Biomedical subjects

O Filhol

Publications and source records attributed to O Filhol.

At least 19 recordsLinked to original sources

PubMed2006

p53-dependent inhibition of mammalian cell survival by a genetically selected peptide aptamer that targets the regulatory subunit of protein kinase CK2.

Based on the perturbation of its expression in human cancers and on its involvement in transformation and tumorigenesis, protein kinase CK2 has recently attracted attention as a potential therapeutic target. To assess the value of CK2 as a target for antiproliferative strategies, we have initiated a program aiming to develop inhibitors targeting specifically the regulatory CK2beta subunit. Here, we use a two-hybrid approach to isolate from combinatorial libraries, peptide aptamers that specifically interact with CK2beta. One of these (P1), which has significant sequence homology to the cytomegalovirus IE2 protein, binds with high affinity to the N-terminal domain of CK2beta without disrupting the formation of the CK2 holoenzyme. Expression of green fluorescent protein (GFP)-P1 in different mammalian cell lines activates p53 phosphorylation on serine 15, induces an upregulation of p21 and the release of the Cyt-C and apoptosis-inducing factor proapoptotic proteins triggering caspase-dependent and caspase-independent apoptosis. GFP-P1-induced apoptosis is associated with a p53-dependent pathway as cell death was abrogated in p53 knocked out cells. In summary, our data show that genetically selected peptide aptamers that specifically target CK2beta can induce apoptosis in mammalian cells through the recruitment of a p53-dependent apoptosis pathway. They also emphasize the critical role of CK2beta for cell survival and might allow the design of novel proapoptotic agents targeting this protein.

3T3 Cells↗

PubMed2001

Visualization and molecular analysis of nuclear import of protein kinase CK2 subunits in living cells.

We have generated fusion proteins between the subunits of CK2 and GFP and characterized their behaviour in living cells. The expressed fusion proteins were functional and interacted with endogenous CK2. Imaging of NIH3T3 cells expressing low level of GFP-CK2alpha or GFP-CK2beta showed that both proteins were mostly nuclear in interphase. Both CK2 subunits contain nuclear localization domains that target them independently to the nucleus. Once in the nucleus, both subunits diffused rapidly in the nucleoplasm. In mitotic cells, CK2 subunits were dispersed throughout the cytoplasm and were not associated to chromatin. Our data are compatible with the idea that each subunit can translocate individually to the nucleus to interact with each other or with important cellular partners. Understanding the molecular mechanisms which regulate the dynamic localization of CK2 subunits will be of central importance.

3T3 Cells↗

PubMed2000

Mitotic phosphorylation of DNA topoisomerase II alpha by protein kinase CK2 creates the MPM-2 phosphoepitope on Ser-1469.

DNA topoisomerase II alpha is required for chromatin condensation during prophase. This process is temporally linked with the appearance of mitosis-specific phosphorylation sites on topoisomerase IIalpha including one recognized by the MPM-2 monoclonal antibody. We now report that the ability of mitotic extracts to create the MPM-2 epitope on human topoisomerase II alpha is abolished by immunodepletion of protein kinase CK2. Furthermore, the MPM-2 phosphoepitope on topoisomerase II alpha can be generated by purified CK2. Phosphorylation of C-truncated topoisomerase II alpha mutant proteins conclusively shows, that the MPM-2 epitope is present in the last 163 amino acids. Use of peptides containing all conserved CK2 consensus sites in this region indicates that only the peptide containing Arg-1466 to Ala-1485 is able to compete with topoisomerase II alpha for binding of the MPM-2 antibody. Replacement of Ser-1469 with Ala abolishes the ability of the phosphorylated peptide to bind to the MPM-2 antibody while a peptide containing phosphorylated Ser-1469 binds tightly. Surprisingly, the MPM-2 phosphoepitope influences neither the catalytic activity of topoisomerase II alpha nor its ability to form molecular complexes with CK2 in vitro. In conclusion, we have identified protein kinase CK2 as a new MPM-2 kinase able to phosphorylate an important mitotic protein, topoisomerase II alpha, on Ser-1469.

