Role of calcineurin and protein phosphatase-2A in the regulation of DARPP-32 dephosphorylation in neostriatal neurons

DARPP-32, a dopamine- and cyclic AMP-regulated phosphoprotein of M-r 32 kDa, is phosphorylated on Thr(34) by cyclic AMP-dependent protein kinase, resulting in its conversion to a potent inhibitor of protein phosphatase-1 (PP-1), Conversely, Thr(34)-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 mu M), a calcineurin inhibitor, maximally increased the level of phosphorylated DARPP-32 by 17 +/- 2-fold. Okadaic acid (1 mu M), 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.