Phenylalanine positively modulates the cAMP-dependent phosphorylation and negatively modulates the vasopressin-induced and okadaic-acid-induced phosphorylation of phenylalanine 4-monooxygenase in intact rat hepatocytes.
The state of phosphorylation of phenylalanine hydroxylase was determined in isolated intact rat hepatocytes. 32P-labeled phenylalanine hydroxylase was immunoisolated from cells loaded with 32Pi or from cell extracts 'back-phosphorylated' with [gamma-32P]ATP by cAMP-dependent protein kinase. The rate of phenylalanine hydroxylase phosphorylation in cells with elevated cAMP was similar to that observed for the isolated enzyme phosphorylated by homogeneous cAMP-dependent protein kinase. The phosphorylation rate in cAMP-stimulated cells was increased up to four times (reaching 0.018 s-1) by the presence of phenylalanine, the phosphate content (mol/mol hydroxylase) increasing to 0.5 from the basal level (0.17) in 50 s. The half maximal effect of phenylalanine was obtained at a physiologically relevant concentration (110 microM). The synthetic phenylalanine hydroxylase cofactor dimethyltetrahydropterin also enhanced the cAMP-stimulated phosphorylation of phenylalanine hydroxylase, presumably by displacing the endogenous cofactor, tetrahydrobiopterin. Phenylalanine was a negative modulator of the phosphorylation of phenylalanine hydroxylase induced by incubating cells with vasopressin or with the phosphatase inhibitor okadaic acid. The same site on the phenylalanine hydroxylase was phosphorylated in response to these two agents as in response to elevated cAMP. The available evidence suggested that not only vasopressin, but also okadaic acid, acted by stimulating the multifunctional Ca2+/calmodulin-dependent protein kinase II or a kinase with closely resembling properties.
Døskeland
A P
AP
Department of Biochemistry, University of Bergen, Norway.
Vintermyr
O K
OK
Flatmark
T
T
Cotton
R G
RG
Døskeland
S O
SO
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism*; D004988:Ethers, Cyclic / Q000494:pharmacology*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D008297:Male; D008969:Molecular Sequence Data; D019319:Okadaic Acid; D009842:Oligopeptides / Q000378:metabolism; D010649:Phenylalanine / Q000494:pharmacology*; D010651:Phenylalanine Hydroxylase / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D014667:Vasopressins / Q000494:pharmacology
D004988:Ethers, Cyclic; D046911:Macromolecular Substances; D009842:Oligopeptides; D014667:Vasopressins; D019319:Okadaic Acid; D010649:Phenylalanine; D000242:Cyclic AMP; D010651:Phenylalanine Hydroxylase; D011494:Protein Kinases; D010749:Phosphoprotein Phosphatases