Bolduc David, Rahdar Meghdad, Tu-Sekine Becky, Sivakumaren Sindhu Carmen, Raben Daniel, Amzel L Mario, Devreotes Peter, Gabelli Sandra B, Cole Philip
Department of Pharmacology and Molecular Sciences , Johns Hopkins University School of Medicine , Baltimore , United States.
Elife. 2013 Jul 9;2:e00691. doi: 10.7554/eLife.00691.
The tumor suppressor PIP3 phosphatase PTEN is phosphorylated on four clustered Ser/Thr on its C-terminal tail (aa 380-385) and these phosphorylations are proposed to induce a reduction in PTEN's plasma membrane recruitment. How these phosphorylations affect the structure and enzymatic function of PTEN is poorly understood. To gain insight into the mechanistic basis of PTEN regulation by phosphorylation, we generated semisynthetic site-specifically tetra-phosphorylated PTEN using expressed protein ligation. By employing a combination of biophysical and enzymatic approaches, we have found that purified tail-phosphorylated PTEN relative to its unphosphorylated counterpart shows reduced catalytic activity and membrane affinity and undergoes conformational compaction likely involving an intramolecular interaction between its C-tail and the C2 domain. Our results suggest that there is a competition between membrane phospholipids and PTEN phospho-tail for binding to the C2 domain. These findings reveal a key aspect of PTEN's regulation and suggest pharmacologic approaches for direct PTEN activation. DOI:http://dx.doi.org/10.7554/eLife.00691.001.
肿瘤抑制因子PIP3磷酸酶PTEN在其C末端尾巴（氨基酸380 - 385）的四个成簇的丝氨酸/苏氨酸上发生磷酸化，并且有人提出这些磷酸化会导致PTEN向质膜募集的减少。目前对于这些磷酸化如何影响PTEN的结构和酶功能还知之甚少。为了深入了解PTEN磷酸化调控的机制基础，我们使用表达蛋白连接技术生成了半合成的位点特异性四磷酸化PTEN。通过结合生物物理和酶学方法，我们发现相对于未磷酸化的对应物，纯化的尾巴磷酸化PTEN显示出降低的催化活性和膜亲和力，并经历构象压缩，这可能涉及其C末端尾巴与C2结构域之间的分子内相互作用。我们的结果表明，膜磷脂和PTEN磷酸化尾巴在与C2结构域结合方面存在竞争。这些发现揭示了PTEN调控的一个关键方面，并提出了直接激活PTEN的药理学方法。DOI:http://dx.doi.org/10.7554/eLife.00691.001
