Biomedical subjects

B Omri

Publications and source records attributed to B Omri.

8 recordsLinked to original sources

PubMed1990

[Modifications of protein kinase C activity and phosphorylation of lipocortin I in cultures of pig thyroid cells].

When cultured in the absence of thyreostimulin (TSH), thyroid cells lose some of their differentiated functions such as iodide transport and its incorporation into thyroglobulin. In the presence of TSH (0.1 mU/ml), these differentiated functions are preserved ("TSH cells"). The addition of tetradecanoyl phorbol 13 acetate (TPA) inhibits some differentiated functions of the cells and provokes important modifications of bio-signalling pathways. The protein kinase C (pKC) activity, unchanged in "control" and "TSH cells", was dramatically modified in TPA treated cells. After translocation, the pKC activity was down-regulated and the phosphorylation of its endogenous substrates (35-38 kDa) disappeared. Among these substrates, we identified the lipocortin I (LC I) (35 kDa), a phospholipase A2 inhibitory protein related to the Ca2+ binding protein family. By monodimensional electrophoresis (PAGE-SDS) and western-blot, we evidenced the presence of LCI in cytosols and particulate extracts. By 2 dimensional electrophoresis (PAGE-SDS and IEF) and western-blot we identified a phosphorylated and unphosphorylated LCI protein. The phosphorylation of LCI by pKC decreased its isoelectric point from 6.9-6.6. The modifications of pKC activity and LCI phosphorylation and the changes in the bio-signalling pathways can partly account for the loss of differentiation observed in control or TPA treated cells.

Animals↗

PubMed1989

Identification of four lipocortin proteins and phosphorylation of lipocortin I by protein kinase C in cytosols of porcine thyroid cell cultures.

Four proteins of the lipocortin family, lipocortin I (35 kDa), lipocortin II (36 kDa), lipocortin V (32 kDa) and lipocortin VI (67-70 kDa), were identified in the cytosols of 2-day-old cultures of thyroid cells. Only lipocortin I was phosphorylated in vitro in fully differentiated, thyroid stimulating hormone-treated cells (0.1 mU/ml). Protein kinase C was the only kinase activity which phosphorylated lipocortin I. Phosphorylation shifted its pI from 6.9 to 6.6. The in vitro phosphorylation of lipocortin I was impaired in cultures exposed for 2 days to phorbol ester (10(-7) M), although it was present in both the cytosol and the particulate fraction of these cells.

Animals↗

PubMed1989

Thyrotropin but not epidermal growth factor down-regulates the isozyme I (PKa I) of cyclic AMP-dependent protein kinases in dog thyroid cells in primary cultures.

The activity of the two cAMP-dependent protein kinases (PKa I and PKa II) was evaluated in dog thyroid cells in primary cultures after a 6-day growth period induced by either thyrotropin (TSH) or epidermal growth factor (EGF). Although the total PKa activity was not affected in cells cultured in the presence of TSH or EGF, their actions on the PKa I and PKa II expressions were significantly different. The activity of PKa I was strongly inhibited by TSH (70-80%) while with EGF it was either stimulated or unaffected with respect to controls. The two mitogens did not have a significant effect on the activity of PKa II. Forskolin (Fk) mimicked the effect of TSH. The expression of the two regulatory subunits (R I and R II), evaluated by the covalent binding of 8-azido-cAMP, was similar to the expression of the corresponding catalytic activities, suggesting a coregulation of the catalytic and regulatory subunits from the same isozyme. After chronic stimulation by TSH, differentiated dog thyroid cells are almost completely deprived of PKa I.

Affinity Labels↗

PubMed1988

Phorbol ester prevents the thyroid-stimulating-hormone-induced but not the forskolin-induced decrease of cAMP-dependent protein kinase activity in thyroid cell cultures.

