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PubMed1999

Phosphorylation of the complement component, C9, by an ecto-protein kinase of human leukemic cells.

Ecto-protein kinases (ecto-PK) are surface constituents of many, if not all, animal cell types; normal, transformed or malignant. The occurrence of ecto-PK on the surface of human leukemia cell lines was described [Paas, Y., Fishelson, Z., 1995. Shedding of tyrosine and serine/threonine ecto-PK from human leukemic cells. Arch. Biochem. Biophys. 316 780-788.]. These ecto-PKs have been shown to phosphorylate several exogenous substrates, including the complement C9 protein, an essential component of the terminal complement system. C9 is phosphorylated by ecto-PK of K562 cells on serine residue(s). Phosphorylation occurs in the N-terminal C9a portion produced by cleavage of phosphorylated C9 with human alpha-thrombin. C9 polymers generated upon incubation of C9 with ZnCl2 do not serve as substrates for the K562 ecto-PK. In contrast, unfolded C9, obtained by reduction and alkylation, serves as a superior substrate for the K562 ecto-PK. Native C9 phosphorylation produced a rather low stoichiometry of incorporated phosphate (around 3%) per C9. Despite that, the phosphorylated C9 expressed reduced hemolytic activity. The complement-sensitive variant of K562 (K562/S) did not express the C9 phosphorylating activity. Various PK inhibitors tested failed to block C9 phosphorylation. Only heparin and 2,3-diphosphoglycerate (dpGA) prevented C9 phosphorylation, indicating that the ecto-PK is related to the casein kinase CK2. C9 can be phosphorylated by ecto-PK from other tumor cells, including Jurkat, SK-OV-3 and BT-474. These results suggest that extracellular phosphorylation of C9 may serve as a protective mechanism against complement in tumor cells.

Animals↗
PubMed1999

The expression of casein kinase 2alpha' and phosphatase 2A activity.

Protein phosphatase 2A (PP2A) activity may be differentially regulated by the expression of proteins containing a related amino acid sequence motif such as the casein kinase 2alpha (CK2alpha) subunit or SV40 small t antigen (SVt). Expression of CK2alpha increases PP2A activity whereas SVt decreases its activity. In this work we have tested for the effect of the expression of a third protein containing a similar motif that could be involved in PP2A regulation, the catalytic casein kinase 2alpha' subunit. Our results show that despite the structural similarity of this protein with the other CK2 catalytic (alpha) subunit, the function of the two subunits with respect to the modulation of PP2A activity is quite different: CK2alpha increases whereas CK2alpha' slightly decreases PP2A activity.

Amino Acid Sequence↗
PubMed2003

Cloning and functional characterization of squid voltage-dependent Ca2+ channel beta subunits: involvement of N-terminal sequences in differential modulation of the current.

cDNAs that encode beta subunits of voltage-dependent Ca(2+) channel were cloned from the optic lobe of the squid Loligo bleekeri. The subunits, LoCa(v)beta(1a) and LoCa(v)beta(1b) are 96% identical in amino acid sequence. The sole sequence differences are in the N-terminal region and in a five amino acid insertion in the central region of LoCa(v)beta(1b). RT-PCR revealed that LoCa(v)beta(1a) and LoCa(v)beta(1b) transcripts were expressed mainly in the optic lobe and stellate ganglion, and more weakly in mantle muscle, systemic heart, gill, branchial heart, stomach and liver. Coexpression of LoCa(v)beta(1a) or LoCa(v)beta(1b) with mammalian Ca(v)2.3 and alpha(2)/delta subunits in the Xenopus oocyte resulted in high-voltage-activated currents, and showed slow current inactivation and moderate steady-state inactivation. Comparison of the squid subunits with four mammalian beta subunits, beta(1b), beta(2a), beta(3) and beta(4), demonstrated that the modulatory effects of the beta subunits on steady-state inactivation kinetics were beta(3)<beta(4) approximately beta(1b)<LoCa(v)beta(1a) approximately LoCa(v)beta(1b)<beta(2a). LoCa(v)beta(1a)-induced current amplitude was about two to four times higher than that of LoCa(v)beta(1b). Experiments with point mutants and chimeras suggest that potential PKC and CK2 phosphorylation sites in the N-terminal region of LoCa(v)beta(1b) affect the current amplitude reciprocally, and may be responsible for regulating current amplitude.

