Molecular mechanisms to amplify dopamine signaling in neostriatal neurons

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DARPP-32 (Dopamine and cyclic AMP-Regulated Phospho-Protein, Mr 32 kDa) plays an obligatory role in most of the actions of dopamine. DARPP-32 is phosphorylated on Thr34 by cAMP-dependent protein kinase (PKA), resulting in its conversion into a potent inhibitor of protein phosphatase-1 (PP-i). When DARPP-32 is phosphorylated on Thr75 by cdk5, it becomes an inhibitor of PKA and thereby modulates dopaminergic signaling (Bibb et al., Nature 402 : 669-671, 1999). In this study, we investigated the regulation of DARPP-32 phosphorylation at Thr75 by dopamine. Using mOuse neostriatal slices, SKF8 1297, a D1 agonist, decreased the level of phospho-Thr75 DARPP-32 through a echanism involving PKA. Quinpirole, a D2 agonist, increased the level of phospho-Thr75 DARPP-32.We have found, in striatal homogenates, that PP-2A plays a prominent role in the dephosphorylation of phospho-Thr75 DARPP-32.The ability of PKA to dephosphorylate phospho-Thr75 DARPP-32 might be explained either by an inhibition of cdk5 activity or by a stimulation of PP-2A activity. Forskolin decreased the level of phospho-Thr75 DARPP-32.This effect of forskolin was abolished in the presence of concentration of okadaic acid (1μM), which selectively inhibited PP-2A, but not in the presence of roscovitine, a selective inhibitor of cdk5.In addition, treatment of slices with forskolin did not alter cdk5 activities measured subsequently in homogenate. These data demonstrate that PKA dephosphorylates phospho-Thr75 DARPP-32, possibly by a mechanism involving the activation of PKA2A. Together, these results indicate that via positive feedback mechanisms Cdk5 signaling and PKA signaling are mutually antagonistic.