 (CK2) is ubiquitous in eukaryotic cells and is apparently involved in the control of cell division. The holoenzyme is a tetramer composed of two catalytic subunits (alpha and/or alpha') and regulatory subunits (beta 2). The alpha and alpha' subunits are encoded by different genes but are very similar in amino acid sequence, except that alpha' is normally considerably shorter. There have been extensive biochemical studies with recombinant alpha and beta subunits of many species, but only one previous description of the activity of an isolated recombinant alpha' subunit from human CK2 (Bodenbach, L., Fauss, J., Robitzki, A., Krehan, A., Lorenz, P., Lozeman, F. J. & Pyerin, W. (1994) Recombinant human casein kinase II. A study with the complete set of subunits (alpha, alpha', and beta), site-directed autophosphorylation mutants and a bicistronically expressed holoenzyme, Eur. J. Biochem. 220, 263-273). In the present work, the isolation and bacterial expression of a cDNA coding for the alpha' subunit of zebrafish (Danio rerio) is reported. The clone covers the complete coding region that generates a protein of 348 amino acids that is 86% identical to the alpha' subunits of human and chicken, and 82% identical to the sequenced portion of the CK2 alpha subunit of zebrafish. The recombinant alpha' subunit has apparent K(m) values for ATP (6 microM), GTP (20 microM), casein (2.0 mg/ml) and the model peptide RRRDDDSEDD (0.3 mM) which are very similar to those of the recombinant alpha subunit of Xenopus laevis. The alpha' subunit kcat was 7.2 min-1 which is again similar to that of Xenopus laevis alpha subunit (7.5 min-1). The alpha' subunit also behaved similarly to CK2 alpha with regard to optimal concentrations for Mg+2 or Mn+2 and to the inhibition by heparin and the poly(Glu80Tyr20) peptide. However alpha' kinase activity was less sensitive to poly(U) inhibition than alpha, it was more heat stable than alpha, and alpha' was slightly more sensitive to KCl inhibition than alpha. The difference in salt sensitivity, however, was enhanced by the presence of the regulatory beta subunit which shifted the optimal salt concentration of the phosphorylating activity. The alpha' 2 beta 2 holoenzyme was inhibited by KCl concentrations above 100 mM, while the alpha 2 beta 2 enzyme was stimulated by KCl concentrations up to 150 mM and required 180 mM for inhibition. Another important difference between alpha and alpha' is seen in the degree of the stimulation of casein phosphorylation activity in the presence of the regulatory beta subunit. When assayed at 100 mM KCl stoichiometric amounts of CK2 beta produced maximal stimulation of both alpha' (D. rerio) and alpha (X. laevis), however the activity levels with alpha' were stimulated 20-fold by beta while the addition of beta stimulated alpha (X. laevis) only 7-8-fold.

Amino Acid Sequence↗
PubMed1997

Rat liver Golgi apparatus contains a protein kinase similar to the casein kinase of lactating mammary gland.

By using a beta-casein-derived specific peptide substrate for mammary gland Golgi-enriched-fraction casein kinase, phosphorylating activity has been detected in the Golgi apparatus of rat liver, spleen and to a lesser extent, kidney and brain, while the other post-nuclear cytoplasmic fractions are totally devoid of such a casein kinase activity. In contrast ubiquitous protein kinases CK1 and CK2 (casein kinases 1 and 2), tested with their specific peptide substrates, display different subcellular distribution and are almost undetectable in the Golgi fraction. The absence of CK2 in the Golgi fraction has been also confirmed using specific antibodies. The relatedness between the liver Golgi apparatus casein kinase (G-CK) and the bona fide mammary gland Golgi-enriched-fraction casein kinase (GEF-CK) is supported by a variety of observations, notably: (a) identical peptide substrate specificity, consistent with an S-X-E-X consensus sequence; (b) preference for Mn2+, and, to a lesser extent, Co2+, over Mg2+, as activating cation; (c) superimposable elution profiles from DEAE-Sepharose, heparin-Sepharose, and Superdex 200, this latter consistent with a molecular mass around 500 kDa; (d) insensitivity to staurosporine and heparin (a potent inhibitor of CK2) and inability to use GTP as phosphate donor (by contrast to CK2). These data provide the evidence for the existence of a third class of ubiquitous casein kinases here termed G-CK, distinct from CK1 and CK2, specifically located to the Golgi apparatus and related to the bona fide casein kinase(s) responsible for the phosphorylation of casein secreted from lactating mammary gland. The possible involvement of G-CK in the phosphorylation of secretory pathways proteins at S-X-E motifs is discussed.

