Biomedical subjects

O Filhol

Publications and source records attributed to O Filhol.

32 records · Page 2Linked to original sources

PubMed1995

[Has protein kinase CK2 a role in the intracellular mitogenic signalling?].

The protein kinase CK2 is an ubiquitous serine-threonine kinase found in all eukaryotic cells. Although well characterized on a biochemical ground, its role and regulation in the intact cell are not clearly understood. Its possible implication in the control of cell proliferation has been examined by several different approaches. (i) Immunocytochemical detection of CK2 revealed that whereas the signal was evenly distributed throughout cycle arrested cells in primary culture, it accumulates rapidly (30-90 min) in the nuclear compartment in cells stimulated to grow. (ii) CK2 biosynthesis is activated as an early response to growth factors in quiescent cells. The neo-synthesized kinase accumulates as the cells progress through the G1 phase. This growth factor-activated biosynthesis concerns in parallel the two kinase subunits. (iii) The kinase is activated in vitro by polyamines, which are increased in cells challenged by growth factors. Spermine binds to a specific domain of the beta subunit of CK2. (iv) In addition to phosphorylation CK2 forms a molecular complex with p53, a major negative regulator of the cell cycle. The complex was demonstrated in intact cells and reconstituted in vitro (Kd 70 nM) with purified components and shown to require the beta subunit and to result in the inhibition of p53 DNA-annealing activity. These observations suggest that CK2 and p53 may play a coordinated role in the cell response to mitogenic stimuli.

Animals↗

PubMed1994

Oligomeric casein kinase II isoforms are expressed in bovine tissues and adrenocortical cells in culture.

Casein kinase II is a ubiquitous serine-threonine kinase made of two different (alpha and beta) subunits, with an alpha 2 beta 2 stoichiometry, alpha bearing the catalytic site of the enzyme. Two different genes encode two different (alpha and alpha') catalytic subunits. The present work shows that at least two forms of CKII (CKIIa and CKIIb) can be isolated from bovine tissues and adrenocortical cells in culture. The use of specific antibodies developed against the alpha and alpha' subunits disclosed that while CKIIa contains both alpha and alpha', the alpha subunit was highly prominent in CKIIb. This suggests that CKIIb may be an alpha 2 beta 2 oligomer whereas CKIIa may be an alpha alpha' beta 2 moiety or an alpha alpha' beta 2 and alpha' 2 beta 2 mixture. Transition from quiescence to proliferation was concomitant with an average two fold increase in total cell CKII activity involving both CKIIa and CKIIb isoforms. The two isoforms were found in cytosolic and particulate adrenocortical subcellular preparations and they similarly increased their association to intracellular organelles in response to growth stimulatory conditions.

Adrenal Cortex↗

PubMed1994

Modulation of the molecular organization and activity of casein kinase 2 by naturally occurring polyamines.

Polyamines have been reported to stimulate casein kinase 2 (CK2) in vitro. We have shown that the phosphorylation of different substrates is diversely stimulated by basic effectors and that the responsiveness of CK2 activity may be influenced by the overall conformation of the protein substrate but also by a specific interaction with the enzyme itself. Our data show that native hetero tetrameric CK2 is a spermine binding protein and a spermine binding site was identified in the N-terminal region of the beta subunit of CK2. We found that recombinant CK2 undergoes a progressive polymerization in low salt conditions, giving rise to three different polymer structures. A ring-like structure formed by the association of four protomers alpha 2 beta 2 was characterized by gel filtration, sucrose density gradient analysis and electron microscopy. Polyamines like spermine, when added under conditions where the enzyme preparation contains a mixture of various oligomers, could trigger their dissociation and their interconversion in the fully active ring structure. These results suggest that in the presence of positive effectors like polyamines, CK2 adopts a ring-like structural organization which may represent the active state of this kinase.

Amino Acid Sequence↗

PubMed1993

Epidermal growth factor stimulates a protein tyrosine kinase which is separable from the epidermal growth factor receptor.

