Quantitative in vivo analyses reveal calcium-dependent phosphorylation sites and identifies a novel component of the Toxoplasma invasion motor complex.
Quantitative in vivo analyses reveal calcium-dependent phosphorylation sites and identifies a novel component of the Toxoplasma invasion motor complex.
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DOI：
10.1371/journal.ppat.1002222
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发表时间：
2011-09
期刊：
影响因子：
6.7
通讯作者：
Tonkin CJ
中科院分区：
文献类型：
--
作者：
Nebl T;Prieto JH;Kapp E;Smith BJ;Williams MJ;Yates JR 3rd;Cowman AF;Tonkin CJ
顶复门寄生虫依赖于宿主细胞的入侵生存和增殖。钙依赖性信号通路似乎是必需的微线释放和滑行运动，但激活激酶的目标仍然在很大程度上未知。我们的特点是钙依赖性磷酸化事件在弓形虫宿主细胞入侵。用Ca 2+动员药物刺激活速殖子导致许多寄生虫蛋白的磷酸化，如32[P]-标记蛋白提取物的差异2-DE显示所示。多维蛋白质鉴定技术（MudPIT）在300多种弓形虫蛋白质上鉴定了1546个磷酸化位点，其中包括10个肌动球蛋白侵袭马达上的位点。使用基于培养物中氨基酸稳定同位素（SILAC）的定量LC-MS/MS分析，我们监测了入侵运动复合物的丰度和磷酸化的变化，并确定了其三种组分-GAP 45，MLC 1和MyoA的Ca 2+依赖性磷酸化模式。此外，跨GAP 45、MLC 1和MyoA的六个残基的钙依赖性磷酸化与侵袭运动活性相关。通过分析蛋白质，似乎更强烈地与入侵电机后钙刺激，我们还确定了一种新的15 kDa的钙调素样蛋白，可能代表的弓形虫入侵电机的MyoA必需轻链。这表明，入侵运动活动不仅可以通过磷酸化，而且还可以通过钙离子与这种新成分的直接结合来调节。顶复门寄生虫是一组具有广泛医学和农业意义的专性胞内病原体。这一门包括疟原虫属，疟疾的病原体和无处不在的寄生虫弓形虫，弓形虫对艾滋病患者、移植受体和未出生的胎儿造成疾病负担。无论它们针对的宿主细胞如何，所有顶复门寄生虫都必须在接触宿主细胞时激活入侵。钙介导的信号转导途径调节这一过程，但分子过程在很大程度上是未知的。使用一系列的蛋白质组学方法，我们揭示了弓形虫的蛋白质磷酸化后钙信号，此外，确定磷酸化位点的一系列蛋白质，可能发挥至关重要的作用，在调节寄生虫的运动和微线分泌。通过定量监测钙信号传导后的磷酸化沉积，我们定义了GAP 45和MLC 1的推定调节结构域，并进一步显示了侵袭马达可能更强烈地与钙信号传导相关的证据。我们还确定了一种新的钙调素样蛋白是入侵马达的一部分，这表明直接的Ca 2+结合也可能调节马达活性。
Apicomplexan parasites depend on the invasion of host cells for survival and proliferation. Calcium-dependent signaling pathways appear to be essential for micronemal release and gliding motility, yet the target of activated kinases remains largely unknown. We have characterized calcium-dependent phosphorylation events during Toxoplasma host cell invasion. Stimulation of live tachyzoites with Ca2+-mobilizing drugs leads to phosphorylation of numerous parasite proteins, as shown by differential 2-DE display of 32[P]-labeled protein extracts. Multi-dimensional Protein Identification Technology (MudPIT) identified ∼546 phosphorylation sites on over 300 Toxoplasma proteins, including 10 sites on the actomyosin invasion motor. Using a Stable Isotope of Amino Acids in Culture (SILAC)-based quantitative LC-MS/MS analyses we monitored changes in the abundance and phosphorylation of the invasion motor complex and defined Ca2+-dependent phosphorylation patterns on three of its components - GAP45, MLC1 and MyoA. Furthermore, calcium-dependent phosphorylation of six residues across GAP45, MLC1 and MyoA is correlated with invasion motor activity. By analyzing proteins that appear to associate more strongly with the invasion motor upon calcium stimulation we have also identified a novel 15-kDa Calmodulin-like protein that likely represents the MyoA Essential Light Chain of the Toxoplasma invasion motor. This suggests that invasion motor activity could be regulated not only by phosphorylation but also by the direct binding of calcium ions to this new component. Apicomplexan parasites are a group of obligate intracellular pathogens of wide medical and agricultural significance. Included within this phylum is Plasmodium spp, the causative agents to malaria and the ubiquitous parasite Toxoplasma, which inflicts disease burden on AIDS patients, transplant recipients and the unborn fetus. No matter the host cell that they target, all apicomplexan parasites must activate invasion upon host cell contact. Calcium-mediated signal transduction pathways modulate this process, yet the molecular processes are largely unknown. Using a range of proteomics approaches we reveal proteins in Toxoplasma that are phosphorylated upon calcium signaling, and furthermore, identify phosphorylation sites on a range of proteins that may play crucial roles in regulating parasite motility and microneme secretion. By quantitatively monitoring phosphorylation deposition upon calcium signaling we define putative regulatory domains of GAP45 and MLC1 and further show evidence that the invasion motor potentially more strongly associates upon calcium signaling. We also identified that a new Calmodulin-like protein is part of the invasion motor and this suggests that direct Ca2+ binding may also modulate motor activity.
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