Amplification of dopaminergic signaling by a positive feedback loop

Abstract

Dopamine and cAMP-regulated phosphoprotein of M(r) 32,000 (DARPP-32) plays an obligatory role in most of the actions of dopamine. In resting neostriatal slices, cyclin-dependent kinase 5 (Cdk5) phosphorylates DARPP-32 at Thr-75, thereby reducing the efficacy of dopaminergic signaling. We report here that dopamine, in slices, and acute cocaine, in whole animals, decreases the state of phosphorylation of striatal DARPP-32 at Thr-75 and thereby removes this inhibitory constraint. This effect of dopamine is achieved through dopamine D1 receptor-mediated activation of cAMP-dependent protein kinase (PKA). The activated PKA, by decreasing the state of phosphorylation of DARPP-32-Thr-75, de-inhibits itself. Dopamine D2 receptor stimulation has the opposite effect. The ability of activated PKA to reduce the state of phosphorylation of DARPP-32-Thr-75 is apparently attributable to increased protein phosphatase-2A activity, with Cdk5 being unaffected. Together, these results indicate that via positive feedback mechanisms, Cdk5 signaling and PKA signaling are mutually antagonistic.

Figure 1

Regulation of DARPP-32 phosphorylation at Thr-34 and Thr-75 by dopamine via D1- and D2-type receptors in neostriatum. (a) Effect of dopamine on the level of phospho-Thr-34 and phospho-Thr-75 DARPP-32. Slices were incubated with dopamine (100 μM) in the presence of a dopamine uptake inhibitor, nomifensine (10 μM), for the indicated times. Immunoblots are shown (Upper) for the detection of phospho-Thr-34 (Left) and phospho-Thr-75 (Right), using phosphorylation state-specific antibodies and the same nitrocellulose membrane. Quantitative results, normalized to values obtained with untreated slices, are shown below each blot. A.U., arbitrary units. Data represent means ± SEM for four experiments. **, P < 0.01 compared with 0 min; †, P < 0.05 compared with 6 min, Student's t test. (b) Regulation of DARPP-32 phosphorylation at Thr-34 (Left) and Thr-75 (Right) by dopamine D1 and D2 receptor agonists and antagonists. Data are shown for untreated neostriatal slices (Con) and slices treated with a D1 agonist, SKF81297 (SKF) (1 μM); a D1 antagonist, SCH23390 (SCH) (1 μM); a D2 agonist, quinpirole (Quin) (1 μM); and a D2 antagonist, raclopride (Rac) (1 μM), as indicated. Data represent means ± SEM for 5–15 experiments. *, P < 0.05, **, P < 0.01 compared with control; ††, P < 0.01 compared with SKF81297 alone; §§, P < 0.01 compared with quinpirole alone, Student's t test. (c) Effect of SCH23390 and raclopride on dopamine regulation of DARPP-32 phosphorylation on Thr-34 (Left) and Thr-75 (Right). Data are shown for treatment of neostriatal slices with dopamine (100 μM), SCH23390 (1 μM), and raclopride (1 μM), as indicated. Data represent means ± SEM for five experiments. **, P < 0.01 compared with control; ††, P < 0.01 compared with dopamine alone, Student's t test.

Figure 2

Regulation by acute cocaine of DARPP-32 phosphorylation at Thr-34 and Thr-75. Data are shown for mice treated with saline (Sal) or cocaine (Coc). (a) Representative immunoblots for detection of each phosphorylation site and for total DARPP-32 are shown. (b and c) The amounts of phospho-Thr-34 DARPP-32 (b) and phospho-Thr-75 DARPP-32 (c) were quantified by densitometry. Data represent means ± SEM for five to six mice per group. *, P < 0.05 compared with saline-injected mice, Student's t test.

Figure 3

Effect of PKA activation on DARPP-32 phosphorylation at Thr-34 and Thr-75 in neostriatum. (a) Immunoblots showing the levels of phospho-Thr-34, phospho-Thr-75, and total DARPP-32 in response to treatment with 10 μM forskolin (FSK), 10 μM 1,9-dideoxyforskolin (dFSK) (an inactive analog of forskolin), or 5 mM 8-bromo-cAMP (cAMP) for 5 min, detected on the same nitrocellulose membrane. (b and c) The amounts of phospho-Thr-34 DARPP-32 (b) and phospho-Thr-75 DARPP-32 (c) were quantified by densitometry, and the data were normalized to values obtained with untreated slices. Data represent means ± SEM for 3–10 experiments. *, P < 0.05; **, P < 0.01 compared with control, Student's t test.

