用56 mM K⁺处理的大鼠嗜铬细胞瘤PC12细胞中酪氨酸羟化酶至少三个位点的磷酸化：确定环磷酸腺苷依赖性蛋白激酶和钙/钙调蛋白依赖性蛋白激酶磷酸化的酪氨酸羟化酶位点
Phosphorylation of tyrosine hydroxylase on at least three sites in rat pheochromocytoma PC12 cells treated with 56 mM K+: determination of the sites on tyrosine hydroxylase phosphorylated by cyclic AMP-dependent and calcium/calmodulin-dependent protein kinases.
作者信息
Tachikawa E, Tank A W, Yanagihara N, Mosimann W, Weiner N
出版信息
Mol Pharmacol. 1986 Nov;30(5):476-85.
Incubation of rat pheochromocytoma PC12 cells with the calcium ionophore, A23187 (10(-5) M), 56 mM K+, or dibutyryl cAMP (2 mM) is associated with increased activity and enhanced phosphorylation of tyrosine hydroxylase in the cells. Both the activation and the increased phosphorylation of tyrosine hydroxylase produced by A23187 and 56 mM K+ are dependent on the presence of extracellular calcium, whereas similar effects produced by dibutyryl cAMP are independent of calcium. The effects of 56 mM K+ plus dibutyryl cAMP or A23187 plus dibutyryl cAMP on the activation and phosphorylation of tyrosine hydroxylase are additive. In contrast, the effects of 56 mM K+ plus A23187 on either the activation or the phosphorylation of the enzyme are not additive. Following stimulation of intact PC12 cells with 32Pi, in order to label ATP stores, and tryptic digestion of the phosphorylated enzyme, separation of the tryptic phosphopeptides by high pressure liquid chromatography yields four distinct 32P-peptide peaks. Incubation of the cells in the presence of either 56 mM K+ or A23187 is associated with increased 32Pi incorporation into three peptides whereas, in the presence of dibutyryl cAMP, increased 32Pi incorporation is observed in only one of these peptides. When tyrosine hydroxylase purified from rat pheochromocytoma tumor is incubated in vitro with [gamma-32P]ATP and either cAMP-dependent or calcium/calmodulin-dependent protein kinase under appropriate conditions, increased phosphorylation of tyrosine hydroxylase is observed. However, even though in vitro phosphorylation by cAMP-dependent protein kinase is associated with activation of tyrosine hydroxylase, in vitro phosphorylation by calcium/calmodulin-dependent protein kinase does not lead to activation of the enzyme. Tryptic digestion of tyrosine hydroxylase phosphorylated by calcium/calmodulin-dependent protein kinase yields three distinct 32P-peptide peaks, which are identical to those phosphorylated by treatment of intact PC12 cells with either high K+ or A23187. In contrast, cAMP-dependent protein kinase phosphorylates only one peptide, which is identical to that phosphorylated by treatment of the intact cells with dibutyryl cAMP. These results indicate that tyrosine hydroxylase is activated and phosphorylated at multiple sites in PC12 cells exposed to 56 mM K+ or A23187. The results suggests that the in situ phosphorylation of these sites is catalyzed by calcium/calmodulin-dependent protein kinase; however, phosphorylation by this protein kinase is not sufficient to activate the enzyme.
用钙离子载体A23187（10⁻⁵ M）、56 mM K⁺或二丁酰环磷腺苷（2 mM）孵育大鼠嗜铬细胞瘤PC12细胞，会使细胞中酪氨酸羟化酶的活性增加且磷酸化增强。A23187和56 mM K⁺所引起的酪氨酸羟化酶的激活及磷酸化增加均依赖于细胞外钙的存在，而二丁酰环磷腺苷产生的类似作用则与钙无关。56 mM K⁺加二丁酰环磷腺苷或A23187加二丁酰环磷腺苷对酪氨酸羟化酶激活和磷酸化的作用是相加的。相比之下，56 mM K⁺加A23187对该酶激活或磷酸化的作用并非相加。在用³²Pi刺激完整的PC12细胞以标记ATP储备并对磷酸化酶进行胰蛋白酶消化后，通过高压液相色谱分离胰蛋白酶磷酸肽可得到四个不同的³²P - 肽峰。在56 mM K⁺或A23187存在的情况下孵育细胞，会使³²Pi掺入三种肽的量增加，而在二丁酰环磷腺苷存在的情况下，仅观察到³²Pi掺入其中一种肽的量增加。当从大鼠嗜铬细胞瘤肿瘤中纯化的酪氨酸羟化酶在体外与[γ - ³²P]ATP以及在适当条件下的环磷腺苷依赖性或钙/钙调蛋白依赖性蛋白激酶一起孵育时，会观察到酪氨酸羟化酶的磷酸化增加。然而，尽管环磷腺苷依赖性蛋白激酶的体外磷酸化与酪氨酸羟化酶的激活相关，但钙/钙调蛋白依赖性蛋白激酶的体外磷酸化并不会导致该酶的激活。对钙/钙调蛋白依赖性蛋白激酶磷酸化的酪氨酸羟化酶进行胰蛋白酶消化会产生三个不同的³²P - 肽峰，这与用高K⁺或A23187处理完整PC12细胞所磷酸化的肽峰相同。相比之下，环磷腺苷依赖性蛋白激酶仅磷酸化一种肽，这与用二丁酰环磷腺苷处理完整细胞所磷酸化后的肽相同。这些结果表明，在暴露于56 mM K⁺或A23187的PC12细胞中，酪氨酸羟化酶在多个位点被激活和磷酸化。结果表明这些位点的原位磷酸化是由钙/钙调蛋白依赖性蛋白激酶催化的；然而，该蛋白激酶的磷酸化不足以激活该酶。
Zotero 插件