Duan H, Tsvetkov L M, Liu Y, Song Y, Swaroop M, Wen R, Kung H F, Zhang H, Sun Y
Department of Molecular Biology, Pfizer Global Research and Development, Ann Arbor Laboratories, Ann Arbor, Michigan 48105, USA.
Mol Carcinog. 2001 Jan;30(1):37-46. doi: 10.1002/1098-2744(200101)30:1<37::aid-mc1011>3.0.co;2-7.
The sensitive-to-apoptosis gene (SAG) was initially identified as a redox-inducible, apoptosis-protective protein and subsequently found to be the second family member of regulator of cullins (ROC)/RING box protein (Rbx)/Hrt, which acts as a component of E3 ubiquitin ligase. We report here that SAG promoted cell growth under serum starvation. Microinjection of SAG mRNA into quiescent NIH/3T3 cells induced S-phase entry as determined by [(3)H]-thymidine incorporation. Likewise, overexpression of SAG by either adenovirus infection of immortalized human epidermal keratinocytes (Rhek-1) or DNA transfection of SY5Y human neuroblastoma cells induced cell proliferation under serum starvation. Because cyclin-dependent kinase inhibitors (CKIs), including p21, p27, and p57, are degraded through the ubiquitin pathway, we tested whether SAG-induced cell growth is associated with CKI degradation. Although there was no significant difference in the levels of p21 and p57 between the vector controls and SAG-overexpressing cells, serum starvation induced 10- to 18-fold accumulation of p27 in control Rhek-1 cells. Accumulation of p27 was remarkably inhibited (only 2 to 5-fold) in SAG-infected cells. Inhibition of p27 accumulation was also observed in stably SAG-overexpressing SY5Y cells. Significantly, SAG-associated inhibition of p27 accumulation was largely abolished by the treatment with a proteasome inhibitor. In vivo binding of SAG and Skp2, an F-box protein that promotes p27 ubiquitination, was detected, and the binding was enhanced in SAG-overexpressing cells grown under serum starvation. Thus, SAG-induced growth with serum withdrawal appears to be associated with SAG-mediated p27 degradation. Mol. Carcinog. 30:37-46, 2001.
凋亡敏感基因（SAG）最初被鉴定为一种氧化还原诱导的凋亡保护蛋白，随后被发现是Cullin调节因子（ROC）/RING盒蛋白（Rbx）/Hrt的第二个家族成员，它作为E3泛素连接酶的一个组成部分发挥作用。我们在此报告，SAG在血清饥饿条件下促进细胞生长。将SAG mRNA显微注射到静止的NIH/3T3细胞中，通过[³H] - 胸腺嘧啶掺入法测定可诱导细胞进入S期。同样，通过腺病毒感染永生化人表皮角质形成细胞（Rhek - 1）或对SY5Y人神经母细胞瘤细胞进行DNA转染来过表达SAG，可在血清饥饿条件下诱导细胞增殖。由于包括p21、p27和p57在内的细胞周期蛋白依赖性激酶抑制剂（CKIs）通过泛素途径降解，我们测试了SAG诱导的细胞生长是否与CKI降解有关。虽然载体对照和SAG过表达细胞之间p21和p57的水平没有显著差异，但血清饥饿诱导对照Rhek - 1细胞中p27积累10至18倍。在SAG感染的细胞中，p27的积累受到显著抑制（仅2至5倍）。在稳定过表达SAG的SY5Y细胞中也观察到p27积累的抑制。重要的是，用蛋白酶体抑制剂处理后，SAG相关的p27积累抑制在很大程度上被消除。检测到SAG与Skp2（一种促进p27泛素化的F - 盒蛋白）在体内的结合，并且在血清饥饿条件下生长的SAG过表达细胞中这种结合增强。因此，血清撤出时SAG诱导的生长似乎与SAG介导的p27降解有关。《分子致癌学》30:37 - 46，2001年。
