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.
Breton
M F
MF
INSERM U.96, Unité de Recherche sur la Glande Thyroide et la Régulation Hormonale, Kremlin-Bicêtre, France.
Haye
B
B
Omri
B
B
Jacquemin
C
C
Pavlovic-Hournac
M
M
D000818:Animals; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology*; D000242:Cyclic AMP / Q000378:metabolism; D007700:Kinetics; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D013552:Swine; D013961:Thyroid Gland / Q000187:drug effects / Q000201:enzymology*; D013972:Thyrotropin / Q000494:pharmacology*
D005576:Colforsin; D013972:Thyrotropin; D000242:Cyclic AMP; D011494:Protein Kinases