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. In cells cultured for two days in the presence of TSH (0.1 mU/ml), the overall PKC activity was stimulated (ca. 200%) in the two compartments. This stimulation was parallel to the increase in protein expression of the three PKC isoforms (as demonstrated by immunoblot analysis) and was accompanied by a redistribution of cPKC alpha and nPKC epsilon toward the particulate fraction. In TSH-treated cells, hydroxyapatite chromatography of cytosolic PKC revealed an additional peak of PKC activity eluted at 195 mM potassium phosphate. Its elution molarity did not correspond to the molarity of any known PKC isozyme, and it did not cross-react with antibodies directed against cPKC isozymes--: alpha, beta, or gamma. When TSH was replaced by forskolin (10(-5) M), identical quantitative and qualitative modifications were obtained, suggesting that, in thyroid cells, the cyclic AMP-dependent regulatory cascade could be involved in the control of PKC isoforms expression by TSH.
Féliers
D
D
INSERM U96, Le Kremlin-Bicêtre, France.
Dang
P M
PM
Haye
B
B
Pavlovic-Hournac
M
M
D000818:Animals; D002478:Cells, Cultured; D002852:Chromatography, Ion Exchange; D005576:Colforsin / Q000494:pharmacology*; D000242:Cyclic AMP / Q000378:metabolism; D015151:Immunoblotting; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D018716:Molecular Mimicry; D011493:Protein Kinase C / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D013552:Swine; D013961:Thyroid Gland / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D013972:Thyrotropin / Q000494:pharmacology*
D007527:Isoenzymes; D005576:Colforsin; D013972:Thyrotropin; D000242:Cyclic AMP; D011493:Protein Kinase C