Restricted Protein Phosphatase 2A Targeting by Merkel Cell Polyomavirus Small T Antigen
Restricted Protein Phosphatase 2A Targeting by Merkel Cell Polyomavirus Small T Antigen
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DOI：
10.1128/jvi.00157-15
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发表时间：
2015-04-01
影响因子：
5.4
通讯作者：
Moore, Patrick S.
中科院分区：
文献类型：
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作者：
Kwun, Hyun Jin;Shuda, Masahiro;Moore, Patrick S.
默克尔细胞多瘤病毒（MCV）是一种新发现的编码小T（sT）癌蛋白的人类肿瘤病毒。我们进行了MCV sT FLAG亲和纯化，然后进行质谱（MS）分析，鉴定了几种蛋白磷酸酶（PP），包括PP 2A A和C亚基和PP 4C，作为潜在的细胞相互作用蛋白。PP 2A靶向对于非人多瘤病毒（例如猿猴病毒40（SV 40））的转化特性是关键的，但对于MCV sT诱导的啮齿动物细胞转化不是必需的。我们比较了MCV和SV 40 sT之间PP 2A结合的异同。SV 40 sT与亚基PP 2A A α和PP 2A C共免疫纯化，而MCV sT与PP 2A A α、PP 2A A β和PP 2A C共免疫纯化。在MCV sT蛋白的29个位点上扫描丙氨酸诱变揭示了PP 2A结合结构域位于与先前描述的结合E3连接酶如Fbw 7的大T稳定结构域（LSD）环相对的分子表面上。MCV sT-PP 2A相互作用可以通过诱变与MCV sT LSD依赖性4 E-BP 1过度磷酸化和病毒DNA复制增强在功能上区分开。MCV sT对PP 2A B亚基取代的范围有限，仅抑制B56 α组装成磷酸酶全酶。相反，SV 40 sT抑制B55 α、B56 α和B56 β组装成PP 2A。我们的结论是，MCV的sT所需的默克尔细胞癌的生长，但其在体外的转化活性取决于LSD的相互作用，而不是PP 2A的targeting.IMPORTANCEMerkel细胞多瘤病毒是一种新发现的人类癌症病毒，促进癌症，部分，通过其小T（sT）癌蛋白的表达。已经发现动物多瘤病毒sT癌蛋白通过阻断一组称为蛋白磷酸酶2A（PP 2A）的磷酸酶的活性而引起实验性肿瘤。我们的结构分析表明，MCV sT也取代了PP 2A的B亚基，以抑制PP 2A活性。然而，MCV sT仅置换PP 2A B亚基的有限子集，这不足以在体外引起肿瘤细胞形成。MCV sT通过另一个称为大T稳定结构域的区域转化肿瘤细胞。PP 2A靶向和转化活性位于MCV sT分子的相对面上，并且可以在遗传上彼此分离。
Merkel cell polyomavirus (MCV) is a newly discovered human cancer virus encoding a small T (sT) oncoprotein. We performed MCV sT FLAG-affinity purification followed by mass spectroscopy (MS) analysis, which identified several protein phosphatases (PP), including PP2A A and C subunits and PP4C, as potential cellular interacting proteins. PP2A targeting is critical for the transforming properties of nonhuman polyomaviruses, such as simian virus 40 (SV40), but is not required for MCV sT-induced rodent cell transformation. We compared similarities and differences in PP2A binding between MCV and SV40 sT. While SV40 sT coimmunopurified with subunits PP2A A alpha and PP2A C, MCV sT coimmunopurified with PP2A A alpha, PP2A A beta, and PP2A C. Scanning alanine mutagenesis at 29 sites across the MCV sT protein revealed that PP2A-binding domains lie on the opposite molecular surface from a previously described large T stabilization domain (LSD) loop that binds E3 ligases, such as Fbw7. MCV sT-PP2A interactions can be functionally distinguished by mutagenesis from MCV sT LSD-dependent 4E-BP1 hyperphosphorylation and viral DNA replication enhancement. MCV sT has a restricted range for PP2A B subunit substitution, inhibiting only the assembly of B56 alpha into the phosphatase holoenzyme. In contrast, SV40 sT inhibits the assembly of B55 alpha, B56 alpha and B56 epsilon into PP2A. We conclude that MCV sT is required for Merkel cell carcinoma growth, but its in vitro transforming activity depends on LSD interactions rather than PP2A targeting.IMPORTANCEMerkel cell polyomavirus is a newly discovered human cancer virus that promotes cancer, in part, through expression of its small T (sT) oncoprotein. Animal polyomavirus sT oncoproteins have been found to cause experimental tumors by blocking the activities of a group of phosphatases called protein phosphatase 2A (PP2A). Our structural analysis reveals that MCV sT also displaces the B subunit of PP2A to inhibit PP2A activity. MCV sT, however, only displaces a restricted subset of PP2A B subunits, which is insufficient to cause tumor cell formation in vitro. MCV sT instead transforms tumor cells through another region called the large T stabilization domain. The PP2A targeting and transforming activities lie on opposite faces of the MCV sT molecule and can be genetically separated from each other.