Firestein R, Feuerstein N
Center for Gerontology, Allegheny University of the Health Sciences and the Division of Rheumatology, Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
J Biol Chem. 1998 Mar 6;273(10):5892-902. doi: 10.1074/jbc.273.10.5892.
Activating transcription factor 2 (ATF2) is regulated by phosphorylation via the Jun N-terminal kinase, and its binding activity is markedly induced at late stages of T and B lymphocyte activation (Feuerstein, N., Firestein, R., Aiyer, N., Xiao, H., Murasko, D., and Cristofalo, V. (1996) J. Immunol. 156, 4582-4593). To identify proteins that interact specifically with ATF2 in lymphocytes, the yeast two-hybrid interaction system was employed using ATF2 cDNA as a "bait." In two separate screenings, a clone was identified that revealed a novel sequence with homology to several members of the ubiquitin-conjugating enzyme family. An identical sequence was recently reported as the human homolog of the yeast UBC9, hUBC9. Northern blot analysis revealed a 1.3-kilobase RNA transcript, which showed differential levels of expression in various human tissues and a moderate induction after a 48-h stimulation of peripheral blood T lymphocytes. An antibody that was generated against the bacterially expressed glutathione S-transferase-hUBC9 detected a approximately 19-kDa protein, which localizes predominantly in the nuclei of T cells. Further quantitative assays using the yeast two-hybrid system confirmed a high and specific level of interaction of hUBC9 with ATF2 and lack of interaction with lamin or control vectors. Two other cyclic AMP-responsive element-binding transcription factors, CREB and ATF1, also showed significant levels of interaction with hUBC9. However, this interaction was severalfold lower as compared with ATF2. Far Western blot analysis confirmed the specific binding of ATF2 and hUBC9 also in vitro. Evidence is presented that indicates a physiological significance for the interaction of hUBC9 with ATF2. (a) We show that ATF2 is ubiquitinated in vivo and in vitro, and (b) ATF2 ubiquitination in vitro is facilitated by addition of purified hUBC9. (c) ATF2 is shown to undergo a proteolytic process, which is rapidly regulated upon T cell activation concomitant with induction of ATF2 phosphorylation. (d) A proteasome inhibitor delays the down-regulation of ATF2 phophorylation after T cell activation. Taken collectively, these results implicate a role for hUBC9 and the ubiquitin/proteasome pathway in regulation of ATF2 in T cells.
活化转录因子2（ATF2）通过Jun N端激酶磷酸化进行调控，其结合活性在T和B淋巴细胞活化后期显著诱导（费尔斯坦，N.，菲尔斯坦，R.，艾耶尔，N.，肖，H.，穆拉斯科，D.，和克里斯托法洛，V.（1996年）《免疫学杂志》156，4582 - 4593）。为了鉴定在淋巴细胞中与ATF2特异性相互作用的蛋白质，以ATF2 cDNA作为“诱饵”采用酵母双杂交相互作用系统。在两次独立筛选中，鉴定出一个克隆，其显示出与泛素结合酶家族的几个成员具有同源性的新序列。最近报道了一个相同序列作为酵母UBC9的人类同源物，即hUBC9。Northern印迹分析显示有一个1.3千碱基的RNA转录本，其在各种人类组织中表达水平不同，在外周血T淋巴细胞经48小时刺激后有适度诱导。针对细菌表达的谷胱甘肽S - 转移酶 - hUBC9产生的抗体检测到一个约19千道尔顿的蛋白质，其主要定位于T细胞核中。使用酵母双杂交系统的进一步定量分析证实hUBC9与ATF2有高水平的特异性相互作用，且与核纤层蛋白或对照载体无相互作用。另外两个环磷酸腺苷反应元件结合转录因子，CREB和ATF1，也显示出与hUBC9有显著水平的相互作用。然而，与ATF2相比，这种相互作用要低几倍。Far Western印迹分析也证实在体外ATF2和hUBC9有特异性结合。有证据表明hUBC9与ATF2的相互作用具有生理意义。（a）我们表明ATF2在体内和体外均被泛素化，（b）体外添加纯化的hUBC9可促进ATF2的泛素化。（c）ATF2显示经历一个蛋白水解过程，该过程在T细胞活化时伴随ATF2磷酸化的诱导而迅速受到调控。（d）蛋白酶体抑制剂延迟T细胞活化后ATF2磷酸化的下调。综合来看，这些结果表明hUBC9和泛素/蛋白酶体途径在T细胞中对ATF2的调控中起作用。
