Department of Molecular Microbiology, Faculty of Medicine, Technion-Israel Institute of Technology, Bat Galim, Haifa 31096, Israel.
J Biol Chem. 2013 May 10;288(19):13718-27. doi: 10.1074/jbc.M112.343756. Epub 2013 Mar 25.
The adenovirus E4orf4 protein must bind protein phosphatase 2A (PP2A) for its functions.
The E4orf4 binding site in PP2A was mapped to the α1,α2 helices of the B55α subunit.
The E4orf4 binding site in PP2A-B55α lies above the substrate binding site and does not overlap it.
A novel functional significance was assigned to the α1,α2 helices of the PP2A-B55α subunit. The adenovirus E4orf4 protein regulates the progression of viral infection and when expressed outside the context of the virus it induces nonclassical, cancer cell-specific apoptosis. All E4orf4 functions known to date require an interaction between E4orf4 and protein phosphatase 2A (PP2A), which is mediated through PP2A regulatory B subunits. Specifically, an interaction with the B55α subunit is required for induction of cell death by E4orf4. To gain a better insight into the E4orf4-PP2A interaction, mapping of the E4orf4 interaction site in PP2A-B55α has been undertaken. To this end we used a combination of bioinformatics analyses of PP2A-B55α and of E4orf4, which led to the prediction of E4orf4 binding sites on the surface of PP2A-B55α. Mutation analysis, immunoprecipitation, and GST pulldown assays based on the theoretical predictions revealed that the E4orf4 binding site included the α1 and α2 helices described in the B55α structure and involved at least three residues located in these helices facing each other. Loss of E4orf4 binding was accompanied by reduced contribution of the B55α mutants to E4orf4-induced cell death. The identified E4orf4 binding domain lies above the previously described substrate binding site and does not overlap it, although its location could be consistent with direct or indirect effects on substrate binding. This work assigns for the first time a functional significance to the α1,α2 helices of B55α, and we suggest that the binding site defined by these helices could also contribute to interactions between PP2A and some of its cellular regulators.
腺病毒 E4orf4 蛋白必须与蛋白磷酸酶 2A（PP2A）结合才能发挥其功能。
PP2A 中的 E4orf4 结合位点被映射到 B55α 亚基的α1、α2 螺旋。
PP2A-B55α 中的 E4orf4 结合位点位于底物结合位点上方，不与其重叠。
PP2A-B55α 的α1、α2 螺旋被赋予了新的功能意义。腺病毒 E4orf4 蛋白调节病毒感染的进展，当它在病毒之外的环境中表达时，会诱导非经典的、癌细胞特异性的细胞凋亡。迄今为止，所有已知的 E4orf4 功能都需要 E4orf4 与蛋白磷酸酶 2A（PP2A）之间的相互作用，这种相互作用是通过 PP2A 调节 B 亚基介导的。具体来说，E4orf4 诱导细胞死亡需要与 B55α 亚基相互作用。为了更深入地了解 E4orf4-PP2A 相互作用，我们对 PP2A-B55α 中的 E4orf4 相互作用位点进行了定位。为此，我们使用了对 PP2A-B55α 的生物信息学分析和 E4orf4 的组合，这导致了在 PP2A-B55α 表面预测 E4orf4 结合位点。基于理论预测的突变分析、免疫沉淀和 GST 下拉实验表明，E4orf4 结合位点包括 B55α 结构中描述的α1 和α2 螺旋，涉及至少三个位于这些螺旋中的相互面对的残基。E4orf4 结合丧失伴随着 B55α 突变体对 E4orf4 诱导的细胞死亡的贡献减少。确定的 E4orf4 结合域位于先前描述的底物结合位点上方，不与其重叠，尽管其位置可能与对底物结合的直接或间接影响一致。这项工作首次赋予了 B55α 的α1、α2 螺旋功能意义，我们认为这些螺旋定义的结合位点也可能有助于 PP2A 与其一些细胞调节剂之间的相互作用。
