Phosphorylation of a Myosin Motor by TgCDPK3 Facilitates Rapid Initiation of Motility during Toxoplasma gondii egress.
Phosphorylation of a Myosin Motor by TgCDPK3 Facilitates Rapid Initiation of Motility during Toxoplasma gondii egress.
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DOI：
10.1371/journal.ppat.1005268
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发表时间：
2015
期刊：
影响因子：
6.7
通讯作者：
Arrizabalaga G
中科院分区：
文献类型：
--
作者：
Gaji RY;Johnson DE;Treeck M;Wang M;Hudmon A;Arrizabalaga G
钙依赖性蛋白激酶 (CDPK) 家族的成员在某些致病寄生虫中含量丰富，但在哺乳动物细胞中不存在，这使得它们成为强有力的候选药物靶点。在专性细胞内寄生虫弓形虫中，TgCDPK3 对于钙依赖性从宿主细胞的排出非常重要。尽管如此，TgCDPK3 在流出过程中发挥其功能的具体底物仍然未知。为了弥补这一知识差距，我们应用了基于邻近性的蛋白质相互作用陷阱 BioID，并鉴定了 13 个与 TgCDPK3 邻近或直接相互作用的蛋白质。其中包括肌球蛋白 A (TgMyoA)，它是一种非常规运动蛋白，对驱动这种寄生虫的滑行运动发挥着重要作用，其丝氨酸 21 被一种未知激酶磷酸化，此前已被证明对运动和外出很重要。通过无偏肽阵列方法，我们确定 TgCDPK3 可以在体外特异性磷酸化 TgMyoA 的丝氨酸 21 和 743。 TgmyoA 无效突变体（表现出出口延迟）与 S21 或 S743 突变为丙氨酸的 TgMyoA 的互补未能挽救出口缺陷。同样，运动蛋白中的拟磷酸化突变克服了对 TgCDPK3 的需要。此外，胞外 Tgcdpk3 突变寄生虫具有运动缺陷，可通过 TgMyoA 的 S21+S743 磷酸模拟物的表达来补充。因此，我们的研究证实，TgCDPK3 对 TgMyoA 的磷酸化负责启动蠕动和寄生虫从宿主细胞中排出，并为这种独特的激酶如何调节弓形虫的裂解周期提供了机制见解。 弓形虫可导致免疫功能低下者和先天性感染者严重疾病和死亡。由于现有药物的局限性，需要研究寄生虫独特且必需的蛋白质。我们最近确定 TgCDPK3 是钙依赖性蛋白激酶家族的成员，存在于植物和一些寄生虫中，但在人类细胞中不存在，可调节寄生虫从宿主细胞的排出。虽然假设 TgCDPK3 通过磷酸化激活运动所需的蛋白质来控制从宿主的快速退出，但该激酶的特定底物仍然未知。我们现在应用了相互作用捕获系统来识别被该激酶修饰的蛋白质，其中包括寄生虫运动蛋白肌球蛋白 A (TgMyoA)。我们发现 TgCDPK3 特异性磷酸化 TgMyoA，这种磷酸化对于寄生虫的排出和运动很重要。
Members of the family of calcium dependent protein kinases (CDPK’s) are abundant in certain pathogenic parasites and absent in mammalian cells making them strong drug target candidates. In the obligate intracellular parasite Toxoplasma gondii TgCDPK3 is important for calcium dependent egress from the host cell. Nonetheless, the specific substrate through which TgCDPK3 exerts its function during egress remains unknown. To close this knowledge gap we applied the proximity-based protein interaction trap BioID and identified 13 proteins that are either near neighbors or direct interactors of TgCDPK3. Among these was Myosin A (TgMyoA), the unconventional motor protein greatly responsible for driving the gliding motility of this parasite, and whose phosphorylation at serine 21 by an unknown kinase was previously shown to be important for motility and egress. Through a non-biased peptide array approach we determined that TgCDPK3 can specifically phosphorylate serines 21 and 743 of TgMyoA in vitro. Complementation of the TgmyoA null mutant, which exhibits a delay in egress, with TgMyoA in which either S21 or S743 is mutated to alanine failed to rescue the egress defect. Similarly, phosphomimetic mutations in the motor protein overcome the need for TgCDPK3. Moreover, extracellular Tgcdpk3 mutant parasites have motility defects that are complemented by expression of S21+S743 phosphomimetic of TgMyoA. Thus, our studies establish that phosphorylation of TgMyoA by TgCDPK3 is responsible for initiation of motility and parasite egress from the host-cell and provides mechanistic insight into how this unique kinase regulates the lytic cycle of Toxoplasma gondii. Toxoplasma gondii can cause severe disease and death in the immunocompromised and in those infected congenitally. Due to limitations of existing drugs there is a need for studying proteins that are unique and essential to the parasite. We recently established that TgCDPK3, a member of a family of calcium dependent protein kinase present in plants and some parasites but absent in human cells, regulates parasite egress from the host cell. While it has been hypothesized that TgCDPK3 controls rapid exit from the host by phosphorylating proteins needed for activating motility, the particular substrates of this kinase remained unknown. We have now applied an interaction trap system to identify the proteins that are modified by this kinase, which include a parasite motor protein Myosin A (TgMyoA). We show that TgCDPK3 specifically phosphorylates TgMyoA and this phosphorylation is important for parasite egress and motility.