Division of Gastroenterology and Liver Diseases, Keck School of Medicine of University of Southern California, Los Angeles, CA 90033, USA.
Hepatology. 2012 Sep;56(3):982-93. doi: 10.1002/hep.25701. Epub 2012 Jul 12.
Ubiquitin-conjugating enzyme 9 (Ubc9) is required for sumoylation and is overexpressed in several malignancies, but its expression in hepatocellular carcinoma (HCC) is unknown. Hepatic S-adenosyl methionine (SAMe) levels decrease in methionine adenosyltransferase 1A (Mat1a) knockout (KO) mice, which develop HCC, and in ethanol-fed mice. We examined the regulation of Ubc9 by SAMe in murine liver and human HCC, breast, and colon carcinoma cell lines and specimens. Real-time polymerase chain reaction and western blotting measured gene and protein expression, respectively. Immunoprecipitation followed by western blotting examined protein-protein interactions. Ubc9 expression increased in HCC and when hepatic SAMe levels decreased. SAMe treatment in Mat1a KO mice reduced Ubc9 protein, but not messenger RNA (mRNA) levels, and lowered sumoylation. Similarly, treatment of liver cancer cell lines HepG2 and Huh7, colon cancer cell line RKO, and breast cancer cell line MCF-7 with SAMe or its metabolite 5'-methylthioadenosine (MTA) reduced only Ubc9 protein level. Ubc9 posttranslational regulation is unknown. Ubc9 sequence predicted a possible phosphorylation site by cell division cycle 2 (Cdc2), which directly phosphorylated recombinant Ubc9. Mat1a KO mice had higher phosphorylated (phospho)-Ubc9 levels, which normalized after SAMe treatment. SAMe and MTA treatment lowered Cdc2 mRNA and protein levels, as well as phospho-Ubc9 and protein sumoylation in liver, colon, and breast cancer cells. Serine 71 of Ubc9 was required for phosphorylation, interaction with Cdc2, and protein stability. Cdc2, Ubc9, and phospho-Ubc9 levels increased in human liver, breast, and colon cancers.
Cdc2 expression is increased and Ubc9 is hyperphosphorylated in several cancers, and this represents a novel mechanism to maintain high Ubc9 protein expression that can be inhibited by SAMe and MTA.
泛素连接酶 9（Ubc9）是 sumoylation 的必需酶，在几种恶性肿瘤中过表达，但在肝细胞癌（HCC）中的表达情况尚不清楚。甲硫氨酸腺苷转移酶 1A（Mat1a）敲除（KO）小鼠和乙醇喂养的小鼠中，肝 S-腺苷甲硫氨酸（SAMe）水平降低，这些小鼠会发展为 HCC。我们在小鼠肝和人 HCC、乳腺癌和结肠癌细胞系和标本中检查了 SAMe 对 Ubc9 的调节。实时聚合酶链反应和蛋白质印迹分别测量基因和蛋白质的表达。免疫沉淀后进行蛋白质印迹检查蛋白-蛋白相互作用。Ubc9 在 HCC 中表达增加，且肝 SAMe 水平降低时表达增加。SAMe 处理 Mat1a KO 小鼠可降低 Ubc9 蛋白，但不降低信使 RNA（mRNA）水平，并降低 sumoylation。同样，SAMe 或其代谢物 5'-甲基硫代腺苷（MTA）处理肝癌细胞系 HepG2 和 Huh7、结肠癌细胞系 RKO 和乳腺癌细胞系 MCF-7，仅降低 Ubc9 蛋白水平。Ubc9 的翻译后调节尚不清楚。Ubc9 序列预测其可能被细胞分裂周期 2（Cdc2）磷酸化的位点，Cdc2 可直接磷酸化重组 Ubc9。Mat1a KO 小鼠的磷酸化（磷酸化）Ubc9 水平较高，SAMe 处理后恢复正常。SAMe 和 MTA 处理降低了肝、结肠和乳腺癌细胞中的 Cdc2 mRNA 和蛋白水平以及磷酸化 Ubc9 和蛋白 sumoylation。Ubc9 的丝氨酸 71 是磷酸化、与 Cdc2 相互作用和蛋白稳定性所必需的。人肝、乳腺和结肠癌症中 Cdc2、Ubc9 和磷酸化 Ubc9 水平增加。
几种癌症中 Cdc2 表达增加，Ubc9 过度磷酸化，这代表了一种维持高 Ubc9 蛋白表达的新机制，SAMe 和 MTA 可抑制该机制。