Amino Acid Sequence↗
PubMed1999

Evidence for alternative splicing of ecto-ATPase associated with termination of purinergic transmission.

Ectonucleotidases provide the signal termination mechanism for purinergic transmission, including fast excitatory neurotransmission by ATP in the CNS. This study provides evidence for ectonucleotidase expression in the rat cochlea, brain and other tissues. In addition to detection of rat ecto-ATPase and ecto-ATPDase in these tissues, we identify a novel ecto-ATPase splice variant arising from the loss of a putative exon (193 bp) in the C-terminal coding region. This is the first evidence of alternative splicing in the ecto-ATPase gene family. Splicing of the 193-bp putative exon containing a stop codon extends the open reading frame and provides translation of an additional 50 amino acids compared with the isoform isolated earlier from the rat brain (rEATPase(A); GenBank accession #Y11835). The splice variant (rEATPase(B); GenBank accession #AF129103) encodes 545 amino acids with a predicted protein molecular mass of 60 kDa. rEATPase(B) contains a long cytoplasmic tail (62 amino acids) with three potential protein kinase CK2 phosphorylation sites not present in rEATPase(A). Co-expression of two ecto-ATPase isoforms with different regulatory sites suggests that the extracellular ATP signal levels may be differently influenced by intracellular feedback pathways.

Adenosine Triphosphatases↗
PubMed2000

A new family of genes and pseudogenes potentially expressing testis- and brain-specific leucine zipper proteins in man and mouse.

We have characterized a new mouse gene highly transcribed in the testis, and a derived intronless gene expressed in the embryo. The latter gene is present in Mus musculus domesticus and in Mus musculus castaneus but is absent in Mus spretus. The sequencing of different clones from a testis cDNA library reveals a complex transcriptional regulation for the intron-containing gene. The use of several promoters, alternative splicing and trans-splicing, and of two different polyadenylation sites account for the diversity. The different cDNAs encode proteins with features of basic helix-loop-helix leucine zipper (bHLH-ZIP) DNA-binding factors with homology to a new brain-specific factor. The presence of multiple CK2 and PKC phosphorylation sites suggests that their activity may be regulated by phosphorylation. In man, a pseudogene, apparently derived from the same transcript as in mouse and showing 90% homology in the coding region, is present within an intron of another gene. Interestingly, although the human pseudogene is highly mutated in human, in the mouse it has only four nucleotide changes compared with the cDNA of origin, and is still capable of encoding a protein.

Alternative Splicing↗
PubMed2002

Biochemical and enzymatic characterization of a partially purified casein kinase-1 like activity from Trypanosoma cruzi.

Two protein kinase activities that use casein as a substrate, Q-I and Q-II, were identified in the epimastigote stage of Trypanosoma cruzi upon chromatography on Q-Sepharose. Q-I was purified further through concanavalin A-sepharose (Q-I*) to remove any trace of the contaminating protease cruzipain. The optimal activity for Q-I* was obtained at pH 8.0, 25 degreesC, 5 mM MgCl(2) and 75 mM NaCl. The size and pI of Q-I* were determined to be 33-36 kDa and 9.6, respectively. When two selective peptide substrates for casein kinases (CKs) (P1: RRKDLHDDEEDEAMSITA for CK1 and P2: RRRADDSDDDDD for CK2) were used, Q-I* was shown to specifically phosphorylate P1. Kinetic studies showed that Q-I* has a K(m) of 5.3 +/- 0.34 mg/ml for casein, 157.6 +/- 5.3 microM for P1 and 35.9 +/- 3.9 microM for ATP. The enzyme was inhibited by N-(2-amino-ethyl)-5-chloroisoquinoline-8-sulfonamide (CKI-7) or 1-(5-chloroisoquinoline-8-sulfonyl) (CKI-8), two inactivators of mammalian CKs. CKI-7 behaved as a competitive inhibitor with respect to ATP, with a K(I) of 75-100 microM. Treatment with high concentrations of polylysine or heparin also resulted in a significant inhibition of Q-I*. Two well-known activators of mammalian CKs, spermine and spermidine, were also tested. Spermine and spermidine activated Q-I* in a dose-dependent manner. Based on the following characteristics: (1) the ionic strength required for elution from anion-exchange resins; (2) its molecular size and monomeric structure; (3) pI; (4) high level of specificity for P1; (5) inactivation by CKI-7 and CKI-8; and (6) insensitivity to GTP and low concentrations of heparin, we conclude that Q-I* belongs to the CK1 family of protein kinases.

