Fisone G, Snyder G L, Aperia A, Greengard P
Laboratory of Molecular and Cellular Neuroscience, Rockefeller University, New York, New York, USA.
Mol Med. 1998 Apr;4(4):258-65.
The ion pump Na+,K(+)-ATPase is responsible for the secretion of cerebrospinal fluid from the choroid plexus. In this tissue, the activity of Na+,K(+)-ATPase is inhibited by serotonin via stimulation of protein kinase C-catalyzed phosphorylation. The choroid plexus is highly enriched in two phosphoproteins which act as regulators of protein phosphatase-1 activity, DARPP-32 and inhibitor-1. Phosphorylation catalyzed by cAMP-dependent protein kinase on a single threonyl residue converts DARPP-32 and inhibitor-1 into potent inhibitors of protein phosphatase-1. Previous work has shown that in the choroid plexus, phosphorylation of DARPP-32 and I-1 is enhanced by isoproterenol and other agents that activate cAMP-PK. We have now examined the possible involvement of the cAMP-PK/protein phosphatase-1 pathway in the regulation of Na+,K(+)-ATPase.
The state of phosphorylation of Na+,K(+)-ATPase was measured by determining the amount of radioactivity incorporated into the ion pump following immunoprecipitation from 32P-prelabeled choroid plexuses incubated with various drugs (see below). Two-dimensional phosphopeptide mapping was employed to identify the protein kinase involved in the phosphorylation of Na+,K(+)-ATPase.
The serotonin-mediated increase in Na+,K(+)-ATPase phosphorylation is potentiated by okadaic acid, an inhibitor of protein phosphatases-1 and -2A, as well as by forskolin or the beta-adrenergic agonist, isoproterenol, activators of cAMP-dependent protein kinase. Two-dimensional phosphopeptide maps suggest that this potentiating action occurs at the level of a protein kinase C phosphorylation site. Forskolin and isoproterenol also stimulate the phosphorylation of DARPP-32 and protein phosphatase inhibitor-1, which in their phosphorylated form are potent inhibitors of protein phosphatase-1.
The results presented here support a model in which okadaic acid, forskolin, and isoproterenol achieve their synergistic effects with serotonin through phosphorylation of DARPP-32 and inhibitor-1, inhibition of protein phosphatase-1, and a reduction of dephosphorylation of Na+,K(+)-ATPase at a protein kinase C phosphorylation site.
离子泵Na +，K（+）-ATP酶负责脉络丛脑脊液的分泌。在该组织中，5-羟色胺通过刺激蛋白激酶C催化的磷酸化作用抑制Na +，K（+）-ATP酶的活性。脉络丛中富含两种作为蛋白磷酸酶-1活性调节剂的磷蛋白，即多巴胺和腺苷酸环化酶调节磷酸蛋白（DARPP-32）和抑制剂-1。由环磷酸腺苷（cAMP）依赖性蛋白激酶催化的单个苏氨酰残基上的磷酸化作用将DARPP-32和抑制剂-1转化为蛋白磷酸酶-1的有效抑制剂。先前的研究表明，在脉络丛中，异丙肾上腺素和其他激活cAMP-PK的药物可增强DARPP-32和I-1的磷酸化作用。我们现在研究了cAMP-PK /蛋白磷酸酶-1途径在Na +，K（+）-ATP酶调节中的可能作用。
通过测定在用各种药物孵育的32P预标记脉络丛中进行免疫沉淀后掺入离子泵的放射性量，来测量Na +，K（+）-ATP酶的磷酸化状态（见下文）。采用二维磷酸肽图谱来鉴定参与Na +，K（+）-ATP酶磷酸化的蛋白激酶。
蛋白磷酸酶-1和-2A的抑制剂冈田酸以及cAMP依赖性蛋白激酶的激活剂福斯高林或β-肾上腺素能激动剂异丙肾上腺素可增强5-羟色胺介导的Na +，K（+）-ATP酶磷酸化增加。二维磷酸肽图谱表明，这种增强作用发生在蛋白激酶C磷酸化位点水平。福斯高林和异丙肾上腺素还刺激DARPP-32和蛋白磷酸酶抑制剂-1的磷酸化，它们的磷酸化形式是蛋白磷酸酶-1的有效抑制剂。
此处给出的结果支持一种模型，其中冈田酸、福斯高林和异丙肾上腺素通过DARPP-32和抑制剂-1的磷酸化、蛋白磷酸酶-1的抑制以及蛋白激酶C磷酸化位点处Na +，K（+）-ATP酶去磷酸化的减少，与5-羟色胺产生协同作用。
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