Biomedical subjects

C Cochet

Publications and source records attributed to C Cochet.

At least 19 recordsLinked to original sources

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↗

PubMed1992

[Bone risk of hormone-suppressing therapy in differentiated thyroid epithelioma. Study by dual photon absorptiometry].

A decreased bone mineral density is a well known complication of thyrotoxicosis. If bone is also adversely affected by a subclinical hyperthyroidism is still debated. Such a situation is deliberately induced for treatment of thyroid differentiated carcinoma. We compared bone mineral measurements in case of thyroid carcinoma exposed to prolonged suppressive hormonal treatment (288 patient year, LT3 in 87%) and in age and weight matched controls. We did not find any difference in both sexes between the two populations in term of fracture rate and vertebral mineral density measured by dual photon absorptiometry, nor any influence of gonadal status in women.

Absorptiometry, Photon↗

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

Structural analysis of the transmembrane domain of the epidermal growth factor receptor.

The ligand-binding domain of the epidermal growth factor (EGF) receptor is separated from the cytoplasmic protein tyrosine kinase domain by a predicted single transmembrane segment. Antipeptide antibodies prepared against the outer portion of the predicted transmembrane segment confirmed this area was exposed only when cells were treated with permeabilizing agents. To investigate structural requirements for signal transduction by the transmembrane domain, three types of mutant EGF receptor were prepared. The first type was designed to shorten the transmembrane domain, the second to place proline substitutions within this domain, and the third to make amino acid substitutions analogous to those present in the transforming c-erbB2/neu oncoprotein. Mutant human receptors were expressed in null recipient mouse B82L and Chinese hamster ovary cells. All receptors bound EGF and exhibited EGF-stimulated protein tyrosine kinase activity in vivo as assayed using a 125I-labeled monoclonal anti-phosphotyrosine antibody. EGF stimulated growth of cells expressing each mutant receptor with similar dose-response characteristics. In contrast to other growth factor receptors, the transmembrane domain of the EGF receptor is tolerant to a variety of changes which neither mimic EGF action by constitutive activation nor interfere with ligand-induced signal transduction.

Amino Acid Sequence↗

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↗

PubMed1991

Increased SOD1 enzymatic activity and gene modifications in orangutans: evolutionary implications.

Superoxide dismutase CuZn (SOD1) enzymatic activity was measured in five orangutans (Pongo pygmaeus, PPY) and compared to that of man, chimpanzee, and gorilla. It was found to be increased by a factor of two in one orangutan (Ralfina) and by a factor of 1.5 in the four others. In situ hybridization of the SOD1 cDNA human probe showed a heterozygous intra-chromosomal rearrangement of pair PPY XXI, possibly an insertion, in Ralfina. Southern blotting showed that the SOD1 gene is modified in the three orangutans that were investigated and that a further modification of the 5'-end of the gene had occurred in Ralfina. The evolutionary implications of these observations are discussed.

Animals↗

PubMed1991

Histiocytosis X of the hypothalamus.

An unusual case of cutaneous and hypothalamic histiocytosis X (HX) is reported. The hypothalamic involvement occurred as a tumor that mimicked a chiasm glioma on computed tomography angioscanning. Magnetic resonance imaging after gadolinium injection localized the tumor within the third ventricle floor. The HX origin of the tumor was confirmed by histological examination of hypothalamic biopsies obtained by transventricular endoscopy. The results of endocrine evaluation were consistent with anterior panhypopituitarism resulting from a multiple releasing-hormone secretory defect, but there was no diabetes insipidus. This unusual endocrine aspect has not been previously described in the field of hypothalamic HX. Lastly, the tumor was insensitive to low dose megavoltage radiation therapy. This unusual case stresses the superiority of magnetic resonance imaging over computed tomography scanning in the assessment of suprasellar tumors and emphasizes the usefulness of transventricular endoscopy in these cases.

Female↗

PubMed1991

Transforming growth factor beta 1: an autocrine regulator of adrenocortical steroidogenesis.

Transforming growth factor beta 1 (TGF beta 1) is a member of a large family of structurally related regulatory polypeptides which comprises both functionally similar (TGF beta 1, TGF beta 2, TGF beta 3, TGF beta 4 and TGF beta 5) and functionally distinct proteins. In the past few years, TGF beta 1 has emerged as a multifunctional protein. One of its remarkable properties is its capacity to negatively modulate the differentiated, steroidogenic adrenocortical functions. We present here a review of the results from our recent work related to the effects of TGF beta 1 on bovine adrenocortical cell (zona fasciculata-reticularis) functions. We identified the steroid 17 alpha-hydroxylase (P-450 17 alpha) biosynthetic enzyme and the angiotensin II receptor as major targets whose expression are negatively regulated by TGF beta 1 in these cells. We characterized TGF beta 1 receptors at the surface of adrenocortical cells (mainly type I and type III receptors) and observed that their number is increased under ACTH treatment. Furthermore, we could detect the presence of immunoreactive TGF beta 1 in the bovine adrenal cortex whereas it was undetectable in the adrenal medulla and in the capsule. We also observed that adrenocortical cells secrete TGF beta 1 under a latent form together with large amounts of alpha 2-macroglobulin, a protease inhibitor known to be implied in the latency of TGF beta in serum. Taken together, these observations led us to a working hypothesis, proposing TGF beta 1 as an autocrine and/or paracrine regulator of adrenocortical steroidogenic functions. This concept points out the physiological activation of the latent TGF beta 1 complex as the important limiting step controlling its action in the adrenal cortex.

Adrenal Cortex↗

PubMed1991

[Autocrine regulation of adrenal steroidogenesis by growth factor transforming TGF-beta 1].

