okadaic-acid and sapropterin

Extracts from rat corpus striatum, or striatal proteins resolved by chromatography on DE-52, were tested for protein phosphatase activity using tyrosine hydroxylase, phosphorylated by cAMP-dependent protein kinase, as substrate. The predominant dephosphorylating activity was independent of divalent cations and was inhibited by low concentrations (100 nM) of okadaic acid, defining the phosphatase as type 2A. Phosphatase type 2C (Mg2+ and Mn2+ stimulated) was evident in the presence of okadaic acid but at a level of approximately 10% of type 2A activity. Phosphatase 2B (Ca2+ and calmodulin dependent) mediated dephosphorylation of tyrosine hydroxylase was not apparent. The dephosphorylation of [32P]-tyrosine hydroxylase was not modulated by tetrahydrobiopterin, ATP, or GTP. These results indicate that tyrosine hydroxylase which has been phosphorylated by cAMP dependent protein kinase is dephosphorylated predominantly by phosphatase type 2A in brain, and the activity of this phosphatase is not modulated by pteridines or nucleotides.

Topics: Adenosine Triphosphate; Animals; Biopterins; Brain; Chromatography, Ion Exchange; Cyclic AMP-Dependent Protein Kinases; Dopamine; Ethers, Cyclic; Guanosine Triphosphate; Isoenzymes; Neostriatum; Okadaic Acid; PC12 Cells; Phosphoprotein Phosphatases; Phosphorylation; Rats; Tyrosine 3-Monooxygenase

Tetrahydrobiopterin (BH4), the obligatory cofactor of the aromatic amino acid hydroxylases, decreased the in situ 32P-phosphorylation of tyrosine hydroxylase (TH) in rat striatal synaptosomes. Incubation of pre-32P-labeled synaptosomes with BH4 in the presence of a permeant analogue of cAMP decreased the cAMP-stimulated level of 32P label incorporation into TH by about 50%, as determined by immunoprecipitation and autoradiography of SDS-polyacrylamide gels. The extent of inhibition mirrored changes in intrasynaptosomal BH4 levels and varied both as a function of BH4 concentration and length of incubation. A similar decrease in the amount of TH 32P-labeling was observed with the precursor of BH4, sepiapterin. This effect, in turn, was reversed by the inhibitor of sepiapterin reductase, N-acetyl-serotonin. Finally, exposure of pre-32P-labeled synaptosomes to the inhibitor of protein phosphatase 2A, okadaic acid, blocked the response to BH4. Collectively, the data suggest that BH4 stimulates the dephosphorylation of TH in situ and thus may play a dual role both as a cofactor for catalysis and a regulator of hydroxylase activity.

Topics: Animals; Biopterins; Bucladesine; Corpus Striatum; Ethers, Cyclic; Male; Okadaic Acid; Phosphoprotein Phosphatases; Phosphorus Radioisotopes; Phosphorylation; Protein Phosphatase 2; Pteridines; Pterins; Rats; Rats, Sprague-Dawley; Synaptosomes; Tyrosine 3-Monooxygenase