The potent tumor promoter 12-O-tetradecanoyl-phorbol 13-acetate (TPA) affects several thyroid cell functions and interacts with thyroid-stimulating hormone (TSH) either by inhibiting or potentiating its action on different cellular parameters. Since phorbol ester acts mainly through the activation of protein kinase C, which is its receptor, we studied this activation and its interaction with TSH and forskolin in suspension cultures of porcine thyroid cells. In thyroid cell cultures, TPA has a dual effect on protein kinase C activity: immediately (2-5 min) after exposure of cells to TPA, it began to be translocated from the cytosol to the particulate fraction. The transfer of the cytosolic enzyme was total and could occur with or without a loss of activity. The translocated enzyme still needed Ca2+ and phospholipids for its activation. The basal activity increased transiently (2-4 h) in both the cytosol and particulate fractions during translocation. The peak activity in the particulate fraction was reached 10-30 min after exposure of cells to TPA, and was followed by down-regulation of protein kinase C and almost complete disappearance of its activity. The residual activity was about 13% of control after a 2-day exposure to TPA. It was unequally distributed between cytosol (4%) and particulate fraction (9%). Prolonged exposure of cells to TPA did not affect either the activity or the subcellular distribution of the cAMP-dependent protein kinase activity. TPA interacted with TSH and prevented the decrease of this activity induced by prolonged exposure of cells to the hormone not only when it was introduced simultaneously with TSH, but also when it was added 24 h after TSH. However, the forskolin-induced decrease in cAMP-dependent protein kinase activity was not prevented by the presence of TPA. TPA also affected the increases in cAMP accumulation mediated by TSH and forskolin. The TSH-induced increase was significantly stimulated by TPA after short contacts (5-15 min), while longer preincubations of cells with TPA provoked a very strong inhibition of the TSH action. However, the forskolin-induced stimulation of the cAMP accumulation was maintained and even further increased in the presence of TPA. Consequently, the actions of TSH and TPA are apparently interdependent, while those of forskolin and TPA seem to be parallel and independent. Neither TSH nor forskolin prevented the TPA-induced down regulation of protein kinase C. The biologically inactive phorbol ester analogue 4 alpha-phorbol 12,13-didecanoate had no effect on protein kinase C activity, and did not interact with either TSH or forskolin.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1988

Decrease in cAMP-dependent protein kinase activity in suspension cultures of porcine thyroid cells exposed to TSH or forskolin.

Suspension cultures of porcine thyroid cells were used to study the action of TSH and forskolin (Fk) on cAMP-dependent (PKa) and Ca2+-phospholipid-dependent (PKc) protein kinase--enzymes which represent the key step in the transduction of extracellular signals. The PKa activity in cells cultured for 2 days in the presence of TSH was decreased to about 50% of control level with a TSH dose of 0.1 mU/ml. This decrease is dose dependent; only traces of PKa activity remained at very high doses of TSH (50 mU/ml). Similar results were obtained with Fk (10(-5) M), the adenylate cyclase activator. It decreased the PKa activity to the level obtained with 0.1-1.0 mU/ml TSH. The loss of the PKa activity was parallel in cytosol and particulate fractions, suggesting that there is no translocation of enzymes under the action of either TSH or Fk. Neither TSH nor Fk had any effect on PKc, which became the predominant activity in cells exposed to either of the regulators. The cAMP-dependent phosphorylation of endogenous proteins was lower in TSH- or Fk-treated cells than in controls, and was dependent, like the PKa activity, on the dose of TSH. Polyacrylamide gel electrophoresis (PAGE) revealed the specific substrates of PKa in cultured thyroid cells. Proteins of 28, 30 and 33 kDa were regularly found, while 58 kDa protein was not present in all experiments. PAGE patterns showed that the decrease in endogenous phosphorylation in TSH- and Fk-treated cells was due to decreased labelling of PKa-specific substrates. The observed down-regulation of PKa activity could have an influence on the expression of thyroid cell differentiation.

Animals↗

PubMed1987

Characteristics of thyroid protein kinase C. Different Ca2 requirement for the phosphorylation of endogenous proteins and of H1 histone.

