The modulation of phenylalanine hydroxylase activity by okadaic acid.

The enzyme phenylalanine hydroxylase (EC 1.14.16.1) catalyses the hydroxylation of phenylalanine to tyrosine in man and other mammals. The hepatic enzyme is controlled by a reversible phosphorylation mechanism [l]. The phosphorylation state of the enzyme is under hormonal control. For example, glucagon stimulates phosphorylation and activation of the enzyme through the action of the cyclic AMP-dependent protein kinase [2,3]. Little is known about the protein phosphatase(s) responsible for the dephosphorylation of the enzyme in the intact liver cell. Work with purified phenylalanine hydroxylase has indicated that protein phosphatase 2A may be responsible for the dephosphorylation of this enzyme i n vivo [4]. In addition, we have previously shown that glucagondependent alterations in the phosphorylation state of the hydroxylase in liver cells are antagonised by polyamines such as spermine [5] which are known activators of protein phosphatase 2A [6]. Okadaic acid has been identified as a potent and specific inhibitor of protein phosphatases 1 and 2A i n v i t r o [7]. It was therefore of interest to investigate the effect of okadaic acid on the phosphorylation state and activity of phenylalanine hydroxylase in isolated liver cells. Rat liver cells were isolated and incubated as described previously 131. Changes in the phosphorylation state of phenylalanine hydroxylase were assessed by an immunoblotting technique as described elsewhere [El. Alterations in the enzymic activity of the hydroxylase were quantified using a 3H20 release assay based on that described in [3] using [ 4-3H] phenylalanine as substrate. Table 1 shows the impact of okadaic acid on phenylalanine hydroxylase phosphorylation state and specific enzymic activity in isolated liver cells. Incubation of liver cells, in the presence of either okadaic acid or glucagon, for 15 min resulted in a significant increase in phenylalanine hydroxylase specific activity. These changes were accompanied by significant elevations in the phosphorylation state of the enzyme. This effect of okadaic acid is consistent with the possibility that protein phosphatase 2A is the major protein phosphatase responsible for the dephosphorylation of phenylalanine hydroxylase in liver cells. When liver cells were incubated in the prescence of both okadaic acid and glucagon a clear additivity of responses was not seen. This is perhaps surprising, in view of the different modes of action of these effectors ie. activation of a protein kinase in the case of glucagon and inhibition of a protein phoshatase in that of okadaic acid.