Amino Acid Sequence↗

PubMed2000

The disruption of adherens junctions is associated with a decrease of E-cadherin phosphorylation by protein kinase CK2.

The down-regulation of E-cadherin is a common event in carcinogenesis. Phosphorylation/dephosphorylation is one posttranscriptional process which may regulate intercellular junctions. Here we show that in okadaic acid-treated keratinocytes, E-cadherin expression is shifted from the membrane to the cytoplasm, preventing cells from forming aggregates. These changes of E-cadherin localization and function are associated with a decrease in its phosphorylation state. The decrease in E-cadherin phosphorylation was essentially detected in okadaic acid-treated cell lysates isolated from 0.5% Triton-soluble fraction and not in the Triton-insoluble fraction linked to the cytoskeleton, suggesting a role of E-cadherin phosphorylation in cell-cell interactions. E-cadherin was markedly phosphorylated by CK2, either the purified recombinant enzyme or the endogenous enzyme. Using specific CK2 inhibitors such as heparin and 5, 6-dichloro-1-beta-d-ribofuranosylbenzimidazole, endogenous CK2 was confirmed as the main enzyme phosphorylating E-cadherin. The decrease in E-cadherin phosphorylation by endogenous CK2 was not restored by the addition of purified CK2, confirming that it is not due to a defect in CK2 expression or to its reduced activity, but rather to the incapacity of CK2 to phosphorylate E-cadherin. The co-immunoprecipitation and colocalization of E-cadherin and CK2 suggests that CK2 may play a critical role in the maintenance of epidermis cohesion.

Cadherins↗

PubMed1999

The multifunctional herpes simplex virus IE63 protein interacts with heterogeneous ribonucleoprotein K and with casein kinase 2.

Herpes simplex virus type 1 (HSV-1), the prototype alpha-herpesvirus, causes several prominent diseases. The HSV-1 immediate early (IE) protein IE63 (ICP27) is the only regulatory gene with a homologue in every mammalian and avian herpesvirus sequenced so far. IE63 is a multifunctional protein affecting transcriptional and post-transcriptional processes, and it can shuttle from the nucleus to the cytoplasm. To identify interacting cellular proteins, a HeLa cDNA library was screened in the yeast two-hybrid system using IE63 as bait. Several interacting proteins were identified including heterogeneous nuclear ribonucleoprotein K (hnRNP K), a multifunctional protein like IE63, and the beta subunit of casein kinase 2 (CK2), a protein kinase, and interacting regions were mapped. Confirmation of interactions was provided by fusion protein binding assays, co-immunoprecipitation from infected cells, and CK2 activity assays. hnRNP K co-immunoprecipitated from infected cells with anti-IE63 serum was a more rapidly migrating subfraction than hnRNP K immunoprecipitated by anti-hnRNP K serum. Using anti-IE63 serum, both IE63 and hnRNP K were phosphorylated in vitro by CK2, while in immunoprecipitates using anti-hnRNP K serum, IE63 but not hnRNP K was phosphorylated by CK2. These data provide important new insights into how this key viral regulatory protein exerts its functions.

Animals↗

PubMed1999

Crystal structure of the human protein kinase CK2 regulatory subunit reveals its zinc finger-mediated dimerization.

Protein kinase CK2 is a tetramer composed of two alpha catalytic subunits and two beta regulatory subunits. The structure of a C-terminal truncated form of the human beta subunit has been determined by X-ray crystallography to 1.7 A resolution. One dimer is observed in the asymmetric unit of the crystal. The most striking feature of the structure is the presence of a zinc finger mediating the dimerization. The monomer structure consists of two domains, one entirely alpha-helical and one including the zinc finger. The dimer has a crescent shape holding a highly acidic region at both ends. We propose that this acidic region is involved in the interactions with the polyamines and/or catalytic subunits. Interestingly, conserved amino acid residues among beta subunit sequences are clustered along one linear ridge that wraps around the entire dimer. This feature suggests that protein partners may interact with the dimer through a stretch of residues in an extended conformation.

