Xu W, Gong L, Haddad M M, Bischof O, Campisi J, Yeh E T, Medrano E E
Roy M. and Phyllis Gough Huffington Center on Aging, Department of Cell Biology, Baylor College of Medicine, One Baylor Plaza M320 and VAMC, Houston, Texas, 77030, USA.
Exp Cell Res. 2000 Mar 15;255(2):135-43. doi: 10.1006/excr.2000.4803.
The basic helix-loop-helix/leucine zipper (bHLH/ZIP) microphthalmia-associated transcription factor (MITF) regulates transcription of genes encoding enzymes essential for melanin biosynthesis in melanocytes and retinal pigmented epithelial cells. To determine how MITF activity is regulated, we used the yeast two-hybrid system to identify proteins expressed by human melanoma cells that interact with MITF. The majority of clones that showed positive interaction with a 158-amino-acid region of MITF containing the bHLH/ZIP domain (aa 168-325) encoded the ubiquitin conjugating enzyme hUBC9. The association of MITF with hUBC9 was further confirmed by an in vitro GST pull-down assay. Although hUBC9 is known to interact preferentially with SENTRIN/SUMO1, in vitro transcription/translation analysis demonstrated greater association of MITF with ubiquitin than with SENTRIN. Importantly, cotransfection of MITF and hUBC9 expression vectors resulted in MITF protein degradation. MITF protein was stabilized by the proteasome inhibitor MG132, indicating the role of the ubiquitin-proteasome system in MITF degradation. Serine 73, which is located in a region rich in proline, glutamic acid, serine, and threonine (PEST), regulates MITF protein stability, since a serine to alanine mutation prevented hUBC9-mediated MITF (S73A) degradation. Furthermore, we identified lysine 201 as a potential ubiquitination site. A lysine to arginine mutation abolished MITF (K201R) degradation by hUBC9 in vivo. Our experiments indicate that by targeting MITF for proteasome degradation, hUBC9 is a critical regulator of melanocyte differentiation.
碱性螺旋-环-螺旋/亮氨酸拉链（bHLH/ZIP）小眼相关转录因子（MITF）可调节黑素细胞和视网膜色素上皮细胞中编码黑色素生物合成所需酶的基因转录。为了确定MITF活性是如何被调节的，我们利用酵母双杂交系统来鉴定人类黑色素瘤细胞中表达的与MITF相互作用的蛋白质。大多数与包含bHLH/ZIP结构域（第168 - 325位氨基酸）的MITF的158个氨基酸区域显示出阳性相互作用的克隆编码泛素结合酶hUBC9。通过体外GST下拉实验进一步证实了MITF与hUBC9的关联。尽管已知hUBC9优先与SENTRIN/SUMO1相互作用，但体外转录/翻译分析表明MITF与泛素的关联比与SENTRIN的关联更强。重要的是，共转染MITF和hUBC9表达载体导致MITF蛋白降解。蛋白酶体抑制剂MG132可使MITF蛋白稳定，这表明泛素-蛋白酶体系统在MITF降解中起作用。位于富含脯氨酸、谷氨酸、丝氨酸和苏氨酸（PEST）区域的丝氨酸73调节MITF蛋白稳定性，因为丝氨酸到丙氨酸的突变可阻止hUBC9介导的MITF（S73A）降解。此外，我们鉴定出赖氨酸201为潜在的泛素化位点。赖氨酸到精氨酸的突变消除了hUBC9在体内对MITF（K201R）的降解作用。我们的实验表明，通过将MITF靶向蛋白酶体降解，hUBC9是黑素细胞分化的关键调节因子。