Thyroid protein kinase C (PKc) from cytosols of porcine and rat thyroid glands has been characterized using histone H1 or endogenous proteins as substrates. As in many other tissues histone H1 is by far the preferred exogenous substrate of thyroid PKc. Kinetic studies with H1 showed that, compared to rat thyroids, porcine glands are particularly rich in PKc, the predominant kinase activity in this tissue. The cAMP-dependent protein kinase (PKa) level, on the contrary, is very similar in both rat and porcine thyroids. Consequently, for the same type of tissue, there may be great species differences in the PKc level and the ratios between PKc and PKa kinase activities. Chromatographic properties of thyroid PKc are similar to those described in other tissues (one major peak followed by a small shoulder) except that elution of the main peak can vary depending on the nature of the salt gradient (approximately 55 mM for NaCl and 15 mM for sodium phosphate). In the first case PKc is completely separated from the PKa activity, in the second it is coeluted with the peak of PKa type I. The one-dimensional PAGE pattern of proteins phosphorylated by porcine PKc is very similar to the pattern obtained by rat enzyme. Protein bands of 18 kDa, 22-25 kDa and 32-36 kDa are specific substrates of the thyroid PKc, after in vitro phosphorylation of cytosol proteins. A great difference in Ca2+ requirement for PKc activation was noted, depending whether histone H1 or endogenous proteins were substrates. As in other tissues, calcium was absolutely necessary for phosphorylation of histone H1 by PKc. The addition of calcium was not absolutely necessary when endogenous proteins were the substrates, either for the activation of the enzyme or for phosphorylation of the PKc-specific substrates. Almost the same rate of phosphorylation was obtained with or without calcium in the incubation medium. However the one-dimensional PAGE pattern of phosphorylated proteins was different in the presence or absence of calcium. While addition of calcium was not absolutely necessary for the phosphorylation of a great number of proteins by the PKc, its presence was indispensable for the phosphorylation of certain endogenous substrates. However, calcium alone, in the absence of phospholipids had no effect on the phosphorylation of these proteins. Endogenous proteins, phosphorylated by the PKc only when calcium was present, were resolved by the two-dimensional PAGE into several distinct spots with molecular masses of 32-35 kDa and pI range of 5-7.5.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1986

Endogenous substrates of protein kinase C in experimentally induced and regressed rat thyroid goitres.

The presence of endogenous substrates of the protein kinase C (PKc) in rat thyroid glands has been demonstrated in in vitro phosphorylated cytosolic proteins by polyacrylamide gel electrophoresis (PAGE). Rat thyroid PKc specifically catalyzes the phosphorylation of the 35 kDa and 18 kDa proteins. These proteins were not labelled in the presence of Ca2+ alone, but they were phosphorylated when phospholipids alone were added. In hyperplastic glands the total phosphorylation of endogenous proteins is stimulated, due to the increased labelling of the 35 kDa and 18 kDa proteins. No extra phosphorylated bands were revealed by PAGE analysis. After suppression of growth activity the labelling of the two PKc-specific substrates was strongly inhibited.

Animals↗

PubMed1985

Protein kinase C activity in experimentally developed and regressed rat thyroid goitres.

The presence of protein kinase C activity in rat thyroid glands was demonstrated by kinetic studies, using lysine-rich histone as substrate. DE 52 cellulose chromatography resolved thyroidal protein kinase C activity into two peaks, the first eluting at 50 mM and the second at 150 mM salt solution. They contained respectively 40% and 60% of the total activity. In cytosols from experimentally induced goitres, which are hyperplastic tissues, protein kinase C activity rose 3-4-fold compared to control glands. In this tissue, protein kinase C seems to be present in reversibly activated form, since its activity was completely inhibited in the absence of calcium and phospholipids. After removal of growth stimulus, rapid involution of goitres occurred, producing a spectacular decrease in protein kinase C activity. In goitres regressed for 5 days, the level of protein kinase C was lower than in untreated control tissue. The protein kinase C activities in control glands and developed and regressed goitres were 1.88, 5.85 and 0.74 pmoles 32P/mg tissue, respectively. These results clearly demonstrate, for the first time, a direct correlation between the protein kinase C level and the thyroid gland growth activity induced by endogenous stimuli.

Animals↗