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cAMP counter-regulates insulin-mediated protein phosphatase-2A inactivation in rat skeletal muscle cells
1Diabetes Research Laboratory, Winthrop University Hospital, Mineola, New York 11501, USA. diabetes96@aol.com
Abstract:
In this study, we examined the mechanism of recently reported inactivation of protein phosphatase-2A (PP-2A) by insulin (Srinivasan, M., and Begum, N. (1994) J. Biol. Chem. 269, 12514-12520) and its counter-regulation by cAMP agonists. Exposure of L6 myotubes to insulin resulted in a rapid inhibition of PP-2A that was accompanied by a 3-fold increase in the phosphotyrosine content of the immunoprecipitated PP-2A catalytic subunit. Pretreatment with (Sp)-cAMP, a cAMP agonist, completely blocked insulin-mediated inhibition of PP-2A activity and decreased the tyrosine phosphorylation of PP-2A catalytic subunit to control levels. To understand the mechanism of counter-regulation of PP-2A by (Sp)-cAMP, cells were pretreated with sodium orthovanadate, an inhibitor of phosphotyrosine phosphatases. Vanadate prevented the effect of (Sp)-cAMP on PP-2A activity and increased the phosphorylation status of PP-2A catalytic subunit to the level observed with insulin. Wortmannin, a phosphatidylinositol 3-kinase inhibitor, and rapamycin, an inhibitor of 70-kDa S6 kinase activation, prevented insulin-mediated inactivation of PP-2A, suggesting that these pathways may participate in insulin-mediated phosphorylation and inactivation of PP-2A. These results show that insulin signaling results in a rapid inactivation of PP-2A by increased tyrosine phosphorylation and cAMP agonists counter-regulate insulin's effect on PP-2A by decreasing phosphorylation, presumably via an activated phosphatase.
Insights
Insulin rapidly inactivates protein phosphatase-2A (PP-2A) by increasing its tyrosine phosphorylation. Cyclic adenosine monophosphate (cAMP) agonists counteract this effect, decreasing PP-2A phosphorylation and restoring its activity.
Area of Science:
- Cellular signaling pathways
- Enzyme regulation
- Molecular biology
Background:
- Insulin signaling is crucial for glucose metabolism.
- Protein phosphatase-2A (PP-2A) plays a key role in dephosphorylation.
- Recent studies indicated insulin inactivates PP-2A.
Purpose of the Study:
- To investigate the mechanism of insulin-induced PP-2A inactivation.
- To explore the counter-regulation of PP-2A by cAMP agonists.
- To identify signaling pathways involved in PP-2A regulation.
Main Methods:
- Incubation of L6 myotubes with insulin and/or cAMP agonists.
- Measurement of PP-2A activity.
- Immunoprecipitation and Western blotting to assess tyrosine phosphorylation of PP-2A catalytic subunit.
- Use of inhibitors: sodium orthovanadate, wortmannin, and rapamycin.
Main Results:
- Insulin treatment rapidly inhibited PP-2A activity and increased its catalytic subunit's tyrosine phosphorylation.
- (Sp)-cAMP pretreatment blocked insulin's inhibitory effect and reduced tyrosine phosphorylation.
- Sodium orthovanadate mimicked insulin's effect on PP-2A phosphorylation when cells were pretreated with (Sp)-cAMP.
- Wortmannin and rapamycin inhibited insulin-mediated PP-2A inactivation.
Conclusions:
- Insulin signaling inactivates PP-2A through increased tyrosine phosphorylation.
- cAMP agonists counteract insulin's effect by decreasing PP-2A phosphorylation, likely via an activated phosphatase.
- Phosphatidylinositol 3-kinase and 70-kDa S6 kinase pathways are implicated in insulin-mediated PP-2A regulation.