Na+,K(+)-ATPase phosphorylation in the choroid plexus: synergistic regulation by serotonin/protein kinase C and isoproterenol/cAMP-PK/PP-1 pathways.
BACKGROUND
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Fisone
G
G
Laboratory of Molecular and Cellular Neuroscience, Rockefeller University, New York, New York, USA.
Snyder
G L
GL
Aperia
A
A
Greengard
P
P
D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000818:Animals; D002352:Carrier Proteins; D002831:Choroid Plexus / Q000201:enzymology*; D005576:Colforsin / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D051937:Dopamine and cAMP-Regulated Phosphoprotein 32; D004357:Drug Synergism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D007545:Isoproterenol / Q000494:pharmacology; D008297:Male; D009419:Nerve Tissue Proteins / Q000378:metabolism; D019319:Okadaic Acid / Q000494:pharmacology; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000032:analysis; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010750:Phosphoproteins; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D054645:Protein Phosphatase 1; D016601:RNA-Binding Proteins / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D012701:Serotonin / Q000494:pharmacology; D015398:Signal Transduction / Q000502:physiology; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*
D000318:Adrenergic beta-Agonists; D002352:Carrier Proteins; D051937:Dopamine and cAMP-Regulated Phosphoprotein 32; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D009419:Nerve Tissue Proteins; D010748:Phosphopeptides; D010750:Phosphoproteins; D016601:RNA-Binding Proteins; C021042:protein phosphatase inhibitor-1; D005576:Colforsin; D019319:Okadaic Acid; D012701:Serotonin; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D000254:Sodium-Potassium-Exchanging ATPase; D007545:Isoproterenol