Amino Acid Sequence↗
PubMed2002

Prointerleukin-16 contains a functional CcN motif that regulates nuclear localization.

The immunomodulatory cytokine interleukin-16 (IL-16) represents the secreted C-terminus of a larger precursor, pro-IL-16. Following cleavage by caspase 3, the residual N-terminal domain translocates into the nucleus, inducing G(0)/G(1) cell cycle arrest. We have previously identified a classical bipartite nuclear localization sequence (NLS) in the N-terminal domain of pro-IL-16. We now show that N-terminal to the NLS domain of pro-IL-16 are protein kinase CK2 substrate and cdc2 kinase substrate sites which, along with the NLS, constitute a dual phosphorylation-regulated CcN motif which regulates nuclear localization of pro-IL-16. In addition, we demonstrate that mutation of either site is associated with impairment of the N-terminal domain's ability to induce G(0)/G(1) cell cycle arrest. This is the first description of a functional CcN motif in a cytokine precursor.

Active Transport, Cell Nucleus↗
PubMed2003

Basal and hydrogen peroxide stimulated sites of phosphorylation in heterogeneous nuclear ribonucleoprotein C1/C2.

Hydrogen peroxide (H2O2) is a recently recognized second messenger, which regulates mammalian cell proliferation and migration. The biochemical mechanisms by which mammalian cells sense and respond to low concentrations of H2O2 are poorly understood. Recently, heterogeneous nuclear ribonucleoprotein C1/C2 (hnRNP-C1/C2) was found to be rapidly phosphorylated in response to the application of low concentrations of H2O2 to human endothelial cells. Here, using tandem mass spectrometry, four sites of phosphorylation are identified in hnRNP-C1/C2, all of which are in the acidic C-terminal domain of the protein. Under resting conditions, the protein is phosphorylated at S247 and S286. In response to low concentrations of H2O2, there is increased phosphorylation at S240 and at one of the four contiguous serine residues from S225-S228. Studies using a recombinant acidic C-terminal domain of hnRNP-C overexpressed in Escherichia coli demonstrate that protein kinase CK2 phosphorylates hnRNP-C1/C2 at S247, while protein kinase A and several protein kinase C isoforms fail to phosphorylate the isolated domain. These findings demonstrate that the acidic C-terminal domain of hnRNP-C1/C2 serves as the site for both basal and stimulated phosphorylation, indicating that this domain may play an important role in the regulation of mRNA binding by hnRNP-C1/C2.

Amino Acid Sequence↗
PubMed2004

Interactions of the basic N-terminal and the acidic C-terminal domains of the maize chromosomal HMGB1 protein.

Maize HMGB1 is a typical member of the family of plant chromosomal HMGB proteins, which have a central high-mobility group (HMG)-box DNA-binding domain that is flanked by a basic N-terminal region and a highly acidic C-terminal domain. The basic N-terminal domain positively influences various DNA interactions of the protein, while the acidic C-terminal domain has the opposite effect. Using DNA-cellulose binding and electrophoretic mobility shift assays, we demonstrate that the N-terminal basic domain binds DNA by itself, consistent with its positive effects on the DNA interactions of HMGB1. To examine whether the negative effect of the acidic C-terminal domain is brought about by interactions with the basic part of HMGB1 (N-terminal region, HMG-box domain), intramolecular cross-linking in combination with formic acid cleavage of the protein was used. These experiments revealed that the acidic C-terminal domain interacts with the basic N-terminal domain. The intramolecular interaction between the two oppositely charged termini of the protein is enhanced when serine residues in the acidic tail of HMGB1 are phosphorylated by protein kinase CK2, which can explain the negative effect of the phosphorylation on certain DNA interactions. In line with that, covalent cross-linking of the two terminal domains resulted in a reduced affinity of HMGB1 for linear DNA. Comparable to the finding with maize HMGB1, the basic N-terminal and the acidic C-terminal domains of the Arabidopsis HMGB1 and HMGB4 proteins interact, indicating that these intramolecular interactions, which can modulate HMGB protein function, generally occur in plant HMGB proteins.

