Division of Microbiology, Graduate School of Medicine, University of Tokyo, Tokyo 113-0033, Japan.
Proc Natl Acad Sci U S A. 2011 Sep 6;108(36):14938-43. doi: 10.1073/pnas.1107740108. Epub 2011 Aug 22.
Several pathogenic bacteria have adopted effector proteins that, upon delivery into mammalian cells, undergo tyrosine phosphorylation at the Glu-Pro-Ile-Tyr-Ala (EPIYA) or EPIYA-like sequence motif by host kinases such as Src family kinases (SFKs). This EPIYA phosphorylation triggers complex formation of bacterial effectors with SH2 domain-containing proteins that results in perturbation of host cell signaling and subsequent pathogenesis. Although the presence of such an anomalous protein interaction suggests the existence of a mammalian EPIYA-containing protein whose function is mimicked or subverted by bacterial EPIYA effectors, no molecule that uses the EPIYA motif for biological function has so far been reported in mammals. Here we show that mammalian Pragmin/SgK223 undergoes tyrosine phosphorylation at the EPIYA motif by SFKs and thereby acquires the ability to interact with the SH2 domain of the C-terminal Src kinase (Csk), a negative regulator of SFKs. The Pragmin-Csk interaction prevents translocalization of Csk from the cytoplasm to the membrane and subsequent inactivation of membrane-associated SFKs. As a result, SFK activity is sustained in cells where Pragmin is phosphorylated at the EPIYA motif. Because EPIYA phosphorylation of Pragmin is mediated by SFKs, cytoplasmic sequestration of Csk by Pragmin establishes a positive feedback regulation of SFK activation. Remarkably, the Helicobacter pylori EPIYA effector CagA binds to the Csk SH2 domain in place of Pragmin and enforces membrane recruitment of Csk and subsequent inhibition of SFKs. This work identifies Pragmin as a mammalian EPIYA effector and suggests that bacterial EPIYA effectors target Pragmin to subvert SFKs for successful infection.
几种病原菌已经采用了效应蛋白，这些效应蛋白在进入哺乳动物细胞后，会被宿主激酶（如Src 家族激酶（SFKs））在 Glu-Pro-Ile-Tyr-Ala（EPIYA）或 EPIYA 样序列基序上进行酪氨酸磷酸化。这种 EPIYA 磷酸化触发细菌效应蛋白与含 SH2 结构域的蛋白质形成复合物，导致宿主细胞信号转导的紊乱，并随后导致发病机制。尽管存在这种异常的蛋白相互作用表明存在一种哺乳动物 EPIYA 蛋白，其功能被细菌 EPIYA 效应蛋白模拟或颠覆，但迄今为止，哺乳动物中没有报道使用 EPIYA 基序进行生物学功能的分子。在这里，我们表明哺乳动物 Pragmin/SgK223 在 EPIYA 基序上被 SFKs 进行酪氨酸磷酸化，从而获得与 C 末端Src 激酶（Csk）的 SH2 结构域相互作用的能力，Csk 是 SFKs 的负调节剂。Pragmin-Csk 相互作用阻止 Csk 从细胞质易位到膜并随后使膜相关 SFKs 失活。结果，在 Pragmin 在 EPIYA 基序上发生磷酸化的细胞中，SFK 活性得以维持。由于 Pragmin 的 EPIYA 磷酸化是由 SFKs 介导的，因此 Pragmin 将 Csk 隔离在细胞质中，从而建立了 SFK 激活的正反馈调节。值得注意的是，幽门螺杆菌 EPIYA 效应物 CagA 代替 Pragmin 结合到 Csk SH2 结构域，并强制募集 Csk 到膜上，随后抑制 SFKs。这项工作鉴定了 Pragmin 作为一种哺乳动物 EPIYA 效应物，并表明细菌 EPIYA 效应物将 Pragmin 作为靶点，以颠覆 SFKs 以实现成功感染。
