Cho G W, Kim M H, Chai Y G, Gilmor M L, Levey A I, Hersh L B
Department of Biochemistry, University of Kentucky, Lexington, Kentucky, 40536-0084, USA.
J Biol Chem. 2000 Jun 30;275(26):19942-8. doi: 10.1074/jbc.M902174199.
Metabolic labeling of a mutant PC12 cell line, A123.7, expressing recombinant rat vesicular acetylcholine transporter (VAChT) with radiolabeled inorganic phosphate was used to demonstrate phosphorylation of the transporter on a serine residue. Mutational analysis was used to demonstrate that serine 480, which is located on the COOH-terminal cytoplasmic tail, is the sole phosphorylation site. Phosphorylation of serine 480 was attributable to the action of protein kinase C. Using a permanently dephosphorylated form of rat VAChT, S480A rVAChT, it was shown that this mutant displays the same kinetics for the transport of acetylcholine and the binding of the inhibitor vesamicol as does the wild type transporter. However, sucrose gradient density centrifugation showed that, unlike wild type VAChT, the S480A mutant did not localize to synaptic vesicles. These results suggest that phosphorylation of serine 480 of VAChT is involved in the trafficking of this transporter.
用放射性标记的无机磷酸盐对表达重组大鼠囊泡乙酰胆碱转运体（VAChT）的突变型PC12细胞系A123.7进行代谢标记，以证明该转运体在丝氨酸残基上发生磷酸化。通过突变分析证明位于COOH末端胞质尾部的丝氨酸480是唯一的磷酸化位点。丝氨酸480的磷酸化归因于蛋白激酶C的作用。使用大鼠VAChT的永久去磷酸化形式S480A rVAChT，结果表明该突变体在乙酰胆碱转运和抑制剂维西溴铵结合方面表现出与野生型转运体相同的动力学。然而，蔗糖梯度密度离心显示，与野生型VAChT不同，S480A突变体未定位于突触小泡。这些结果表明，VAChT丝氨酸480的磷酸化参与了该转运体的运输过程。