Amino Acid Sequence↗
PubMed2000

Paralogous stellate and Su(Ste) repeats: evolution and ability to silence a reporter gene.

The X-linked Stellate repeats, encoding a putative regulatory subunit of protein kinase CK2, are expressed in XO male testes. The Y-linked, testes-expressed paralogous Su(Ste) repeats are thought to be suppressors of Stellate transcription. The unique, testis-expressed euchromatic gene was suggested to be an ancestor of the both types of amplified paralogous repeats. A Su(Ste)-like orphon was localized on a Y chromosome, outside of the Su(Ste) cluster. Several diagnostic molecular markers peculiar for the both types of diverged Stellate and Su(Ste) units were detected in the orphon sequence. The orphon was suggested to be a close relative of the immediate ancestor of both types of paralogous repeats which initiated evolution on the Y chromosome. Selection pressure on the level of translation was shown as a driving force in the evolution of Su(Ste) repeats, which are considered as more ancient derivatives of the ancestor euchromatic gene than Stellate repeats. In a vicinity of 12E Stellate cluster the undamaged, recently originated euchromatic Stellate orphon was found at 12D, providing the poly(A) signal for the bendless gene. P-element mediated transformations reveal that the fragments of cloned Stellate and Su(Ste) clusters are able to induce variegation of a reporter mini-white gene. The observed variegation phenomenon has peculiar features: a significant increase of trans-activation of a reporter mini-white gene in homozygous state; absence of effects of several conventional modifiers of position effect variegation (PEV) and independence of a severity of variegation on a distance between insertion and centromere region.

Amino Acid Sequence↗
PubMed2003

Protein kinase CK1 from Trypanosoma cruzi.

A protein kinase activity, which uses casein as a substrate, has been purified to homogeneity from the epimastigote stage of Trypanosoma cruzi, by sequential chromatography on Q sepharose, heparin sepharose, phenyl sepharose, and alpha-casein agarose. An apparent molecular weight of 36,000 was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel filtration chromatography and sedimentation analyses demonstrated that the purified native enzyme is a monomer with a sedimentation coefficient of 2.9 S. The hydrodynamic parameters indicated that the shape of the protein is globular with a frictional ratio f/f(o) = 1.36 and a Stokes radius of 27.7 A. When two selective peptide substrates for protein kinases CK1 and CK2 were used (RRKDLHDDEEDEAM. SITA and RRRADDSDDDDD, respectively), the purified kinase was shown to predominantly phosphorylate the CK1-specific peptide. Additionally, the enzyme was inhibited by N-(2-amino-ethyl)-5-chloroisoquinoline-8-sulfonamide, a specific inactivator of CK1s from mammals. Based on these results, we concluded that the purified kinase corresponds to a parasite CK1.

Amino Acid Sequence↗
PubMed1996

Cytokeratin-positive cells in bone marrow for identifying distant micrometastasis of gastric cancer.