The intrinsic protein tyrosine kinase activity of the epidermal growth factor (EGF) receptor is necessary for ligand-induced signaling. To determine whether cellular protein tyrosine kinases are substrates for EGF-activated receptors, phosphotyrosine-containing proteins were isolated from EGF-treated cells and assayed for tyrosine kinase activity using peptide substrates. A tyrosine kinase activity that is distinct from the EGF receptor was adsorbed to monoclonal anti-phosphotyrosine antibody columns and eluted with phenyl phosphate. Near-maximal tyrosine phosphorylation of this kinase occurred within 1 min of cell stimulation with an ED50 for EGF of 2.5 nM. The kinase was deactivated by incubation with purified CD45 tyrosine phosphatase in vitro, but activity could be restored by incubation with purified EGF receptor and Mn2+ ATP. These results suggest a cascade of tyrosine kinase signaling analogous to well characterized serine/threonine kinase cascades.

Amino Acid Sequence↗

PubMed1993

DNA topoisomerase II and casein kinase II associate in a molecular complex that is catalytically active.

Immunoprecipitation of DNA topoisomerase II from yeast results in a preparation that contains casein kinase II; this suggests that the two proteins may associate in the intact cell. Purified recombinant topoisomerase II and casein kinase II associate to form a complex in vitro which is stable after topoisomerase II becomes phosphorylated by the kinase. Studies with isolated recombinant casein kinase II subunits disclosed that although the alpha (catalytic) subunit alone can efficiently phosphorylate topoisomerase II, the formation of a stable topoisomerase II-casein kinase II association requires the presence of the beta subunit of the kinase. Both proteins engaged in this complex retain their catalytic activities. Naturally occurring polyamines and polyanionic compounds appear to be crucial factors governing the interaction between the two proteins. Although the biological significance of a stable catalytically active topoisomerase II-casein kinase II molecular complex remains to be defined, these observations suggest the possibility of a novel mechanism regulating topoisomerase II and casein kinase II activities.

Amino Acid Sequence↗

PubMed1993

Baculovirus-directed expression of human prostatic steroid 5 alpha-reductase 1 in an active form.

In the human prostate, the enzyme steroid 5 alpha-reductase (h5 alpha R) catalyses the conversion of testosterone into the more potent androgen, dihydrotestosterone. Two distinct cDNAs coding for h 5 alpha R in the human prostate have been previously characterized. Enzyme h5 alpha R1 shows a maximum activity at basic pH whereas h5 alpha R2 has an acidic pH optimum activity. We report here the expression of the human steroid h5 alpha R1 in a eukaryotic expression system: the baculovirus-directed-insect cell expression system. The full length cDNA was inserted into the Autographa californica nuclear polyhedrosis virus genome and expressed in Spodoptera frugiperda, Sf9, insect cells. Sf9 cells were infected with the recombinant baculovirus and homogenates used in h5 alpha R activity assays by high pressure liquid chromatography showed that a catalytically active enzyme was produced. The recombinant enzyme showed an apparent Km for testosterone of 2.07 microM and a V(max) of 10.1 nmol of dihydrotestosterone/ min/mg of protein. Recombinant h 5 alpha R1 activity was inhibited by specific h 5 alpha R inhibitors such as 4-MA (Ki = 2.6 nM). Subcellular distribution in Sf9 cells demonstrated that the enzyme was associated with the nuclear membrane.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗

PubMed1992

Casein kinase II and the tumor suppressor protein P53 associate in a molecular complex that is negatively regulated upon P53 phosphorylation.

Selective immunoisolation of P53 from Sf9 cells coexpressing wild-type P53 and casein kinase II yielded a preparation containing casein kinase II, thus suggesting that the two proteins may associate in a molecular complex in the intact cell. Such a complex could indeed be demonstrated in vitro between purified recombinant P53 and oligomeric casein kinase II and was shown to dissociate when P53 became phosphorylated by the kinase. This suggested that the P53 C-terminal domain, which contains the casein kinase II phosphorylation site was involved in the protein-protein interaction; this was confirmed by the fact that an anti-P53 monoclonal antibody directed to that domain inhibited the P53-casein kinase II association. Studies with isolated recombinant casein kinase II subunits disclosed that although the alpha (catalytic) subunit could phosphorylate P53, the formation of a stable P53-casein kinase II association required the presence of the beta subunit of the kinase. This was confirmed by immunoisolation of a P53-beta subunit complex from cells expressing both polypeptides. Although the biological significance of a reversible P53-casein kinase II molecular complex in the control of cell proliferation processes remains to be defined, these observations suggest the possibility of a novel mechanism regulating P53 and casein kinase II activities in the intact cell.

Adenosine Triphosphate↗

PubMed1992

A site of tyrosine phosphorylation in the C terminus of the epidermal growth factor receptor is required to activate phospholipase C.

