The B''/PR72 subunit mediates Ca2+-dependent dephosphorylation of DARPP-32 by protein phosphatase 2A.
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 Ca(2+)-dependent protein phosphatase, PP2B (calcineurin), whereas dephosphorylation of Thr-75 involves primarily PP2A, the latter being subject to control by both cAMP- and Ca(2+)-dependent regulatory mechanisms. In the present study, we have investigated the mechanism of Ca(2+)-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 Ca(2+)-dependent dephosphorylation at Thr-75 of DARPP-32. The PR72 subunit contains two Ca(2+) 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 Ca(2+), 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 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 Ca(2+) can activate serine/threonine phosphatase activity.
Ahn
Jung-Hyuck
JH
Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, NY 10021, USA.
Sung
Jee Young
JY
McAvoy
Thomas
T
Nishi
Akinori
A
Janssens
Veerle
V
Goris
Jozef
J
Greengard
Paul
P
Nairn
Angus C
AC
D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D051937:Dopamine and cAMP-Regulated Phosphoprotein 32 / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D009474:Neurons / Q000378:metabolism*; D009841:Oligonucleotides; D010766:Phosphorylation; D054648:Protein Phosphatase 2 / Q000378:metabolism*; D021122:Protein Subunits / Q000378:metabolism; D034622:RNA Interference
D051937:Dopamine and cAMP-Regulated Phosphoprotein 32; D009841:Oligonucleotides; C497928:PPP1R1B protein, human; D021122:Protein Subunits; D054648:Protein Phosphatase 2; D002118:Calcium