Direct evidence of tumour seeding in distant organs at the time of surgery for gastric cancer is not available. An immunocytochemical assay for epithelial cytokeratin protein may fill this gap since it is a feature of epithelial cells that would not normally be present in bone marrow. The bone marrow of 46 patients with primary gastric cancer was examined for tumour cells, using immunocytochemical techniques and antibody reacting with cytokeratin, a component of the intracytoplasmic network of intermediate filaments. The monoclonal antibody CK2 recognises a single cytokeratin polypeptide (human cytokeratin no. 18) commonly present in epithelial cells. The expression of tumour-suppressor genes p53 and RB for the primary lesion was also determined using the monoclonal antibodies PAb 1801 and 3H9 respectively, and the proliferating activity was determined by the Ki-67 antigen labelling index for MIB-1 antibody staining. Of these 46 patients, 15 (32.6%) presented with cytokeratin-positive cells at the time of primary surgery. The positive findings were related to the undifferentiated tissue type and to the prominent depth of invasion, but not to other clinicopathological factors. In 2 of 15 (13.3%) patients, the depth of invasion was limited to the mucosa. The metastatic potential to bone marrow did not relate to expressions of p53 and RB genes, or to the proliferating activity of MIB-1 staining for the primary lesion of gastric cancer. As tumour cells in bone marrow are indicative of the general disseminative capability of an individual tumour, this technique may be useful for identifying patients at high risk of metastasis from a gastric tumour.

Antibodies, Monoclonal↗
PubMed1997

Tumor cell contamination of peripheral blood stem cell transplants and bone marrow in high-risk breast cancer patients.

Twenty-one high-risk patients with primary stage II/III breast cancer were treated with high-dose chemotherapy comprising etoposide, ifosfamide, carboplatin and epirubicin (VIC-E). Tumor cells of epithelial origin were analyzed using the monoclonal antibodies CK2 (IgG1) and A45-B/B3 (IgG1) against cytokeratin (CK) components in bone marrow (BM) aspirates prior to chemotherapy, and in peripheral blood stem cell transplants (PBSCT). They were separated after the first (21/21 patients) and the second cycle (16/21 patients) of induction chemotherapy with VIP-E (etoposide, ifosfamide, cisplatin, epirubicin). Preliminary results showed CK positive tumor cells in 40% (14/35) of the analyzed transplants. In 7/12 (58.3%) patients, CK positive tumor cells were detectable in BM prior to treatment. Sixteen patients were separated after the 1st and 2nd cycle of VIP-E. PBSCT of 14/16 patients were assessable for presence of CK positive tumor cells. Our preliminary results demonstrate a lower tumor cell contamination of PBSCT separated after the 2nd cycle of induction therapy (14.3%) compared to contamination after the first induction therapy (64.3%). To date, 4/21 patients have experienced a relapse, and three of these patients had tumor cell positive transplants. Due to the small patient number only a trend towards a superior relapse-free survival in the patient group with CK negative transplants can be shown by Kaplan-Meier analysis.

Adult↗
PubMed2004

BMP-2 decreases Mash1 stability by increasing Id1 expression.

In neural development, bone morphogenetic proteins (BMPs) restrict neuronal differentiation, thereby promoting the maintenance of progenitor cells or even inducing astrocytogenesis. We report that exposure of neuroendocrine lung carcinoma cells to BMP-2 leads to a rapid decline in steady-state levels of Mash1 protein and some neuron-specific markers. BMP-2 induces a post-transcriptional decrease in Mash1 levels through enhanced degradation. We demonstrate that Mash1 protein stability is tightly regulated by the E47/Id1 expression ratio. Transient induction of Id1 by BMP-2 negatively correlates with Mash1 levels. Furthermore, an ectopic increase in Id1 levels is sufficient to induce degradation of either ectopic or endogenous Mash1, whereas expression of Mash1 in Id1-deficient cells or overexpression of E47 makes Mash1 levels refractory to the addition of BMP-2. Furthermore, we show that the E47/Id1 expression ratio also regulates CK2-mediated phosphorylation of Mash1 on Ser152, which increases interaction of Mash1-E47 heterodimers. We propose a novel mechanism in which the balance between Id and E protein levels regulates not only the transcriptional function but also protein stability of the neurogenic bHLH transcription factor Mash1.

Animals↗
PubMed2004

Xrcc4 physically links DNA end processing by polynucleotide kinase to DNA ligation by DNA ligase IV.

