title: Inhibitions of protein kinases and protein phosphatases have opposite effects on thyrotropin-stimulated cAMP accumulation in human thyroid cells.
abstract: We investigated the effects of inhibitions of protein phosphatases and protein kinases on thyrotropin (TSH) stimulation of cAMP accumulation in human thyroid cells. Okadaic acid (OA) and calyculin-A (CL-A), two potent inhibitors of type-1 (PP-1) and type-2A (PP-2A) protein phosphatases, had a biphasic concentration-dependent response on cAMP formation. An inhibitory effect (41.3% and 47.2% inhibition with OA and CL-A) was first observed at 1 microM OA and 10 nM CL-A, followed by a reduction of this effect with OA (24% inhibition) or by a complete reversal of inhibition with CL-A, at 10-fold higher concentrations of both products. Addition of purified PP-1 and PP-2A to crude membranes from cells preincubated with OA, reversed OA-induced adenylyl cyclase inhibition, confirming that these protein phosphatases regulate TSH-mediated cAMP production. Levels of protein incorporation of 32P were higher with 10 microM OA than with 1 microM OA and did not correlate with the biphasic effect of OA on cAMP production. These results support a dual action of protein phosphorylation in the control of adenylyl cyclase activity stimulated by TSH. H-7, an inhibitor of nucleotide- and calcium/phospholipid-dependent protein kinase (PKC), increased by 197% the stimulation of cAMP accumulation by TSH in thyroid cells. Phorbol 12-myristate 13-acetate (PMA) counteracted the effect of H-7 on cAMP levels, which suggests that PKC is involved in the action of H-7. Moreover, KT5926, an inhibitor of calcium/calmodulin-dependent protein kinase II and myosin light chain kinase, increased basal cAMP levels rather than cAMP levels stimulated by TSH. In light of these results, we suggest that phosphorylation/dephosphorylation cycles regulate basal and TSH-stimulated adenylyl cyclase activities in human thyroid.
authors.affiliation: Laboratoire de Biophysique, Hôpital Jean Bernard, Poitiers, France.
authors.forename: T
authors.initials: T
authors.lastname: Métayé
authors.forename: D
authors.initials: D
authors.lastname: Sarrouilhe
authors.forename: J L
authors.initials: JL
authors.lastname: Kraimps
authors.forename: J
authors.initials: J
authors.lastname: Barbier
authors.forename: F
authors.initials: F
authors.lastname: Begon
mesh_terms: D002478:Cells, Cultured; D002772:Cholera Toxin / Q000494:pharmacology; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism*; D006801:Humans; D008387:Marine Toxins; D019319:Okadaic Acid / Q000494:pharmacology*; D010080:Oxazoles / Q000494:pharmacology*; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors*; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors; D013961:Thyroid Gland / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D013972:Thyrotropin / Q000494:pharmacology*; D013997:Time Factors
chemical_list: D008387:Marine Toxins; D010080:Oxazoles; D047428:Protein Kinase Inhibitors; D005576:Colforsin; D019319:Okadaic Acid; C059041:calyculin A; D013972:Thyrotropin; D002772:Cholera Toxin; D000242:Cyclic AMP; D010749:Phosphoprotein Phosphatases