Amino Acid Sequence↗

PubMed1999

Crystallization and preliminary x-ray diffraction analysis of the regulatory subunit of human protein kinase CK2.

Protein kinase CK2 is a tetramer composed of two alpha catalytic subunits and two beta regulatory subunits. A C-terminal truncated form of the beta subunit has been overproduced in Escherichia coli and purified to homogeneity. Two crystal forms of the truncated protein which diffract to at least 2 A resolution have been obtained. Form I belongs to the monoclinic space group P21, with unit-cell parameters a = 49.9, b = 92.9, c = 53.7 A, beta = 96.3 degrees, and yields plate-like crystals. Form II belongs to the tetragonal space group P42212, with unit-cell parameters a = 132.19, b = 132.19, c = 63.79 A, and produces rod-shaped crystals. Both crystal forms have a functional dimer in the crystal asymmetric unit.

Casein Kinase II↗

PubMed1999

Dissecting subdomains involved in multiple functions of the CK2beta subunit.

We have characterized several subdomains of the beta subunit of protein kinase CK2. The N-terminal half of the protein exhibits a pseudo-substrate segment in tandem with a polyamine binding domain responsible for the activation of the kinase by these polybasic compounds. Study of the chemical features of this polyamine binding site showed that polyamine analogs exhibiting the highest affinity for CK2 are the best CK2 activators. Mutational analysis disclosed that glutamic residues lying in the polyacidic region of the CK2beta subunit are involved in the interaction with polyamine molecules and allowed the delineation of an autonomous binding domain. Furthermore, this regulatory domain was shown to mediate the association of CK2 with plasma membrane. The C-terminal domain of the CK2beta subunit plays a role in the oligomerization of the kinase since it was observed that a truncated form of this subunit lacking its 33-last amino acids was incompetent for the assembly of polymeric forms of CK2. Altogether, our results support the notion that the beta subunit of CK2 is a modular protein made by the association of interdependent domains that are involved in its multiple functions.

Affinity Labels↗

PubMed1999

Searching interaction partners of protein kinase CK2beta subunit by two-hybrid screening.

To date, the intracellular regulation of protein kinase CK2 is unknown. However it was observed that the enzyme associates with several intracellular proteins and the formation of such molecular complexes may represent a mechanism for the control of CK2 activity. Using the Interaction Trap system in yeast, with the CK2beta as a bait, we looked for CK2 partners. We present the identification of new potential partners of CK2beta and it is hoped that their classification will help in understanding the physiological roles and the regulation of CK2 in the cell.

Base Sequence↗

PubMed1998

The tight association of protein kinase CK2 with plasma membranes is mediated by a specific domain of its regulatory beta-subunit.

Previous immunocytochemical studies have shown that protein kinase CK2 is mostly detected both in the cytoplasm and the nucleus of most cells. In the present study, CK2 was detected in highly purified plasma membrane preparations from rat liver. The protein kinase could be released from the membranes by high salt extraction (>1 M NaCl). Plasma membranes prepared from SF9 insect cells expressing the alpha- and beta-subunits of CK2 also contained a significant amount of oligomeric CK2. Furthermore, it was demonstrated in this cell system as well as in rat liver plasma membranes, that the beta-subunit of the kinase is the targeting subunit which mediates the tight association of the enzyme to plasma membrane components. Binding studies using membranes and recombinant proteins corresponding to different regions of the beta-subunit suggest that a functional domain previously shown to be involved in the binding of polyamines may also participate to the binding of CK2 to membranes. Modification of membranes by trypsin and phospholipases indicated that the binding process may require both membrane protein(s) and phospholipids. Interestingly, it was observed that the amount of membrane-bound CK2 in liver of embryos and new born rats increases dramatically after birth and persists during the postnatal stages of development.

