Department of Microbiology and Tumor Virology Program, Abramson Comprehensive Cancer Center, University of Pennsylvania Medical School, Philadelphia, Pennsylvania, United States of America.
PLoS Pathog. 2011 Feb 10;7(2):e1001275. doi: 10.1371/journal.ppat.1001275.
EBNA3C, one of the Epstein-Barr virus (EBV)-encoded latent antigens, is essential for primary B-cell transformation. Cyclin D1, a key regulator of G1 to S phase progression, is tightly associated and aberrantly expressed in numerous human cancers. Previously, EBNA3C was shown to bind to Cyclin D1 in vitro along with Cyclin A and Cyclin E. In the present study, we provide evidence which demonstrates that EBNA3C forms a complex with Cyclin D1 in human cells. Detailed mapping experiments show that a small N-terminal region which lies between amino acids 130-160 of EBNA3C binds to two different sites of Cyclin D1- the N-terminal pRb binding domain (residues 1-50), and C-terminal domain (residues 171-240), known to regulate Cyclin D1 stability. Cyclin D1 is short-lived and ubiquitin-mediated proteasomal degradation has been targeted as a means of therapeutic intervention. Here, we show that EBNA3C stabilizes Cyclin D1 through inhibition of its poly-ubiquitination, and also increases its nuclear localization by blocking GSK3β activity. We further show that EBNA3C enhances the kinase activity of Cyclin D1/CDK6 which enables subsequent ubiquitination and degradation of pRb. EBNA3C together with Cyclin D1-CDK6 complex also efficiently nullifies the inhibitory effect of pRb on cell growth. Moreover, an sh-RNA based strategy for knock-down of both cyclin D1 and EBNA3C genes in EBV transformed lymphoblastoid cell lines (LCLs) shows a significant reduction in cell-growth. Based on these results, we propose that EBNA3C can stabilize as well as enhance the functional activity of Cyclin D1 thereby facilitating the G1-S transition in EBV transformed lymphoblastoid cell lines.
EBNA3C 是 Epstein-Barr 病毒 (EBV) 编码的潜伏抗原之一，对原发性 B 细胞转化至关重要。细胞周期蛋白 D1 是 G1 期到 S 期进展的关键调节因子，与许多人类癌症密切相关且异常表达。先前已经证明 EBNA3C 在体外与细胞周期蛋白 A 和细胞周期蛋白 E 结合结合到细胞周期蛋白 D1 上。在本研究中，我们提供了证据，证明 EBNA3C 在人类细胞中与细胞周期蛋白 D1 形成复合物。详细的映射实验表明，EBNA3C 位于氨基酸 130-160 之间的小 N 端区域与细胞周期蛋白 D1 的两个不同结合位点结合 - N 端 pRb 结合结构域（残基 1-50）和 C 端结构域（残基 171-240），已知调节细胞周期蛋白 D1 的稳定性。细胞周期蛋白 D1 是短暂的，泛素介导的蛋白酶体降解已被靶向作为治疗干预的一种手段。在这里，我们表明 EBNA3C 通过抑制其多泛素化来稳定细胞周期蛋白 D1，并且还通过阻断 GSK3β 活性增加其核定位。我们进一步表明，EBNA3C 增强了细胞周期蛋白 D1/CDK6 的激酶活性，从而使随后的 pRb 泛素化和降解。EBNA3C 与细胞周期蛋白 D1/CDK6 复合物一起也有效地消除了 pRb 对细胞生长的抑制作用。此外，基于 sh-RNA 的策略对 EBV 转化的淋巴母细胞系 (LCL) 中的 cyclin D1 和 EBNA3C 基因进行敲低，显示出细胞生长显著减少。基于这些结果，我们提出 EBNA3C 可以稳定和增强细胞周期蛋白 D1 的功能活性，从而促进 EBV 转化的淋巴母细胞系中的 G1-S 过渡。
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