TSH control of PKA catalytic subunit activity in thyroid cell cultures.

The protein expression and the enzyme activity of the catalytic subunit (C) of the cAMP-dependent protein kinases were studied in porcine thyroid cell primary cultures stimulated with two doses of TSH (0.1 mU/ml and 1 mU/ml) for 1 to 3 days. In TSH-stimulated cells the desensitization of the catalytic subunit activity was accompanied by a simultaneous and parallel decrease of its immunoreactivity.

Forskolin mimics TSH action on the expression of protein kinase C isozymes in pig thyroid cell cultures.

In porcine thyroid cell cultures, phospholipid-dependent protein kinases (PKCs) have the same characteristics as intact glands. The overall PKC activity, presence of PKC isozymes, chromatographic pattern and endogenous substrates specificity were not modified during the two-day culture period. Three PKC isozymes (cPKC epsilon, nPKC epsilon and aPKC zeta) were identified by immunoblot analysis in the two subcellular fractions: cytosol and particulate extract, both in intact glands and two-day-old cultures.

TSH action on cAMP binding to the regulatory subunits of cAMP-dependent protein kinases in pig thyroid cell cultures.

This study examines the mechanism of TSH action on the cAMP-dependent protein kinases (PKA) by measuring the catalytic activity of the two PKA isozymes (PKA I and PKA II) and their capacity to bind cAMP to the regulatory subunits (RI and RII) in thyroid cell cultures exposed for two days to different doses of TSH. In TSH-treated cell cultures a selective down regulation (up to 60%) of the catalytic activity was found; the PKA I was down regulated at lower TSH doses (0.1 mU/ml and even 0.

Species differences of the thyroid protein kinase C heterogeneity.

Protein kinase C (PKC), the mediator of the phosphoinositide transduction pathway, is a family of at least 11 isozymes and its heterogeneity has been described in many tissues and cells. We studied here the heterogeneity of PKC in thyroid glands from three different species, rat, pig, and dog. By combining immunological and biochemical approaches, we identified in rat thyroids, the PKC alpha, beta II, delta, epsilon, and zeta subspecies, in pig thyroids, the alpha, epsilon, and zeta isozymes, and in dog thyroids, only the alpha and zeta isozymes.

Immunological identification of protein kinase C-alpha and protein kinase C-delta in cultured rat mesangial cells: differential sensitivity of the two isoforms towards the protein kinase inhibitor H7.

Rat mesangial cells contain both calcium-dependent protein kinase C (PKC) activity, which phosphorylates histone H1 and endogenous proteins, and calcium-independent, phospholipid-dependent PKC activity, which phosphorylates only endogenous proteins. The calcium-dependent PKC was identified as PKC alpha by immunoblot analysis and hydroxyapatite chromatography (HPLC). The calcium-insensitive, phospholipid-dependent isoform was identified as PKC delta using similar techniques.

Heterogeneity of protein kinase C in cultured rat mesangial cells.

Two forms of protein kinase C (PKC) activity in cytosol of cultured rat mesangial cells have been characterized in vitro by using histone H1 or endogenous proteins as substrates. Histones H1-phosphorylation was significantly increased only when calcium, phosphatidylserine (PS) and 1,2-diacylglycerol (DAG) or phorbol myristate acetate (PMA) were present together in the incubation medium. EGTA, a calcium chelator, completely inhibited this activity.

Epidermal growth factor and phorbol ester actions on the TSH induced down regulation of the isoenzyme I (PKA I) of cyclic AMP-dependent protein kinases in dog thyroid cell primary cultures.

In dog thyroid cell primary cultures the prolonged presence (up to 4-6 days) of TSH induced down regulation of the isoenzyme I (PKA I) of cAMP-dependent protein kinases. In the simultaneous presence of TSH and EGF this down regulation of PKA I was maintained, although it was slightly smaller than in assays without EGF. In contrast, the simultaneous presence of TPA, totally inhibited the TSH induced down regulation of PKA I.

Changes in cAMP-dependent and Ca2(+)-phospholipid-dependent protein kinase activities in suspension cultures of porcine thyroid cells.

The morphological and functional characteristics and the activities of cyclic AMP- (PKA I and PKA II) and calcium and phospholipid-dependent (PKC) protein kinases were studied in 2-day-old suspension cultures of porcine thyroid cells and were compared with those in freshly dissociated cells and intact glands. Thyroid cell morphology changed during the 2-day culture in the absence of specific regulators. This is characterized by a loss of cellular polarity, exo- and endocytotic vesicles and membranes of the rough endoplasmic reticulum, and an increase in the number of lysosomes, pseudomyelinic structures, lipidic inclusions and free ribosomes.

[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").

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).

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.

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.

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.

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.

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.

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.

Modification of protein kinase pattern in human placentae during gestation.

The cAMP-dependent and cAMP-independent histone kinases have been studied in the two subcellular compartments (cytosol and particulate fraction) from placentae of different gestational age. The total protein kinase activity, as well as its distribution between the two compartments, changes during the period of gestation. The total activity is significantly increased in full-term placentae.

Protein kinase patterns in hyperplastic rat thyroids and in human non-toxic nodular goitres.

Patterns of cAMP-dependent protein kinases and cAMP-independent histone and casein kinases of hyperplastic rat thyroid glands and of human nodular non-toxic goitres have been analysed in two subcellular compartments, cytosol and particulate fraction. In hyperplastic rat glands the different compartmentalization of the two cAMP-dependent isoenzymes was preserved and the pattern of cAMP-independent protein kinases was not changed qualitatively, but the activities of the three classes of protein kinases were enhanced to different degrees: the highest increase was observed for the cAMP-dependent enzymes and the lowest for the cAMP-independent casein kinases. Analysis of individual peaks of cAMP-dependent kinases showed selective stimulation of the cytosolic Type II form and independent control of the Type I activity in the two subcellular compartments.

Some characteristics and hormonal control of thyroidal cAMP-independent protein kinases.

Cyclic AMP-independent protein kinases in cytosol from rat thyroid glands were evaluated using histone and casein as exogenous substrates. Compared with other rat tissues, thyroid gland is rich in histone kinases, while its casein kinase activity is lower than that of liver and brain. Thyroidal cAMP-independent protein kinases can be resolved by sucrose gradient ultracentrifugation into two distinct peaks of histone (HKi-1 and HKi-2) and two peaks of casein (CKi-1 and CKi-2) kinases.

Hormonal regulation of thyroidal protein phosphokinase activities. II. Differential sensitivity of type I and type II cAMP-dependent enzymes to the treatment of rats with thyroxine.

Two cAMP-dependent protein kinase activities can be separated from rat thyroid cytosol. Their elution properties during the gel chromatography, as well as their sedimentation coefficients after sucrose-gradient ultracentrifugation and their capacity to dissociate in the presence of histones, suggest they are like the Type I and Type II protein kinases described in many other tissues. The sensitivity of the two types of kinases to thyroxine treatment is different.

Hormonal regulation of cAMP-independent protein phosphokinase activities: thyroxine and cortisol control of enzymes from rat liver cytosol.

Thyroxine control of cAMP-independent histone and casein phosphokinase activities was studied in thyroidectomized rats treated with thyroxine. All activities were evaluated in the presence of a thermostable inhibitor of cAMP-dependent enzymes. Cytosol enzymes can be resolved by sucrose gradient ultracentrifugation into three peaks of histone kinase activity (3.