Lee K Y, Seeley P J, Müller T H, Helmer-Matyjek E, Sabban E, Goldstein M, Greene L A
Mol Pharmacol. 1985 Aug;28(2):220-8.
A specific antiserum was used to compare phosphorylation of tyrosine hydroxylase (TH) (EC 1.14.16.2, tyrosine 3-monooxygenase) as regulated by elevated K+ and nerve growth factor (NGF) in cultured PC12 pheochromocytoma cells. Exposure of cultures to either elevated K+ or to NGF significantly enhanced the incorporation of [32P]orthophosphate into TH. The effect of elevated K+ was evident at 10 mM and was maximal by 40-80 mM. Increased phosphorylation of TH was detected at 0.1 nM (3 ng/ml) NGF and reached a maximal level by 0.3-1 nM (10-30 ng/ml) NGF. Elevated K+ showed a biphasic time course of action with one maximum of phosphorylation at about 30 sec of exposure and a second after about 10 min of exposure. In contrast, the NGF effect showed an initial lag of several minutes followed by a monophasic increase in phosphorylation to reach a plateau. Both treatments enhanced TH activity, but in each case the time courses of this did not strictly correlate with that of phosphorylation. The effect of elevated K+ on TH phosphorylation required the presence of extracellular Ca2+ and was suppressed by trifluoperazine (100 microM). N-(6-Aminohexyl)-5-(chloronaphthalene)-1-sulfonamide (W-7) (100 microM), a potent inhibitor of calmodulin activity, also blocked the enhancement of phosphorylation by elevated K+, whereas N-(6-aminohexyl)-1-(naphthalene)sulfonamide (W-5) (100 microM), a less potent analogue of W-7, did not. In contrast to these findings, the increase in TH phosphorylation brought about by NGF did not require extracellular Ca2+, and was only slightly affected by trifluoperazine or W-7. When TH phosphorylated under various conditions (control medium, elevated K+, NGF) was subjected to peptide mapping after exposure to Staphylococcus aureus protease V8, multiple phosphorylated peptides were observed. Elevated K+ and NGF each produced increases in labeling of each of the peptides. However, the relative degree of labeling of different peptides was distinct for each condition. These data suggest that elevated K+ and NGF bring about rapid enhancement of the phosphorylation of TH by means of different mechanisms.
使用一种特异性抗血清来比较培养的PC12嗜铬细胞瘤细胞中，酪氨酸羟化酶（TH）（EC 1.14.16.2，酪氨酸3-单加氧酶）的磷酸化情况，该磷酸化受细胞外高钾和神经生长因子（NGF）调控。将培养物暴露于高钾或NGF中，均能显著增强[32P]正磷酸盐掺入TH的量。高钾在10 mM时作用明显，40 - 80 mM时作用达到最大。在0.1 nM（3 ng/ml）NGF时可检测到TH磷酸化增加，0.3 - 1 nM（10 - 30 ng/ml）NGF时达到最大水平。高钾表现出双相作用时间进程，暴露约30秒时有一个磷酸化峰值，暴露约10分钟后有第二个峰值。相比之下，NGF的作用表现为最初几分钟的延迟，随后磷酸化呈单相增加并达到平台期。两种处理均增强了TH活性，但在每种情况下，TH活性的时间进程与磷酸化的时间进程并不严格相关。高钾对TH磷酸化的作用需要细胞外Ca2+的存在，并被三氟拉嗪（100 microM）抑制。N-（6-氨基己基）-5-（氯萘）-1-磺酰胺（W-7）（100 microM），一种钙调蛋白活性的强效抑制剂，也能阻断高钾引起的磷酸化增强，而N-（6-氨基己基）-1-（萘）磺酰胺（W-5）（100 microM），一种W-7的低效类似物，则不能。与这些发现相反，NGF引起的TH磷酸化增加不需要细胞外Ca2+，且仅受到三氟拉嗪或W-7的轻微影响。当在不同条件（对照培养基、高钾、NGF）下磷酸化的TH在暴露于金黄色葡萄球菌蛋白酶V8后进行肽图谱分析时，观察到多个磷酸化肽段。高钾和NGF均使每个肽段的标记增加。然而，不同肽段的相对标记程度在每种条件下是不同的。这些数据表明，高钾和NGF通过不同机制快速增强TH的磷酸化。
