Data demonstrating the direct phosphorylation of
tyrosine hydroxylase [
tyrosine 3-monooxygenase;
L-tyrosine,
tetrahydropteridine:
oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2] purified from rat
pheochromocytoma by
ATP, Mg2+ and
cyclic AMP-dependent protein kinase catalytic subunit are presented. The incorporation of
phosphate is highly correlated with the activation of the
hydroxylase when either the time of preincubation or the amount of
protein kinase subunit is varied. The rate of phosphorylation of
tyrosine hydroylase compares favorably with that of H1
histone, a known substrate of
protein kinase. Lineweaver-Burk analysis of crude or purified rat
pheochromocytoma tyrosine hydroxylase activity, as a function of
pterin cofactor concentration, in the absence of
ATP, Mg2+, and
protein kinase catalytic subunit, yields a curvilinear relationship which can be resolved into two lines, suggesting two
enzyme forms with different affinities for
pterin cofactor. A fraction of the
hydroxylase present in the
tumor exists in the activated state, presumably due to the presence of
ATP and endogenous
protein kinase activity. When the solubl
enzyme is activated by
cyclic AMP,
ATP, Mg2+, and
protein kinase, virtually all of the
enzyme is converted to the low Km state. We conclude that
tyrosine hydroxylase is a substrate of
cyclic AMP-dependent protein kinase in vitro and, presumably, in vivo.