Nerve growth factor and other agents mediate phosphorylation and activation of tyrosine hydroxylase. A convergence of multiple kinase activities.
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A rapid phosphorylation of tyrosine hydroxylase occurs in the PC12 nerve-like clonal cell line in response to nerve growth factor (NGF), epidermal growth factor (EGF), dibutyryl-cAMP, cholera toxin, phorbol- 12-myristate-13-acetate (PMA), or potassium depolarization in the presence of calcium ions. Complete tryptic digestion and two-dimensional peptide mapping reveals four available sites of phosphorylation in the enzyme. Phosphoamino acid analysis demonstrates that serine is the amino acid residue phosphorylated in each peptide. Specific phosphorylation of each of the four sites is achieved by different subsets of the above agents. One peptide site is phosphorylated in response to EGF alone. A second site is phosphorylated only in response to NGF, cholera toxin or dibutyryl-cAMP. A third site is phosphorylated only in response to potassium depolarization and requires the presence of extracellular Ca2+. The fourth site is the only site phosphorylated in response to PMA. These data indicate that at least 4 distinct kinase systems can act to phosphorylate tyrosine hydroxylase in PC12 cells. The PMA-stimulated peptide site is also phosphorylated in response to every one of the other agents. Further proteolytic digestions and phosphopeptide mapping of this common peptide, using Staphylococcus V8 protease and thermolysin, did not generate different phosphopeptides resulting from the different agents. These data suggest that the phosphorylation of this common peptide in response to all of the agents may be mediated by a common kinase, and, hence, that tyrosine hydroxylase phosphorylation by some agents may be mediated by two kinases. Although phosphopeptide maps of tyrosine hydroxylase resulting from cAMP elevation or NGF are qualitatively similar, quantitative differences exist, suggesting differential regulation of the same kinases by these agents. Tyrosine hydroxylase was found to be activated 2--4-fold in response to each phosphorylating agent. Thus, NGF and EGF present novel, natural means of regulating the activation state of tyrosine hydroxylase in responsive neurons.(ABSTRACT TRUNCATED AT 400 WORDS)
