Singaravelu Pavithra, Lee Wei Lin, Wee Sheena, Ghoshdastider Umesh, Ding Ke, Gunaratne Jayantha, Grimes Jonathan M, Swaminathan Kunchithapadam, Robinson Robert C
From the Institute of Molecular and Cell Biology, A*STAR (Agency for Science, Technology and Research), Singapore 138673.
Department of Biological Sciences, National University of Singapore, Singapore 117543.
J Biol Chem. 2017 May 12;292(19):8092-8100. doi: 10.1074/jbc.M116.757971. Epub 2017 Mar 9.
Pathogenic bacteria cause a range of human diseases. To modulate and evade host immune systems, these yersiniae inject effector proteins into host macrophages. One such protein, the serine/threonine kinase YopO (YpkA in ), uses monomeric actin as bait to recruit and phosphorylate host actin polymerization-regulating proteins, including the actin-severing protein gelsolin, to disrupt actin filaments and thus impair phagocytosis. However, the YopO phosphorylation sites on gelsolin and the consequences of YopO-mediated phosphorylation on actin remodeling have yet to be established. Here we determined the effects of YopO-mediated phosphorylation on gelsolin and identified its phosphorylation sites by mass spectrometry. YopO phosphorylated gelsolin in the linker region between gelsolin homology domains G3 and G4, which, in the absence of calcium, are compacted but adopt an open conformation in the presence of calcium, enabling actin binding and severing. Using phosphomimetic and phosphodeletion gelsolin mutants, we found that YopO-mediated phosphorylation partially mimics calcium-dependent activation of gelsolin, potentially contributing to a reduction in filamentous actin and altered actin dynamics in phagocytic cells. In summary, this work represents the first report of the functional outcome of serine/threonine phosphorylation in gelsolin regulation and provides critical insight into how YopO disrupts normal gelsolin function to alter host actin dynamics and thus cripple phagocytosis.
致病细菌会引发一系列人类疾病。为了调节和逃避宿主免疫系统，这些耶尔森氏菌会将效应蛋白注入宿主巨噬细胞。其中一种蛋白，丝氨酸/苏氨酸激酶YopO（在[具体物种]中为YpkA），利用单体肌动蛋白作为诱饵，招募并磷酸化宿主肌动蛋白聚合调节蛋白，包括肌动蛋白切割蛋白凝溶胶蛋白，以破坏肌动蛋白丝，从而损害吞噬作用。然而，凝溶胶蛋白上的YopO磷酸化位点以及YopO介导的磷酸化对肌动蛋白重塑的影响尚未确定。在这里，我们确定了YopO介导的磷酸化对凝溶胶蛋白的影响，并通过质谱鉴定了其磷酸化位点。YopO在凝溶胶蛋白同源结构域G3和G4之间的连接区域磷酸化凝溶胶蛋白，在没有钙的情况下，该区域是紧密的，但在有钙的情况下会呈现开放构象，从而实现肌动蛋白的结合和切割。使用磷酸模拟和磷酸缺失的凝溶胶蛋白突变体，我们发现YopO介导的磷酸化部分模拟了钙依赖性的凝溶胶蛋白激活，这可能有助于减少丝状肌动蛋白，并改变吞噬细胞中的肌动蛋白动力学。总之，这项工作首次报道了凝溶胶蛋白调节中丝氨酸/苏氨酸磷酸化的功能结果，并为YopO如何破坏正常凝溶胶蛋白功能以改变宿主肌动蛋白动力学从而削弱吞噬作用提供了关键见解。