Figure 4

PKA activation reduces phospho-Thr-75 DARPP-32 even when Cdk5 activity is inhibited. (a) Slices were incubated in the absence or presence of roscovitine (Rosc) (10 μM or 50 μM) for 60 min, followed by the addition of forskolin (FSK) (2 μM) for 5 min. The amounts of phospho-Thr-75 DARPP-32 were quantified by densitometry, and the data were normalized to the values obtained with untreated slices. Data represent means ± SEM for 4–15 experiments. **, P < 0.01 compared with no addition; †, P < 0.05, ††, P < 0.01 compared with forskolin alone; §§, P < 0.01 compared with roscovitine (10 μM) alone; ¶, P < 0.05 compared with roscovitine (50 μM) alone, Student's t test. (b) Homogenates of neostriatal slices treated with forskolin at the indicated concentrations were subjected to immunoprecipitation (IP) with either a Cdk5 antibody (C-8) or a normal rabbit IgG. The immunoprecipitates, containing Cdk5 and its cofactor p35, were used to phosphorylate histone H1 in the absence or presence of roscovitine. Results similar to those shown here were obtained in two other experiments.

Figure 5

Dephosphorylation of phospho-Thr-75 DARPP-32 in vitro (a) and in intact neostriatal neurons (b). (a) In vitro dephosphorylation of phospho-Thr-75 DARPP-32 by phosphatases in neostriatal homogenates in the presence of thio-phospho-Thr-34 DARPP-32 (P-S-D32, 0.1 μM) (a highly selective inhibitor of PP-1), 2 nM okadaic acid (OKA) (predominantly an inhibitor of PP-2A), 1 μM okadaic acid (an inhibitor of both PP-1 and PP2A), Ca²⁺/calmodulin (CaM) (an activator of PP-2B), and Mg²⁺ (an activator of PP-2C). Data represent means ± SEM for four to six experiments. **, P < 0.01 compared with control, Student's t test. (b) Effects of phosphatase inhibition and PKA activation on phospho-Thr-75 DARPP-32 levels. Neostriatal slices were incubated in the presence of the indicated concentrations of okadaic acid or 10 μM cyclosporin A (CyA) for 60 min, followed by 5 min in the absence (closed bars) or presence (open bars) of 10 μM forskolin. The amount of phospho-Thr-75 DARPP-32 was quantified by densitometry, and the data were normalized to the values obtained with untreated slices. Okadaic acid, but not cyclosporin A, abolished the ability of forskolin to reduce the level of phospho-Thr-75. Data represent means ± SEM for four to five experiments. **, P < 0.01 compared with no addition; ††, P < 0.01 compared with okadaic acid (200 nM) alone; §§, P < 0.01 compared with cyclosporin A alone, Student's t test.

Figure 6

Model illustrating the regulation of dopamine signaling by Cdk5/phospho-Thr-75 DARPP-32 and regulation of the Cdk5/phospho-Thr-75 DARPP-32 pathway by dopamine. The Cdk5 signaling and PKA signaling pathways, through a positive feedback loop, are mutually antagonistic. (Left) Under basal conditions, phosphorylation of DARPP-32 at Thr-75 by Cdk5 causes, successively, inhibition of PKA, inactivation of PP-2A, and decreased dephosphorylation of phospho-Thr-75 DARPP-32. Inhibition of PKA also results in decreased phosphorylation of DARPP-32-Thr-34 and, therefore, in activation of PP-1. Inhibition of PKA and activation of PP-1 synergistically reduce the phosphorylation of various substrates. (Right) Dopamine, by sequentially activating dopamine D1 receptors, PKA and PP-2A, reduces the level of phospho-Thr-75 DARPP-32. Dephosphorylation of DARPP-32 at Thr-75 by PP-2A removes the inhibition of PKA. Activation of PKA also results in increased phosphorylation of DARPP-32-Thr-34 and in inhibition of PP-1. Activation of PKA and inhibition of PP-1 synergistically increase the phosphorylation of various substrates.