Mitchell J P, Hardie D G, Vulliet P R
Department of Veterinary Pharmacology and Toxicology, University of California, Davis 95616.
J Biol Chem. 1990 Dec 25;265(36):22358-64.
The phosphorylation and activation of tyrosine hydroxylase was examined in PC12 cells following depolarization with KCl or treatment with nerve growth factor. Both treatments activate tyrosine hydroxylase (TH) and increase enzyme phosphorylation. Site-specific analysis of the tryptic phosphopeptides of TH isolated from [32P]phosphate-labeled PC12 cells demonstrated that the major phosphorylated peptide (termed "H25") did not contain any of the previously reported phosphorylation sites. Phosphoamino acid analysis of this peptide demonstrated that the phosphorylated residue was a serine. Synthetic tryptic peptides containing putative phosphorylation sites were prepared, and subjected to high performance liquid chromatography analysis and isoelectric focusing. The tryptic phosphopeptide containing serine 31 comigrated with the H25 peptide during both of these analytical techniques. The tryptic phosphopeptide produced by the phosphorylation of tyrosine hydroxylase by the recently discovered proline-directed protein kinase and the phosphorylated synthetic phosphopeptide TH2-12 are clearly separated from H25 by this analysis. We conclude that serine 31 is phosphorylated during KCl depolarization and nerve growth factor treatment of PC12 cells and that this phosphorylation is responsible for the activation of tyrosine hydroxylase. Since this site is not located in a sequence selective for any of the "classical" protein kinases, we suggest that a novel protein kinase may be responsible for the phosphorylation of this site. Since serine 31 has a proline residue on the carboxyl-terminal side, the possibility that this kinase may be related to the recently reported proline-directed protein kinase is discussed. Other sites that are also phosphorylated on TH during KCl depolarization include serine 19, which is known to be phosphorylated by calmodulin-dependent protein kinase II. A schematic model for the regulation of tyrosine hydroxylase activity by phosphorylation of the NH2-terminal regulatory domain is presented.
在用氯化钾去极化或用神经生长因子处理后的PC12细胞中，检测了酪氨酸羟化酶的磷酸化和激活情况。这两种处理均激活酪氨酸羟化酶（TH）并增加酶的磷酸化。对从[32P]磷酸盐标记的PC12细胞中分离出的TH的胰蛋白酶磷酸肽进行位点特异性分析表明，主要的磷酸化肽（称为“H25”）不包含任何先前报道的磷酸化位点。对该肽进行磷酸氨基酸分析表明，磷酸化残基是丝氨酸。制备了含有假定磷酸化位点的合成胰蛋白酶肽，并进行了高效液相色谱分析和等电聚焦。在这两种分析技术中，含有丝氨酸31的胰蛋白酶磷酸肽与H25肽共迁移。通过该分析，最近发现的脯氨酸定向蛋白激酶使酪氨酸羟化酶磷酸化产生的胰蛋白酶磷酸肽和磷酸化的合成磷酸肽TH2-12与H25明显分离。我们得出结论，在PC12细胞的氯化钾去极化和神经生长因子处理过程中，丝氨酸31被磷酸化，并且这种磷酸化负责酪氨酸羟化酶的激活。由于该位点不在任何“经典”蛋白激酶的选择性序列中，我们认为一种新型蛋白激酶可能负责该位点的磷酸化。由于丝氨酸31在羧基末端侧有一个脯氨酸残基，讨论了这种激酶可能与最近报道的脯氨酸定向蛋白激酶相关的可能性。在氯化钾去极化过程中，TH上也被磷酸化的其他位点包括丝氨酸19，已知其被钙调蛋白依赖性蛋白激酶II磷酸化。提出了通过NH2末端调节域的磷酸化来调节酪氨酸羟化酶活性的示意图模型。
