Molecular mechanisms to amplify dopamine signaling in neostriatal neurons

DARPP-32（多巴胺和环腺苷酸调节磷酸蛋白，Mr 32 kDa）在多巴胺的大多数作用中起着强制性作用。DARPP-32通过cAMP依赖性蛋白激酶（PKA）在Thr 34上磷酸化，导致其转化为蛋白磷酸酶-1（PP-i）的有效抑制剂。当DARPP-32在Thr 75上被cdk 5磷酸化时，它成为PKA的抑制剂，从而调节多巴胺能信号传导（Bibb et al.，Nature 402：669-671，1999）。在这项研究中，我们研究了多巴胺对DARPP-32在Thr 75磷酸化的调节。使用mOuse新纹状体切片，SKF 8 1297，一种D1激动剂，通过PKA机制降低磷酸化Thr 75 DARPP-32的水平。D2激动剂Quinpirole可增加纹状体组织匀浆中磷酸化Thr 75 DARPP-32的水平。我们发现，在纹状体匀浆中，PP-2A在磷酸化Thr 75 DARPP-32的去磷酸化过程中起重要作用。PKA对磷酸化Thr 75 DARPP-32的去磷酸化作用可能与抑制cdk 5活性或刺激PP-2A活性有关。毛喉素可降低磷酸化Thr 75 DARPP-32的水平。毛喉素的这种作用在一定浓度的冈田酸（1μM）存在下被消除，冈田酸选择性抑制PP-2A，但在存在roscovitine（一种cdk 5的选择性抑制剂）的情况下则不然。此外，用毛喉素处理切片并不改变随后在匀浆中测量的cdk 5活性。这些数据表明PKA可能通过涉及PKA 2A活化的机制使磷酸化Thr 75 DARPP-32去磷酸化。总之，这些结果表明，通过正反馈机制Cdk 5信号和PKA信号是相互拮抗的。

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.