Inhibitions of protein kinases and protein phosphatases have opposite effects on thyrotropin-stimulated cAMP accumulation in human thyroid cells
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T Métayé1, D Sarrouilhe, J L Kraimps
1Laboratoire de Biophysique, Hôpital Jean Bernard, Poitiers, France.
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.
Insights
Protein phosphatases and kinases influence thyroid cell responses to TSH. Inhibiting phosphatases showed a dual effect on cAMP, while kinase inhibitors altered TSH-stimulated cAMP levels, suggesting phosphorylation cycles regulate thyroid activity.
Area of Science:
- Endocrinology
- Molecular Biology
- Cell Signaling
Background:
- Thyrotropin (TSH) regulates thyroid function through cAMP signaling.
- Protein phosphorylation and dephosphorylation are key cellular regulatory mechanisms.
Purpose of the Study:
- To investigate the role of protein phosphatases and protein kinases in TSH-stimulated cAMP accumulation in human thyroid cells.
- To elucidate the involvement of specific signaling pathways in thyroid hormone regulation.
Main Methods:
- Utilized specific inhibitors: Okadaic acid (OA) and calyculin-A (CL-A) for protein phosphatases (PP-1, PP-2A).
- Employed H-7 and KT5926 to inhibit protein kinases (PKC, CaMKII, MLCK).
- Measured cAMP levels and 32P incorporation in human thyroid cells under various treatment conditions.
Main Results:
- Protein phosphatase inhibition by OA and CL-A exhibited a biphasic concentration-dependent effect on cAMP production.
- Purified PP-1 and PP-2A reversed OA-induced adenylyl cyclase inhibition, confirming their regulatory role.
- PKC inhibition (H-7) significantly potentiated TSH-stimulated cAMP accumulation, while CaMKII/MLCK inhibition (KT5926) affected basal cAMP levels.
Conclusions:
- Protein phosphorylation/dephosphorylation cycles play a crucial role in modulating both basal and TSH-stimulated adenylyl cyclase activity in human thyroid cells.
- These findings highlight a complex regulatory network involving phosphatases and kinases in thyroid cell signaling.