B''/PR72 subunit mediates Ca²⁺-dependent dephosphorylation of DARPP-32 by protein phosphatase 2A

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 104; no. 23; pp. 9876 - 9881
• Main Authors: Ahn, Jung-Hyuck, Sung, Jee Young, McAvoy, Thomas, Nishi, Akinori, Janssens, Veerle, Goris, Jozef, Greengard, Paul, Nairn, Angus C
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 05.06.2007; National Acad Sciences
• Subjects: Antibodies; binding sites; Biological Sciences; calcium; Calcium - metabolism; Cell Line; cyclic AMP; cyclin-dependent kinase; dephosphorylation; dopamine; Dopamine and cAMP-Regulated Phosphoprotein 32 - metabolism; Down regulation; glutamic acid; HEK293 cells; Humans; Immunoblotting; Immunoprecipitation; N methyl D aspartate receptors; Neurons; Neurons - metabolism; neurotransmitters; Oligonucleotides; Phosphatases; phosphoproteins; Phosphorylation; Physiological regulation; Protein Phosphatase 2 - metabolism; Protein Subunits - metabolism; receptors; RNA Interference; serine; signal transduction; Substrate specificity; threonine
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.0703589104

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 PR72-containing form of PP2A is necessary for the ability of glutamate acting at α-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.