Amino Acid Sequence↗
PubMed1999

Casein kinase 2-mediated phosphorylation of respiratory syncytial virus phosphoprotein P is essential for the transcription elongation activity of the viral polymerase; phosphorylation by casein kinase 1 occurs mainly at Ser(215) and is without effect.

The major site of in vitro phosphorylation by casein kinase 2 (CK2) was the conserved Ser(232) in the P proteins of human, bovine, and ovine strains of respiratory syncytial virus (RSV). Enzymatic removal of this phosphate group from the P protein instantly halted transcription elongation in vitro. Transcription reconstituted in the absence of P protein or in the presence of phosphate-free P protein produced abortive initiation products but no full-length transcripts. A recombinant P protein in which Ser(232) was mutated to Asp exhibited about half of the transcriptional activity of the wild-type phosphorylated protein, suggesting that the negative charge of the phosphate groups is an important contributor to P protein function. Use of a temperature-sensitive CK2 mutant yeast revealed that in yeast, phosphorylation of recombinant P by non-CK2 kinase(s) occurs mainly at Ser(215). In vitro, P protein could be phosphorylated by purified CK1 at Ser(215) but this phosphorylation did not result in transcriptionally active P protein. A triple mutant P protein in which Ser(215), Ser(232), and Ser(237) were all mutated to Ala was completely defective in phosphorylation in vitro as well as ex vivo. The xanthate compound D609 inhibited CK2 but not CK1 in vitro and had a very modest effect on P protein phosphorylation and RSV yield ex vivo. Together, these results suggest a role for CK2-mediated phosphorylation of the P protein in the promoter clearance and elongation properties of the viral RNA-dependent RNA polymerase.

Animals↗
PubMed2006

siRNA knock down of casein kinase 2 increases force and cross-bridge cycling rates in vascular smooth muscle.

Contraction of smooth muscle involves myosin light chain (MLC) kinase catalyzed phosphorylation of the regulatory MLC, activation of myosin, and the development of force. However, this cannot account for all aspects of a smooth muscle contraction, suggesting that other regulatory mechanisms exist. One potentially important technique to study alternative sites of contractile regulation is the use of small interfering RNA (siRNA). The goal of this study was to determine whether siRNA technology can decrease the levels of a specific protein and allow for the determination of how that protein affects contractile regulation. To achieve this goal, we tested the hypothesis that casein kinase 2 (CK2) is part of the complex regulatory scheme present in vascular smooth muscle. Using intact strips of swine carotid artery, we determined that siRNA against CK2 produced a tissue that resulted in a approximately 60% knockdown after 4 days in organ culture. Intact strips of vascular tissue depleted of CK2 produced greater levels of force and exhibited an increased sensitivity to all stimuli tested. This was accompanied by an increase in cross-bridge cycling rates but not by a change in MLC phosphorylation levels. alpha-Toxin-permeabilized vascular tissue depleted of CK2 also showed an increased sensitivity to calcium compared with control tissues. Our results demonstrate that siRNA is a viable technique with which to study regulatory pathways in intact smooth muscle tissue. Our results also demonstrate that CK2 plays an important role in the mechanism(s) responsible for the development of force and cross-bridge cycling by a MLC phosphorylation-independent pathway.

