Proceedings of the National Academy of Sciences of the United States of America vol:104 issue:23 pages:9876-9881
In dopaminoceptive neurons, dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32) plays a central role in integrating the effects of dopamine and other neurotransmitters. Phosphorylation of DARPP-32 at Thr-34 by protein kinase A results in inhibition of protein phosphatase 1 (PP1), and phosphorylation at Thr-75 by Cdk5 (cyclin-dependent kinase 5) results in inhibition of protein kinase A. Dephosphorylation at Thr-34 involves primarily the Ca2+-dependent protein phosphatase, PP2B (calcineurin), whereas dephosphorylation of Thr-75 involves primarily PP2A, the latter being subject to control by both cAMP- and Ca2+-dependent regulatory mechanisms. In the present study, we have investigated the mechanism of Ca2+-dependent regulation of Thr-75 by PP2A. We show that the PR72 (or B" or PPP2R3A) regulatory subunit of PP2A is highly expressed in striatum. Through the use of overexpression and down-regulation by using RNAi, we show that PP2A, in a heterotrimeric complex with the PR72 subunit, mediates Ca2+-dependent dephosphorylation at Thr-75 of DARPP-32. The PR72 subunit contains two Ca2+ binding sites formed by E and F helices (EF-hands 1 and 2), and we show that the former is necessary for the ability of PP2A activity to be regulated by Ca2+, both in vitro and in vivo. Our studies also indicate that the PP72-containing form of MA is necessary for the ability of glutamate acting at alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid and NMDA receptors to regulate Thr-75 dephosphorylation. These studies further our understanding of the complex signal transduction pathways that regulate DARPP-32. In addition, our studies reveal an alternative intracellular mechanism whereby Ca2+ can activate serine/threonine phosphatase activity.