Animals↗

PubMed1997

Binding of polyamines to an autonomous domain of the regulatory subunit of protein kinase CK2 induces a conformational change in the holoenzyme. A proposed role for the kinase stimulation.

The means by which the cell regulates protein kinase CK2 remain obscure. However, natural polyamines, cellular compounds required for cell proliferation, have been reported to strongly stimulate CK2-mediated phosphorylation of a number of substrates. Using spermine analogs, we have shown that polyamines directly interact with the CK2 beta subunit, and the chemical features of the highly acidic binding site (Asp51-Tyr80) have been determined. In the present study, we show that the isolated beta subunit region extending from residue Asp51 to Pro110 exhibits a specific and efficient polyamine binding activity similar to that of the entire beta subunit. Moreover, the replacement of Glu60, Glu61, and Glu63 of the beta subunit by 3 alanine residues leads to a loss of the spermine-induced stimulation of CK2 activity which correlates with a decrease in spermine binding affinity. Thermal stability studies indicate that the binding of spermine induces a 4 degrees C decrease of the Tm value for the holoenzyme. This was confirmed by circular dichroism analyses, which show that the 6 degrees C negative shift of the CK2 Tm value provoked by spermine binding, reflects a conformational change in the kinase. Together, these observations strongly suggest that this newly defined polyamine-binding domain is involved in the intrasteric regulation of CK2 activity.

Animals↗

PubMed1997

Chemical features of the protein kinase CK2 polyamine binding site.

Protein kinase CK2 is a ubiquitous eukaryotic Ser/Thr kinase whose catalytic activity is enhanced several times by polyamines. We have shown previously that the regulatory beta-subunit of CK2 bears a polyamine binding site located in the region Asp51-Tyr110. In the present study, we have used spermine analogs to investigate the structural requirements of the CK2 polyamine binding site. We have observed a strong correlation between the stimulations of CK2 activity by all tested polyamines and their binding efficiencies to the enzyme. As a result, spermine was found to be the most efficient stimulator of the kinase activity and the best CK2 ligand. The effect of the pH on the stimulation of CK2 activity by spermine strongly suggests the involvement of ionic interactions between the positive charges of spermine and the negative charges of acidic amino acids of the beta-subunit. Using a fusion protein made of MBP and the beta-subunit region encompassing amino acid residues Asp51-Pro110, we have studied the binding of spermine as a function of the ionic strength. We show that this region delineates a functional and autonomous domain containing a binding site involved in the interaction with the four positive charges of spermine. Altogether, these results led to the elaboration of the first model defining the crucial structural parameters of a polyamine-protein interaction at the molecular level.

Animals↗

PubMed1997

Characterization of two different cytoplasmic protein tyrosine kinases from human breast cancer.

Two different protein tyrosine kinases were detected in the cytosolic fraction of different human tumor tissues. After partial purification, the two enzymes, which were highly active in breast tumor tissues, were characterized. One of them, soluble tyrosine kinase-1 (STK-1), represents a soluble form of the c-Src protein, which is apparently underphosphorylated on its C-terminal tyrosine residue whereas the other (STK-2) is a 48-kDa protein tyrosine kinase (PTK), which is molecularly and functionally related to the C-terminal Src kinase (Csk). These two protein tyrosine kinases clearly exhibit a different substrate specificity, and are responsible for the high tyrosine kinase activity present in the cytosolic fraction of human breast cancer. In addition, it was observed that STK-1 and STK-2 are also expressed in the breast cancer cell line, CAL-51.

Adenocarcinoma↗

PubMed1996

Fibroblast growth factor-2 binds to the regulatory beta subunit of CK2 and directly stimulates CK2 activity toward nucleolin.

