Apicomplexan parasites like Toxoplasma gondii grow and replicate within a specialized organelle called the parasitophorous vacuole. The vacuole is decorated with parasite proteins that integrate into the membrane after trafficking through the parasite secretory system as soluble, chaperoned complexes. A regulator of this process is an atypical protein kinase called WNG1. Phosphorylation by WNG1 appears to serve as a switch for membrane integration. However, like its substrates, WNG1 is secreted from the parasite dense granules, and its activity must, therefore, be tightly regulated until the correct membrane is encountered. Here, we demonstrate that, while another member of the WNG family can adopt multiple multimeric states, WNG1 is monomeric and therefore not regulated by multimerization. Instead, we identify two phosphosites on WNG1 that are required for its kinase activity. Using a combination of in vitro biochemistry and structural modeling, we identify basic residues that are also required for WNG1 activity and appear to recognize the activating phosphosites. Among these coordinating residues are the ‘HRD’ Arg, which recognizes activation loop phosphorylation in canonical kinases. WNG1, however, is not phosphorylated on its activation loop, but rather on atypical phosphosites on its C-lobe. We propose a simple model in which WNG1 is activated by increasing ATP concentration above a critical threshold once the kinase traffics to the parasitophorous vacuole.

中文翻译：

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发散激酶 WNG1 通过非典型激活子结构域的磷酸化进行调节

像弓形虫这样的顶复门寄生虫在一种称为寄生液泡的特殊细胞器中生长和复制。液泡上装饰有寄生虫蛋白，这些蛋白质在通过寄生虫分泌系统作为可溶性陪伴复合物运输后整合到膜中。该过程的调节者是一种称为 WNG1 的非典型蛋白激酶。WNG1 的磷酸化似乎充当膜整合的开关。然而，与其底物一样，WNG1 是从寄生虫致密颗粒中分泌出来的，因此必须严格调节其活性，直到遇到正确的膜。在这里，我们证明，虽然 WNG 家族的另一个成员可以采用多种多聚体状态，但 WNG1 是单体，因此不受多聚化调节。相反，我们确定了 WNG1 上其激酶活性所需的两个磷酸位点。通过结合体外生物化学和结构建模，我们鉴定了 WNG1 活性所需的基本残基，并且似乎能够识别激活的磷酸位点。这些协调残基中有“HRD”精氨酸，它识别典型激酶中的激活环磷酸化。然而，WNG1 并未在其激活环上被磷酸化，而是在其 C 叶上的非典型磷酸位点上被磷酸化。我们提出了一个简单的模型，其中一旦激酶运输到寄生液泡，就通过将 ATP 浓度增加到临界阈值以上来激活 WNG1。