Nonhomologous end joining (NHEJ) is the major DNA double-strand break (DSB) repair pathway in mammalian cells. A critical step in this process is DNA ligation, involving the Xrcc4-DNA ligase IV complex. DNA end processing is often a prerequisite for ligation, but the coordination of these events is poorly understood. We show that polynucleotide kinase (PNK), with its ability to process ionizing radiation-induced 5'-OH and 3'-phosphate DNA termini, functions in NHEJ via an FHA-dependent interaction with CK2-phosphorylated Xrcc4. Analysis of the PNK FHA-Xrcc4 interaction revealed that the PNK FHA domain binds phosphopeptides with a unique selectivity among FHA domains. Disruption of the Xrcc4-PNK interaction in vivo is associated with increased radiosensitivity and slower repair kinetics of DSBs, in conjunction with a diminished efficiency of DNA end joining in vitro. Therefore, these results suggest a new role for Xrcc4 in the coordination of DNA end processing with DNA ligation.

Animals↗
PubMed2005

Phosducin-like protein acts as a molecular chaperone for G protein betagamma dimer assembly.

Phosducin-like protein (PhLP) is a widely expressed binding partner of the G protein betagamma subunit dimer (Gbetagamma). However, its physiological role is poorly understood. To investigate PhLP function, its cellular expression was blocked using RNA interference, resulting in inhibition of Gbetagamma expression and G protein signaling. This inhibition was caused by an inability of nascent Gbetagamma to form dimers. Phosphorylation of PhLP at serines 18-20 by protein kinase CK2 was required for Gbetagamma formation, while a high-affinity interaction of PhLP with the cytosolic chaperonin complex appeared unnecessary. PhLP bound nascent Gbeta in the absence of Ggamma, and S18-20 phosphorylation was required for Ggamma to associate with the PhLP-Gbeta complex. Once Ggamma bound, PhLP was released. These results suggest a mechanism for Gbetagamma assembly in which PhLP stabilizes the nascent Gbeta polypeptide until Ggamma can associate, resulting in membrane binding of Gbetagamma and release of PhLP to catalyze another round of assembly.

Carrier Proteins↗
PubMed2005

Caspase-2 primes cancer cells for TRAIL-mediated apoptosis by processing procaspase-8.

Although caspase-2 is believed to be involved in death receptor-mediated apoptosis, the exact function, mode of activation, and regulation of caspase-2 remain unknown. Here we show that protein kinase (PK) CK2 phosphorylates procaspase-2 directly at serine-157. When intracellular PKCK2 activity is low or downregulated by specific inhibitors, procaspase-2 is dephosphorylated, dimerized, and activated in a PIDDosome-independent manner. The activated caspase-2 then processes procaspase-8 monomers between the large and small subunits, thereby priming cancer cells for TNF-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis. The processed procaspase-8 that is recruited to death-inducing signaling complex by TRAIL engagement becomes fully activated, and cancer cells undergo apoptosis. PKCK2 activity is low in TRAIL-sensitive cancer cell lines but high in TRAIL-resistant cancer cell lines. Thus, downregulating PKCK2 activity is required for TRAIL-mediated apoptosis to occur in TRAIL-resistant cancer cells. Our data provide novel insights into the regulation, mode of activation, and function of caspase-2 in TRAIL-mediated apoptosis.

Apoptosis↗
PubMed2006

A novel leptin signalling pathway via PTEN inhibition in hypothalamic cell lines and pancreatic beta-cells.

In obesity and diabetes, the ability of hypothalamic neurons to sense and transduce changes in leptin and insulin levels is compromised. The effects of both hormones require intracellular signalling via the PI3-kinase pathway, which is inhibited by the phosphatase PTEN. We show that leptin-stimulated F-actin depolymerization in mouse hypothalamic cells is inhibited by PTEN, a process involving independent effects of both its lipid and protein phosphatase activities. Potentially mediating this F-actin depolymerization, leptin, but not insulin, stimulated the phosphorylation of PTEN in a CK2 dependent manner, and inhibited its phosphatase activity. Similarly, hyperpolarization of mouse pancreatic beta-cells by leptin also requires coincident PtdIns(3,4,5)P3 generation and actin depolymerization, and could be inhibited by mechanisms requiring both the lipid and protein phosphatase activities of PTEN. These results demonstrate a critical role for PTEN in leptin signalling and indicate a mechanism by which leptin and insulin can produce PI3K dependent differential cellular outputs.

Actins↗