Molecular mechanisms to amplify dopamine signaling in neostriatal neurons

DARPP-32 （Dopamine and cyclic AMP-Regulated Phospho-Protein, mr32 kDa）在多巴胺的大部分作用中起着必不可少的作用。DARPP-32在Thr34上被camp依赖性蛋白激酶（PKA）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Thr75上被cdk5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）。在本研究中，我们研究了多巴胺对DARPP-32 Thr75位点磷酸化的调控。通过小鼠新纹状体切片，D1激动剂SKF8 1297通过涉及PKA的机制降低了phospho-Thr75 DARPP-32的水平。D2激动剂喹匹罗可增加磷酸thr75 DARPP-32的水平。我们发现，在纹状体匀浆中，PP-2A在磷酸化thr75 DARPP-32的去磷酸化中起着突出的作用。PKA使phospho-Thr75 DARPP-32去磷酸化的能力可能是通过抑制cdk5活性或刺激PP-2A活性来解释的。Forskolin降低了phospho-Thr75 - DARPP-32的表达水平。在选择性抑制PP-2A的冈田酸浓度（1μM）存在时，福斯克林的这种作用被消除，但在cdk5的选择性抑制剂罗斯科维汀存在时则没有这种作用。此外，用福斯克林处理切片没有改变随后在匀浆中测量的cdk5活性。这些数据表明，PKA可能通过PKA2A激活的机制使phospho-Thr75 DARPP-32去磷酸化。总之，这些结果表明，通过正反馈机制，Cdk5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.