Cells expressing mutant epidermal growth factor (EGF) receptors have been used to study mechanisms through which EGF increases phospholipase C (PLC) activity. C-terminal truncation mutant EGF receptors are markedly impaired in their ability to increase inositol phosphate formation compared with wild-type EGF receptors. Mutation of the single tyrosine self-phosphorylation site at residue 992 to phenylalanine in an EGF receptor truncated at residue 1000 abolished the ability of EGF to increase inositol phosphate formation. C-terminal deletion mutant receptors that are impaired in their ability to increase inositol phosphate formation effectively phosphorylate PLC-gamma at the same tyrosine residues as do wild-type EGF receptors. EGF enhances PLC-gamma association with wild-type EGF receptors but not with mutant receptors lacking sites of tyrosine phosphorylation. These results indicate that formation of a complex between self-phosphorylated EGF receptors and PLC-gamma is necessary for enzyme activation in vivo. We propose that both binding of PLC-gamma to activated EGF receptors and tyrosine phosphorylation of the enzyme are necessary to elicit biological responses. Kinase-active EGF receptors lacking sites of tyrosine phosphorylation are unable to signal increased inositol phosphate formation and increases in cytosolic Ca2+ concentration.

Animals↗

PubMed1991

Coexpression of both alpha and beta subunits is required for assembly of regulated casein kinase II.

Casein kinase II is an ubiquitous serine-threonine kinase whose functional significance and regulation in the living cell are not clearly understood. The native enzyme has an oligomeric structure made of two different (alpha and beta) subunits with an alpha 2 beta 2 stoichiometry. To facilitate the study of the structure-activity relationship of the kinase, we have expressed its isolated subunits in a baculovirus-directed insect cell expression system. The resulting isolated recombinant alpha subunit exhibited a protein kinase catalytic activity, in agreement with previous observations [Cochet, C., & Chambaz, E. M. (1983) J. Biol. Chem. 258, 1403-1406]. Coinfection of insect cells with recombinant viruses encoding the two kinase subunits resulted in the biosynthesis of a functional enzyme. Active recombinant oligomeric kinase was purified to near homogeneity with a yield of about 5 mg of enzymatic protein per liter, showing that, in coinfected host cells, synthesis was followed, at least in part, by recombination of the two subunits with an alpha 2 beta 2 stoichiometry. The catalytic properties of the recombinant enzyme appeared highly similar to those previously observed for casein kinase II purified from bovine tissue. Access to the isolated subunits and to their alpha 2 beta 2 association disclosed that the beta subunit is required for optimal catalytic activity of the kinase. In addition, the beta subunit is suggested to play an essential role in the regulated activity of the native casein kinase II. This is clearly illustrated by the observation of the effect of spermine which requires the presence of the beta subunit to stimulate the kinase catalytic activity which is borne by the alpha subunit.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1991

Casein kinase II and polyamines may interact in the response of adrenocortical cells to their trophic hormone.

The ubiquitous casein kinase II (CKII) has been shown to accumulate in the cell nuclear compartment, following exposure to extracellular growth stimuli (O. Filhol et al., Biochemistry, 1990, 29, 9928-9936). The aim of the present study was to examine whether intracellular polyamines, whose levels are increased under similar conditions, could be related to this process. It is shown that (i) CKII accumulates in nuclei of adrenocortical cells exposed to their trophic hormone ACTH; (ii) this CKII nuclear translocation is concomitant with an increase in nuclear polyamine content resulting from ACTH-induced polyamine synthesis; (iii) selective inhibition of polyamine biosynthesis by DFMO results in the inhibition of both ACTH-induced cellular polyamine increase and CKII nuclear accumulation. These observations suggest that polyamines may be examined as intracellular messengers in the regulation of CKII activity and subcellular distribution in the cell response to growth factors and trophic hormones.

Adrenal Cortex↗

PubMed1991

Polyamine binding activity of casein kinase II.

Protein phosphorylation by the ubiquitous casein kinase II (CKII) is known to be sensitive to naturally occurring polyamines. Using isolated recombinant alpha and beta subunits of the kinase, as well as the alpha 2 beta 2 oligomeric enzyme, it is shown that (i) CKII binds [3H]-spermine with Kds in the micromolar range. (ii) The beta subunit appears mostly responsible for this binding activity. (iii) The isolated alpha (catalytic) subunit is not activated by polyamines. (iv) The polyamine-dependent activation of the oligomeric CKII requires the beta subunit, which appears as a regulatory component in the native kinase. These observations suggest that there may be a functional interaction between polyamines and CKII in living cells, especially in the response to cell growth factors and trophic hormones.

