Brown Victor M, Krynetski Eugene Y, Krynetskaia Natalia F, Grieger Dara, Mukatira Suraj T, Murti Kuruganti G, Slaughter Clive A, Park Hee-Won, Evans William E
Department of Pharmaceutical Sciences, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.
J Biol Chem. 2004 Feb 13;279(7):5984-92. doi: 10.1074/jbc.M307071200. Epub 2003 Nov 14.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional protein with glycolytic and non-glycolytic functions, including pro-apoptotic activity. GAPDH accumulates in the nucleus after cells are treated with genotoxic drugs, and it is present in a protein complex that binds DNA modified by thioguanine incorporation. We identified a novel CRM1-dependent nuclear export signal (NES) comprising 13 amino acids (KKVVKQASEGPLK) in the C-terminal domain of GAPDH, truncation or mutation of which abrogated CRM1 binding and caused nuclear accumulation of GAPDH. Alanine scanning of the sequence encompassing the putative NES demonstrated at least two regions important for nuclear export. Site mutagenesis of Lys259 did not affect oligomerization but impaired nuclear efflux of GAPDH, indicating that this amino acid residue is essential for proper functioning of this NES. This novel NES does not contain multiple leucine residues unlike other CRM1-interacting NES, is conserved in GAPDH from multiple species, and has sequence similarities to the export signal found in feline immunodeficiency virus Rev protein. Similar sequences (KKVV*7-13PLK) were found in two other human proteins, U5 small nuclear ribonucleoprotein, and transcription factor BT3.
甘油醛-3-磷酸脱氢酶（GAPDH）是一种具有糖酵解和非糖酵解功能的多功能蛋白质，包括促凋亡活性。在用基因毒性药物处理细胞后，GAPDH会在细胞核中积累，并且它存在于一种与硫鸟嘌呤掺入修饰的DNA结合的蛋白质复合物中。我们在GAPDH的C末端结构域中鉴定出一个由13个氨基酸组成的新型CRM1依赖性核输出信号（NES）（KKVVKQASEGPLK），其截短或突变会消除CRM1结合并导致GAPDH在细胞核中积累。对包含推定NES的序列进行丙氨酸扫描显示至少有两个区域对核输出很重要。Lys259的位点诱变不影响寡聚化，但损害了GAPDH的核外流，表明该氨基酸残基对于该NES的正常功能至关重要。与其他与CRM1相互作用的NES不同，这个新型NES不包含多个亮氨酸残基，在多个物种的GAPDH中保守，并且与猫免疫缺陷病毒Rev蛋白中的输出信号具有序列相似性。在另外两种人类蛋白质U5小核核糖核蛋白和转录因子BT3中发现了相似序列（KKVV*7-13PLK）。
