Soond Surinder M, Everson Bethany, Riches David W H, Murphy Gillian
University of Cambridge, Department of Oncology, Cambridge Institute of Medical Research, Hills Road, Cambridge, CB2 2XY, UK.
J Cell Sci. 2005 Jun 1;118(Pt 11):2371-80. doi: 10.1242/jcs.02357.
Tumor necrosis factor alpha-converting enzyme (TACE/ADAM17) has been implicated in the inducible shedding of numerous cell surface proteins. In light of this, the regulation of TACE catalytic activation and protein maturation has recently received considerable interest in the context of extracellular signal-regulated kinase activation and the subsequent phosphorylation of TACE at residue Thr735. In this study, we analysed the subcellular localisation of TACE derivatives using laser-scanning confocal microscopy and cell surface biotinylation. Whereas WT.TACE- and T735A.TACE-enhanced green fluorescent protein (-eGFP) fusion derivatives of TACE were both found to localise with the endoplasmic reticulum, a phosphomimicking form of TACE (T735E.TACE-eGFP) was found to colocalise predominantly with components of the protein secretory pathway (COPII vesicles and trans-Golgi network). Additionally, upon analysis of biotinylated ectopic TACE, we observed that inducible trafficking of TACE to the cell surface was dependent upon extracellular signal-regulated kinase activation and phosphorylation of Thr735. We conclude from our observations that phosphorylation of TACE at Thr735 highlights a key step in inducible TACE protein trafficking and maturation.
肿瘤坏死因子α转化酶（TACE/ADAM17）与多种细胞表面蛋白的诱导性脱落有关。鉴于此，TACE催化激活和蛋白质成熟的调控最近在细胞外信号调节激酶激活以及随后TACE第735位苏氨酸残基磷酸化的背景下受到了相当大的关注。在本研究中，我们使用激光扫描共聚焦显微镜和细胞表面生物素化分析了TACE衍生物的亚细胞定位。虽然野生型TACE和T735A.TACE的增强型绿色荧光蛋白（-eGFP）融合衍生物均定位于内质网，但一种模拟磷酸化形式的TACE（T735E.TACE-eGFP）主要与蛋白质分泌途径的成分（COPII囊泡和顺式高尔基体网络）共定位。此外，在分析生物素化的异位TACE时，我们观察到TACE向细胞表面的诱导性转运依赖于细胞外信号调节激酶激活和第735位苏氨酸的磷酸化。我们从观察结果中得出结论，TACE第735位苏氨酸的磷酸化是诱导性TACE蛋白转运和成熟的关键步骤。