Transforming growth factor beta (TGF beta) is a multifunctional polypeptide presenting various biological activities depending on the cellular context where it is studied. In the adrenal cortex, TGF beta is a potent inhibitor of corticosteroïdogenesis. The sum of data that we have collected in the past years has allowed us to establish that TGF beta (in particular TGF beta 1) is implicated in a physiological autocrine/paracrine regulatory loop. We determined that primary cultures of bovine fasciculatareticularis cells synthesize and secrete about 5 ng/24 h x 10(6) cells of TGF beta but that most of this activity is under a latent form. In the adrenal gland, TFG beta 1 could be localized in the fasciculata-reticularis zone by immunohistochemistry. We also reported that adrenocortical cells possess type I and type III (betaglycan) receptors, whose number is positively regulated by ACTH. Moreover, we have identified 3 major targets of TGF beta in our cell model: cytochrome P-450(17) alpha (17 alpha-hydroxylase) whose gene expression is decreased, angiotensin II receptors and LDL receptors whose number is reduced after TGF beta treatment. As a conclusion, TGF beta appears to participate together with other regulatory peptides (IGF-1, TNF alpha) in the fine tuning of corticosteroid secretion levels which are under the major control of the hypothalamo-pituitary axis.

Adrenal Cortex↗

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↗

PubMed1990

Saliva from cystic fibrosis patients contains an unusual form of epidermal growth factor.

Epidermal Growth Factor (EGF) was assayed in saliva collected from control subjects and cystic fibrosis (CF) patients, using both radioimmuno (RIA) and radioreceptor (RRA) assays. An intriguing finding was that the average ratio of the values found by RRA over those obtained by RIA was of 1.7 for normal subjects and of about 0.4 for CF patients. This observation could be understood following gel filtration analysis of EGF-like material in these salivary fluids. Whereas control saliva contained the expected 6 kDa EGF active peptide, the immunoreactive EGF material from CF patients eluted as a polydisperse macromolecular moiety. The poor biological reactivity of this material as assessed by radioreceptor assay suggests that this EGF anomaly may contribute to the physiopathology of cystic fibrosis, especially as the upper gastrointestinal tract differentiated functions may be the target of normal salivary EGF.

Adolescent↗

PubMed1990

Transforming growth factor-beta stimulates the expression of alpha 2-macroglobulin by cultured bovine adrenocortical cells.

Adrenocortical cell major secreted protein was purified from the conditioned medium of primary cultures of bovine adrenocortical (BAC) cells. Immunochemical analysis and N-terminal sequencing of the purified protein identified it to alpha 2-macroglobulin (alpha 2-M). It appeared that 15 out of the 17 N-terminal amino acids were conserved between adrenocortical cell major secreted protein and human alpha 2-M. Study of alpha 2-M production by BAC cells revealed that its secretion was stimulated severalfold by transforming growth factor-beta 1 (TGF-beta 1). The stimulation occurred in a time-dependent (reaching a plateau at 24 h) and dose-dependent (ED50 = 0.1 ng/ml TGF-beta 1) manner. It was blocked when BAC cells were exposed to 5,6-dichlorobenzimidazole riboside, a potent inhibitor of RNA polymerase II, suggesting that TGF-beta 1 acts as an activator of alpha 2-M gene expression at the transcriptional level. Northern blot analysis confirmed that the alpha 2-M mRNA level was increased (4-fold) in BAC cells following TGF-beta 1 treatment. TGF-beta 2, TGF-beta 1,2, basic fibroblast growth factor, and angiotensin II also appeared able to stimulate alpha 2-M secretion in BAC cells, whereas adrenocorticotropin was strongly inhibitory. Given the previous reports that TGF-beta 1 is a potent inhibitor of adrenocortical steroidogenesis (Feige J.J., Cochet, C., Rainey, W.E., Madani, C., and Chambaz, E. M. (1987) J. Biol. Chem. 262, 13491-13495) and that alpha 2-M is a TGF-beta 1-binding protein, these observations suggest that alpha 2-M may play an important role in conjunction with hormones and growth factors in the homeostatic regulation of adrenocortical functions.

Adrenal Cortex↗

PubMed1989

Dendrotoxin-binding brain membrane protein displays a K+ channel activity that is stimulated by both cAMP-dependent and endogenous phosphorylations.

The purified protein that binds the K+ channel ligands dendrotoxin I and mast cell degranulating peptide can be phosphorylated by cAMP-dependent protein kinase and by an endogenous protein kinase, which may be a specific K+ channel kinase. Phosphorylations take place on the toxin-binding subunit, a polypeptide of 76-80 kDa. Phosphorylation by both kinases leads to activation of the reconstituted dendrotoxin-sensitive K+ channel.

Animals↗

PubMed1989

Tyrosine protein kinase activity of the EGF receptor is required to induce activation of receptor-operated calcium channels.

We have investigated the effects of epidermal growth factor (EGF) on calcium ion channels in A431 epidermoid carcinoma cells. We have found that: -1- EGF stimulates Ca2+ channels. -2- EGF stimulated Ca2+ channels are voltage independent, exhibit a low conductance (8 pS) and a bursting multichannels activity (BMC). -3- Activation of the tyrosine-kinase function of the EGF receptor is required to generate Ca2+ current. -4- Inositol (1,4,5) triphosphate (Ins (1,4,5) P3) and EGF have similar effect on the channel activation. These results suggest that: stimulation of tyrosine-kinase activity of the EGF receptor, production of Ins (1,4,5)P3 and calcium entry via voltage independent channels are important connected steps in mediating the mitogenic effect of this growth factor.

Calcium↗