AMP-activated protein kinase phosphorylates Golgi-specific brefeldin A resistance factor 1 at Thr1337 to induce disassembly of Golgi apparatus
AMP-activated protein kinase phosphorylates Golgi-specific brefeldin A resistance factor 1 at Thr1337 to induce disassembly of Golgi apparatus
复制标题
DOI：
10.1074/jbc.m708296200
复制
发表时间：
2008-02-15
影响因子：
4.8
通讯作者：
Yonezawa, Kazuyoshi
中科院分区：
文献类型：
--
作者：
Miyamoto, Takafumi;Oshiro, Noriko;Yonezawa, Kazuyoshi
营养物质的充足和消耗通过蛋白质磷酸化反应调节细胞活动;然而，许多蛋白质底物仍有待澄清。GBF 1（高尔基体特异性布雷菲德菌素A抗性因子1），一种与高尔基体相关的ADP-核糖基化因子家族的鸟嘌呤核苷酸交换因子，通过使用磷酸化-Akt-底物抗体从葡萄糖耗尽的细胞中分离为磷蛋白，该抗体识别几种蛋白激酶的底物蛋白。GBF 1的磷酸化由2-脱氧葡萄糖（2-DG）诱导，其阻断葡萄糖利用并增加细胞内AMP浓度，并且由AMP活化的蛋白激酶（AMPK）激活剂AICAR诱导。在表达组成型活性AMPK的细胞中观察到这种磷酸化。2-DG诱导的GBF 1磷酸化被AMPK抑制剂化合物C和激酶阴性AMPK的过表达抑制。使用缺失突变体和点突变体的分析鉴定了Thr（1337）作为GBF 1中2-DG诱导的磷酸化位点，其在体外被AMPK磷酸化。已知ATP耗竭会引起高尔基体解体。免疫荧光显微镜分析与高尔基体标记表明，GBF 1与破碎的高尔基体在2-DG和AICAR处理的细胞。激酶阴性AMPK和GBF 1突变体的表达用Ala取代Thr（1337）阻止了2-DG诱导的高尔基体解体。这些结果表明，GBF 1是一种新的AMPK底物，AMPK介导的GBF 1在Thr 1337的磷酸化具有关键作用，可能是通过减弱GBF 1的功能，在降低细胞内ATP浓度的应激条件下诱导的高尔基体解体。
Sufficiency and depletion of nutrients regulate the cellular activities through the protein phosphorylation reaction; however, many protein substrates remain to be clarified. GBF1 (Golgi-specific brefeldin A resistance factor 1), a guanine nucleotide exchange factor for the ADP-ribosylation factor family associated with the Golgi apparatus, was isolated as a phosphoprotein from the glucose-depleted cells by using the phospho-Akt-substrate antibody, which recognizes the substrate proteins of several protein kinases. The phosphorylation of GBF1 was induced by 2-deoxyglucose (2-DG), which blocks glucose utilization and increases the intracellular AMP concentration, and by AICAR, an AMP-activated protein kinase (AMPK) activator. This phosphorylation was observed in the cells expressing the constitutively active AMPK. The 2-DG-induced phosphorylation of GBF1 was suppressed by Compound C, an AMPK inhibitor, and by the overexpression of the kinase-negative AMPK. Analysis using the deletion and point mutants identified Thr(1337) as the 2-DG-induced phosphorylation site in GBF1, which is phosphorylated by AMPK in vitro. ATP depletion is known to provoke the Golgi apparatus disassembly. Immunofluorescent microscopic analysis with the Golgi markers indicated that GBF1 associates with the fragmented Golgi apparatus in the cells treated with 2-DG and AICAR. The expression of the kinase-negative AMPK and the GBF1 mutant replacing Thr(1337) by Ala prevented the 2-DG-induced Golgi disassembly. These results indicate that GBF1 is a novel AMPK substrate and that the AMPK-mediated phosphorylation of GBF1 at Thr1337 has a critical role, presumably by attenuating the function of GBF1, in the disassembly of the Golgi apparatus induced under stress conditions that lower the intracellular ATP concentration.