The presence of fibroblast growth factor-2 (FGF-2) in the nucleus has now been reported both in vitro and in vivo, but its nuclear functions are unknown. Here, we show that FGF-2 added to nuclear extract binds to protein kinase CK2 and nucleolin, a CK2 natural substrate. Added to baculovirus-infected cell extracts overexpressing CK2 or its isolated subunits, FGF-2 binds to the enzyme through its regulatory beta subunit. Using purified proteins, FGF-2 is shown to directly interact with CK2 and to stimulate CK2 activity toward nucleolin. Furthermore, a mitogenic-deficient FGF-2 mutant protein has an impaired ability to interact with CK2 and to stimulate CK2 activity using nucleolin as substrate. We propose that in growing cells, one function of nuclear FGF-2 is to modulate CK2 activity through binding to its regulatory beta subunit.

Animals↗

PubMed1996

Casein kinase 2 inhibits the renaturation of complementary DNA strands mediated by p53 protein.

Considerable effort is currently being devoted to understand the functions of protein p53, a major regulator of cell proliferation. The protein p53 has been reported to catalyse the annealing of complementary DNA or RNA strands. We report that this activity is inhibited in the presence of the serine/threonine protein kinase CK2. It is shown that this inhibition can be explained by the occurrence of a high-affinity molecular association between p53 and CK2. The molecular complex involves an interaction between the C-terminal domain of p53 and the beta subunit of the oligomeric kinase. Accordingly, the isolated alpha subunit of the kinase was without effect. In addition, after phosphorylation by CK2, phosphorylated p53 lost its DNA annealing activity. Because the C-terminal domain of p53 is both involved in the association with CK2 and phosphorylated by it, our results suggest that either protein-protein interaction or phosphorylation of this domain might control the base pairing of complementary sequences promoted by p53 in processes related to DNA replication and repair.

Animals↗

PubMed1996

Identification of a cryptic protein kinase CK2 phosphorylation site in human complement protease Clr, and its use to probe intramolecular interaction.

Treatment of human (activated)C1r by CK2 resulted in the incorporation of [32P]phosphate into the N-terminal alpha region of its non-catalytic A chain. Fragmentation of 32P-labelled (activated)C1r followed by N-terminal sequence and mass spectrometry analyses allowed identification of Ser189 as the phosphorylation site. Accessibility of Ser189 was low in intact C1r, due in part to the presence of one of the oligosaccharides borne by the alpha region, further reduced in the presence of calcium, and abolished when C1r was incorporated into the C1s-C1r-C1r-C1s tetramer or the C1 complex. In contrast, phosphorylation was enhanced in the isolated alpha fragment and insensitive to calcium. Taken together, these data provide support for the occurrence of a (Ca2+)-dependent interaction between the alpha region and the remainder of the C1r molecule.

Amino Acid Sequence↗

PubMed1996

A structural basis for substrate specificities of protein Ser/Thr kinases: primary sequence preference of casein kinases I and II, NIMA, phosphorylase kinase, calmodulin-dependent kinase II, CDK5, and Erk1.

We have developed a method to study the primary sequence specificities of protein kinases by using an oriented degenerate peptide library. We report here the substrate specificities of eight protein Ser/Thr kinases. All of the kinases studied selected distinct optimal substrates. The identified substrate specificities of these kinases, together with known crystal structures of protein kinase A, CDK2, Erk2, twitchin, and casein kinase I, provide a structural basis for the substrate recognition of protein Ser/Thr kinases. In particular, the specific selection of amino acids at the +1 and -3 positions to the substrate serine/threonine can be rationalized on the basis of sequences of protein kinases. The identification of optimal peptide substrates of CDK5, casein kinases I and II, NIMA, calmodulin-dependent kinases, Erk1, and phosphorylase kinase makes it possible to predict the potential in vivo targets of these kinases.

Amino Acid Sequence↗

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↗