Role of Calcineurin and Protein Phosphatase‐2A in the Regulation of DARPP‐32 Dephosphorylation in Neostriatal Neurons

Abstract: DARPP‐32, a dopamine‐ and cyclic AMP‐regulated phosphoprotein of Mr 32 kDa, is phosphorylated on Thr34 by cyclic AMP‐dependent protein kinase, resulting in its conversion to a potent inhibitor of protein phosphatase‐1 (PP‐1). Conversely, Thr34‐phosphorylated DARPP‐32 is dephosphorylated and inactivated in vitro by calcineurin and protein phosphatase‐2A (PP‐2A). We have investigated the relative contributions of these protein phosphatases to the regulation of DARPP‐32 dephosphorylation in mouse neostriatal slices. Cyclosporin A (5 μM), a calcineurin inhibitor, maximally increased the level of phosphorylated DARPP‐32 by 17 ± 2‐fold. Okadaic acid (1 μM), an inhibitor of PP‐1 and PP‐2A, had a smaller effect, increasing phospho‐DARPP‐32 by 5.1 ± 1.3‐fold. The effect of okadaic acid on DARPP‐32 phosphorylation was shown to be due to inhibition of PP‐2A activity. Incubation of slices in the presence of cyclosporin A plus either okadaic acid or calyculin A, another PP‐1/PP‐2A inhibitor, caused a synergistic increase in the level of phosphorylated DARPP‐32. The use of Ca2+‐free/EGTA medium mimicked the effects of cyclosporin A on DARPP‐32 phosphorylation, supporting the conclusion that the action of cyclosporin on DARPP‐32 phosphorylation was attributable to blockade of the Ca2+‐dependent activation of calcineurin. The results indicate that calcineurin and PP‐2A, but not PP‐1, act synergistically to maintain a low level of phosphorylated DARPP‐32 in neostriatal slices.