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 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 microM), a calcineurin inhibitor, maximally increased the level of phosphorylated DARPP-32 by 17+/-2-fold. Okadaic acid (1 microM), 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.
Nishi
A
A
Department of Physiology, Kurume University School of Medicine, Fukuoka, Japan.
Snyder
G L
GL
Nairn
A C
AC
Greengard
P
P
D000818:Animals; D019703:Calcineurin / Q000502:physiology*; D065095:Calcineurin Inhibitors; D016572:Cyclosporine / Q000494:pharmacology; D051937:Dopamine and cAMP-Regulated Phosphoprotein 32; D004338:Drug Combinations; D004357:Drug Synergism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D066298:In Vitro Techniques; D008297:Male; D008387:Marine Toxins; D051379:Mice; D008810:Mice, Inbred C57BL; D017072:Neostriatum / Q000166:cytology / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism*; D019319:Okadaic Acid / Q000494:pharmacology; D010080:Oxazoles / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000502:physiology*; D010750:Phosphoproteins; D010766:Phosphorylation; D054645:Protein Phosphatase 1; D054648:Protein Phosphatase 2
D065095:Calcineurin Inhibitors; D051937:Dopamine and cAMP-Regulated Phosphoprotein 32; D004338:Drug Combinations; D004791:Enzyme Inhibitors; D008387:Marine Toxins; D009419:Nerve Tissue Proteins; D010080:Oxazoles; D010750:Phosphoproteins; D019319:Okadaic Acid; C059041:calyculin A; D016572:Cyclosporine; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D054648:Protein Phosphatase 2