Actinin↗
PubMed2006

Protein kinase casein kinase 2 mediates inhibitor-kappaB kinase and aberrant nuclear factor-kappaB activation by serum factor(s) in head and neck squamous carcinoma cells.

We showed previously that the signal transcription factor nuclear factor-kappaB (NF-kappaB) is aberrantly activated and that inhibition of NF-kappaB induces cell death and inhibits tumorigenesis in head and neck squamous cell carcinomas (HNSCC). Thus, identification of specific kinases underlying the activation of NF-kappaB could provide targets for selective therapy. Inhibitor-kappaB (IkappaB) kinase (IKK) is known to activate NF-kappaB by inducing NH(2)-terminal phosphorylation and degradation of its endogenous inhibitor, IkappaB. Casein kinase 2 (CK2) was previously reported to be overexpressed in HNSCC cells and to be a COOH-terminal IKK, but its relationship to NF-kappaB activation in HNSCC cells is unknown. In this study, we examined the contribution of IKK and CK2 in the regulation of NF-kappaB in HNSCC in vitro. NF-kappaB activation was specifically inhibited by kinase-dead mutants of the IKK1 and IKK2 subunits or small interfering RNA targeting the beta subunit of CK2. CK2 and IKK kinase activity, as well as NF-kappaB transcriptional activity, was shown to be serum responsive, indicating that these kinases mediate aberrant activation of NF-kappaB in response to serum factor(s) in vitro. Recombinant CK2alpha was shown to phosphorylate recombinant IKK2 as well as to promote immunoprecipitated IKK complex from HNSCC to phosphorylate the NH(2)-terminal S32/S36 of IkappaBalpha. We conclude that the aberrant NF-kappaB activity in HNSCC cells in response to serum is partially through a novel mechanism involving CK2-mediated activation of IKK2, making these kinases candidates for selective therapy to target the NF-kappaB pathway in HNSCC.

Carcinoma, Squamous Cell↗
PubMed2004

Extracellular phosphorylation converts pigment epithelium-derived factor from a neurotrophic to an antiangiogenic factor.

The pigment epithelium-derived factor (PEDF) belongs to the superfamily of serine protease inhibitors (serpin). There have been 2 distinct functions attributed to this factor, which can act either as a neurotrophic or as an antiangiogenic factor. Besides its localization in the eye, PEDF was recently reported to be present also in human plasma. We found that PEDF purified from plasma is a phosphoprotein, which is extracellularly phosphorylated by protein kinase CK2 (CK2) and to a lesser degree, intracellularly, by protein kinase A (PKA). CK2 phosphorylates PEDF on 2 main residues, Ser24 and Ser114, and PKA phosphorylates PEDF on one residue only, Ser227. The physiologic relevance of these phosphorylations was determined using phosphorylation site mutants. We found that both CK2 and PKA phosphorylations of PEDF markedly affect its physiologic function. The fully CK2 phosphorylation site mutant S24, 114E abolished PEDF neurotrophic activity but enhanced its antiangiogenic activity, while the PKA phosphorylation site mutant S227E reduced PEDF antiangiogenic activity. This is a novel role of extracellular phosphorylation that is shown here to completely change the nature of PEDF from a neutrophic to an antiangiogenic factor.

Casein Kinase II↗
PubMed2005

Identification of casein kinase 1, casein kinase 2, and cAMP-dependent protein kinase-like activities in Trypanosoma evansi.

