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

Abstract

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.

Fig. 1.

PP2A containing the PR72 subunit selectively regulates dephosphorylation of Thr-75 of DARPP-32. (a) Protein (50 μg per lane) from frontal cortex, striatum, and hippocampus of 7- to 8-week-old Sprague–Dawley rats, or from N2a cells, was separated by SDS/PAGE (4–20% acrylamide), and PR72 expression was measured by immunoblotting using a polyclonal anti-PR72 antibody. (b) N2a cells were transfected with Myc-tagged DARPP-32 without (vector) or with PR72 or B56δ subunit. Cells were treated without (−) or with (+) ionomycin (5 μM) for 10 min. (Upper) Phosphorylation at Thr-34 or Thr-75 of DARPP-32 was analyzed by immunoblotting using phosphospecific antibodies corresponding to each site. Total DARPP-32 expression was analyzed by using anti-Myc antibody. (Lower) Phosphorylation levels of Thr-34 (Left) and Thr-75 (Right) were normalized to values obtained for untreated cells expressing only DARPP-32. Data represent means ± SEM (n = 3). ∗, P < 0.001 compared with vehicle-treated vector control by two-way ANOVA with Bonferroni's posttest.

Fig. 2.

Ca²⁺-dependent regulation of PR72-containing PP2A in cortical neurons. Rat cortical neurons were cotransfected with Myc-DARPP-32 and either vector, PR72, B56δ, or Bα subunit by electroporation. Cells were pretreated without or with BAPTA-AM (50 μM) for 30 min as indicated and then treated with DMSO vehicle, AMPA (50 μM) plus NMDA (100 μM), or ionomycin (10 μM) for an additional 5 min. (Upper) The phosphorylation at Thr-75 of DARPP-32 was analyzed by immunoblotting using a phosphospecific antibody. Total DARPP-32 expression was analyzed by using anti-Myc antibody. (Lower) Phosphorylation levels of Thr-75 were normalized to values obtained for untreated cells in each transfection set. Data represent means ± SEM (n = 3). ∗, P < 0.001 compared with vehicle-treated control by two-way ANOVA with Bonferroni's posttest.

Fig. 3.

AMPA- and NMDA-mediated dephosphorylation at Thr-75 of DARPP-32 requires the PR72 subunit. (a) N2a cells were transfected with vector, FLAG-PR72 or FLAG-B56δ, and a pAAV-H1/PR72 RNAi construct as indicated. Expression levels of PR72 or B56δ were measured by immunoblotting using anti-Flag antibody. Note that PR72 is slightly smaller than B56δ (529 vs. 602 aa excluding the Flag tag), but the difference was hardly visible using SDS/PAGE (4–20% acrylamide). (b) Rat hippocampal neurons were infected with AAV containing myc-tagged DARPP-32 in the absence (no RNAi) or after coinfection with control AAV (control) or AAV containing PR72 RNAi (PR72 RNAi) (for coinfection, viruses were used at a 1:1 pfu ratio). After 5 days of infection, cells were treated without or with AMPA (50 μM), NMDA (100 μM), or forskolin (10 μM) for 5 min. The phosphorylation at Thr-34 or Thr-75 of DARPP-32 was analyzed by immunoblotting using phosphospecific antibodies corresponding to each site. PR72 and DARPP-32 immunoblots were performed with anti-PR72 antibody and anti-myc antibody, respectively. The phosphorylation levels of Thr-34 (Upper) and Thr-75 (Lower) were normalized to values obtained for untreated cells. Data represent means ± SEM (n = 3). ∗, P < 0.001; ∗∗, P < 0.01 compared with vehicle-treated control by two-way ANOVA with Bonferroni's posttest.

Fig. 4.

The EF-hands of PR72 are required for Ca²⁺-dependent regulation of PP2A. (a) Amino acid sequences of EF-hands 1 and 2 in human PR72. The EF-hands were mutated by changing the underlined aspartate and glutamate residues to alanine. (b) N2a cells were transfected with vector, or wild-type or mutant FLAG-tagged PR72 subunit (as indicated). Cells were lysed and immunoprecipitated with agarose-conjugated anti-Flag antibody. Immune complexes were analyzed by SDS/PAGE (4–20% acrylamide) and immunoblotting. PP2A C subunit was detected by using anti-PP2A C antibody, and PR72 subunits were detected with anti-PR72 antibody. (c) Cortical neurons were cotransfected with Myc-DARPP-32 and either control vector, or wild-type (Wt) or mutant PR72, as indicated. After 5 days, cells were treated without or with AMPA (50 μM) plus NMDA (100 μM) for 5 min. The phosphorylation at Thr-34 or Thr-75 of DARPP-32 was analyzed by immunoblotting using phosphospecific antibodies corresponding to each site. Total DARPP-32 expression was analyzed by using anti-Myc antibody. Phosphorylation levels of Thr-34 (Upper) and Thr-75 (Lower) were normalized to values obtained for vector-transfected, untreated cells. Data represent means ± SEM (n = 3). ∗, P < 0.001 compared with vehicle-treated vector control by two-way ANOVA with Bonferroni's posttest.

Fig. 5.

In vitro analysis of Ca²⁺-dependent regulation of PP2A containing the PR72 subunit. HEK293 cells were transfected with either wild-type PR72 or PR72 in which the EF1-hand was mutated. Cells were lysed and wild-type (Wt) or mutant PR72 (EF1 mt) was immunoprecipitated with agarose-conjugated anti-Flag antibody (Inset shows immunoblot showing approximately equal amounts of C subunit in the immunoprecipitated samples). PP2A immune complexes were incubated with each substrate [³²P-Thr-75 DARPP-32 (Left), ³²P-Histone H1 (Center), nonradioactive phosphopeptide (P-peptide) (Right)] in the absence or presence of various concentrations of Ca²⁺ as indicated. Phosphatase activity was normalized to that measured in the absence of Ca²⁺. Data represent means ± SEM (n = 3). ∗, P < 0.001; ∗∗, P < 0.01 compared with zero-calcium by two-way ANOVA with Bonferroni's posttest.