Some aspects of the phosphorylation of phenylalanine 4-monooxygenase by a calcium-dependent and calmodulin-dependent protein kinase

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Abstract
A calmodulin-dependent protein kinase purified from liver catalyzed the incorporation of up to 0.7 moll of phosphate per mol subunit of [rat liver] phenylalanine 4-monooxygenase. The phosphorylation was accompanied by a proportional increase in the hydroxylase activity. The reaction was Ca2+-dependent and was inhibited by physiological concentrations of phenylalanine. Phenylalanine 4-monooxygenase was also a substrate for the cGMP-dependent protein kinase, but in this system phenylalanine stimulated the rate of phosphorylation to a similar extent as that observed in the reaction catalyzed by cAMP-dependent protein kinase. The hydroxylase was not a substrate for phosphorylase kinase. The calmodulin-dependent reversal of the kinase reaction in the presence of MgADP, was also inhibited by phenylalanine. Since the kinetics of the reverse reaction was the same using 32P-hydroxylase phosphorylated by calmodulin-dependent and cAMP-dependent kinases, it is likely that both kinases phosphorylate the same site on the enzyme. This conclusion was further supported by peptide mapping of tryptic and peptic digests of 32P-hydroxylase, which revealed 1 major phosphopeptide with enzyme phosphorylated by either kinase. The Ca2+-dependent and calmodulin-dependent phosphorylation described above may mediate the increased phosphorylation of the hydrolase and its increased activity recently observed in hepatocytes exposed to Ca2+-elevating agents.

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