Animals↗

PubMed1991

Interaction between the epidermal growth factor receptor and phosphoinositide kinases.

Ligand-activated epidermal growth factor (EGF) receptors are coupled to the phosphatidylinositol (PtdIns) pathway to stimulate formation of two second messengers, inositol trisphosphate and diacylglycerol. Investigation of the interaction between EGF receptors and phosphoinositide kinases identified PtdIns and PtdIns(4)P kinase activities in extensively washed EGF receptor immunoprecipitates. Studies using COOH-terminal truncation mutant EGF receptors and immunoisolation by an EGF receptor peptide anti-serum in the presence of peptide (residues 644-666) indicated that the phosphoinositide kinases were associated with the region located between the inner membrane boundary and the kinase domain of the EGF receptor. In vivo cross-linking identified four tyrosine phosphorylated proteins of approximately 135, 62, 55, and 47 kDa associated with the EGF receptor. After EGF stimulation, PtdIns and PtdIns(4)P kinase activities were markedly increased among proteins isolated by monoclonal antiphosphotyrosine antibodies. The activities associated with the EGF receptor and with tyrosine-phosphorylated proteins were identified as PtdIns4-and PtdIns(4)P 5-kinase. Tyrosine dephosphorylation did not alter the activity of the prominent PtdIns(4)P 5-kinase activity. These results indicate that the phosphoinositide kinases are associated with and tyrosine phosphorylated by the EGF receptor as part of the mechanism coordinating responses between signal transduction pathways but do not demonstrate that tyrosine phosphorylation of PtdIns(4)P 5-kinase is sufficient to activate the enzyme.

1-Phosphatidylinositol 4-Kinase↗

PubMed1990

DNA binding activity of casein kinase II.

Casein kinase II, an ubiquitous, oligomeric, messenger-independent protein kinase has previously been shown to concentrate in the nuclear compartment when cells are stimulated to proliferate. The present communication reports that purified mammalian CKII interacts with genomic DNA preparations in vitro. This interaction led to an apparent activation of the kinase, most likely explained by prevention of its aggregation and subsequent denaturation. Binding of CKII was optimum with double stranded DNA preparations; duplex lambda phage DNA exhibited at least two types of binding sites and the high affinity system (Kd approximately equal to 6 x 10(-13) M) represented a binding capacity of about 1 mol CKII per mol DNA. CKII-DNA interaction was stimulated in the presence of a polyamine and inhibited by heparin. Blotting experiments disclosed that DNA binds CKII through its alpha subunit. These observations are in line with the hypothesis that casein kinase II may be examined as a component in the transduction of the mitogenic signal from the cell membrane to the nucleus, in response to growth factors.

Animals↗

PubMed1990

Cytoplasmic and nuclear distribution of casein kinase II: characterization of the enzyme uptake by bovine adrenocortical nuclear preparation.

Casein kinase II (CK II) is a ubiquitous protein kinase that has been found in both nuclear and soluble subcellular fractions and whose precise cellular functions and mechanisms of control remain to be clarified. Using immunocytochemical localization, it was observed that the intracellular distribution of CK II exhibited a striking shift toward an increased nuclear concentration during active proliferation of bovine adrenocortical cells in primary culture. The interaction of CK II with purified adrenocortical cell nuclear preparation was thus examined in vitro. CK II was found to rapidly associate with nuclei in a temperature-dependent and saturable process, resulting in a tight binding of the kinase to nuclear components, as shown by various extraction procedures. This association resulted in a concentration of the kinase in the nuclear preparation about 100-fold that in the medium and exhibited two types of binding sites with Ka of 10(9) and 10(7) M-1, respectively. The nuclear CK II uptake was dependent upon the presence of ATP and was stimulated by a kinase activator such as spermine, although the enzyme activity did not appear to be required for the process. These observations would be in line with a pore-mediated, energy-dependent nuclear uptake of the kinase. Since a number of potential nuclear CK II targets have been reported, including the oncoprotein myc, it is suggested that the nuclear translocation of the kinase as characterized in vitro may have a biological significance in living cell, especially in the control of nuclear activities related to cell proliferation and the mechanism of action of growth factors.

Adrenal Cortex↗