Trypanosoma evansi contains protein kinases capable of phosphorylating endogenous substrates with apparent molecular masses in the range between 20 and 205 kDa. The major phosphopolypeptide band, pp55, was predominantly localized in the particulate fraction. Anti-alpha and anti-beta tubulin monoclonal antibodies recognized pp55 by Western blot analyses, suggesting that this band corresponds to phosphorylated tubulin. Inhibition experiments in the presence of emodin, heparin, and 2,3-bisphosphoglycerate indicated that the parasite tubulin kinase was a casein kinase 2 (CK2)-like activity. GTP, which can be utilized instead of ATP by CK2, stimulated rather than inactivated the phosphorylation of tubulin in the parasite homogenate and particulate fraction. However, GTP inhibited the cytosolic CK2 responsible for phosphorylating soluble tubulin and other soluble substrates. Casein and two selective peptide substrates, P1 (RRKDLHDDEEDEAMSITA) for casein kinase (CK1) and P2 (RRRADDSDDDDD) for CK2, were recognized as substrates in T. evansi. While the enzymes present in the soluble fraction predominantly phosphorylated P1, P2 was preferentially labeled in the particulate fractions. These results demonstrated the existence of CK1-like and CK2-like activities primarily located in the parasite cytosolic and membranous fractions, respectively. Histone II-A and kemptide (LRRASVA) also behaved as suitable substrates, implying the existence of other Ser/Thr kinases in T. evansi. Cyclic AMP only increased the phosphorylation of histone II-A and kemptide in the cytosol, demonstrating the existence of soluble cAMP-dependent protein kinase-like activities in T. evansi. However, no endogenous substrates for this enzyme were identified in this fraction. Further evidences were obtained by using PKI (6-22), a reported inhibitor of the catalytic subunit of mammalian cAMP-dependent protein kinases, which specifically hindered the cAMP-dependent phosphorylation of histone II-A and kemptide in the parasite soluble fraction. Since the sum of the values obtained in the parasite cytosolic and particulate fractions were always higher than the values observed in the total T. evansi lysate, the kinase activities examined here appeared to be inhibited in the original extract.

Animals↗
PubMed2006

A European interlaboratory testing of three well-known procedures for immunocytochemical detection of epithelial cells in bone marrow. Results from analysis of normal bone marrow.

BACKGROUND: This investigation intended to study the unspecific background to be expected in normal bone marrow (BM), comparing three well recognized protocols for immunocytochemical detection of disseminated carcinoma cells. The interlaboratory variation in screening and evaluation of stained cells was analyzed and different screening methods were compared. METHODS: BM mononuclear cells (BM MNC) from 48 normal BMs were immunostained in parallel by three participating laboratories. The protocols, based on three different anti-cytokeratin antibodies, have all been in common use for detection of disseminated carcinoma cells: the A45-B/B3 protocol (Hamburg), the CK2 protocol (Augsburg) and the AE1AE3 protocol (Oslo). For all protocols, the immunostained cells were visualized by the same alkaline-phosphatase (AP) detection system (APAAP) followed by detection of the cells by manual screening and by two different automated screening systems (ACIS from Chromavision and MDS1 from Applied Imaging). Detected AP-visualized cells were morphologically classified into unambiguous hematopoietic (Uhc) and questionable cells (Qc, potentially interpreted as tumor cells). RESULTS: Seven of 48 BMs (15%) harbored > or = 1 AP-visualized cell(s) among 1 x 10(6) BM MNC, both for the A45-B/B3- and for the AE1AE3 protocol, while for CK2 a higher proportion of BMs (21 BMs; 44%) harbored AP-visualized cells (P < 0.01, McNemar's test). The number of Qc was, for all protocols, 1 log lower than the total number of AP-visualized cells. On average, the frequency of Qc was 0.04, 0.08, and 0.02 per 10(6) BM MNC with A45-B/B3, CK2 and AE1AE3, respectively, and the number of Qc-positive BMs 1, 4, and 1. The MDS1 screening sensitivity was similar to manual screening, while ACIS detected fewer cells (P < 0.001, McNemar's test). CONCLUSIONS: All protocols resulted in AP-visualization of occasional hematopoietic cells. However, morphological classification brings the specificity to a satisfactory high level. Approximately 10% of AP-visualized cells were categorized "questionable". The CK2 protocol turned out less specific than the A45-B/B3 and AE1AE3 protocols.

Adult↗