title: Yersiniaeffector protein (YopO)-mediated phosphorylation of host gelsolin causes calcium-independent activation leading to disruption of actin dynamics.
abstract: PathogenicYersiniabacteria 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 inYersinia pestis), 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.
authors.affiliation: 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.
authors.forename: Pavithra
authors.identifier: 0000-0002-4799-807X
authors.initials: P
authors.lastname: Singaravelu
authors.affiliation: From the Institute of Molecular and Cell Biology, A*STAR (Agency for Science, Technology and Research), Singapore 138673, weilin@smart.mit.edu.
authors.forename: Wei Lin
authors.identifier: 0000-0003-1048-7556
authors.initials: WL
authors.lastname: Lee
authors.affiliation: From the Institute of Molecular and Cell Biology, A*STAR (Agency for Science, Technology and Research), Singapore 138673.
authors.forename: Sheena
authors.identifier: 0000-0003-2536-3433
authors.initials: S
authors.lastname: Wee
authors.affiliation: From the Institute of Molecular and Cell Biology, A*STAR (Agency for Science, Technology and Research), Singapore 138673.
authors.forename: Umesh
authors.initials: U
authors.lastname: Ghoshdastider
authors.affiliation: From the Institute of Molecular and Cell Biology, A*STAR (Agency for Science, Technology and Research), Singapore 138673.
authors.forename: Ke
authors.initials: K
authors.lastname: Ding
authors.affiliation: From the Institute of Molecular and Cell Biology, A*STAR (Agency for Science, Technology and Research), Singapore 138673.
authors.forename: Jayantha
authors.identifier: 0000-0002-5377-6537
authors.initials: J
authors.lastname: Gunaratne
authors.affiliation: From the Institute of Molecular and Cell Biology, A*STAR (Agency for Science, Technology and Research), Singapore 138673.|Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, United Kingdom.|Diamond Light Source Ltd., Diamond House, Harwell Science & Innovation Campus, Didcot, Oxfordshire OX11 0DE, United Kingdom, and.
authors.forename: Jonathan M
authors.identifier: 0000-0001-9698-0389
authors.initials: JM
authors.lastname: Grimes
authors.affiliation: Department of Biological Sciences, National University of Singapore, Singapore 117543 dbsks@nus.edu.sg.
authors.forename: Kunchithapadam
authors.identifier: 0000-0001-7940-1090
authors.initials: K
authors.lastname: Swaminathan
authors.affiliation: From the Institute of Molecular and Cell Biology, A*STAR (Agency for Science, Technology and Research), Singapore 138673.
authors.forename: Robert C
authors.identifier: 0000-0001-6367-6903
authors.initials: RC
authors.lastname: Robinson
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism; D000199:Actins / Q000737:chemistry*; D001426:Bacterial Proteins / Q000378:metabolism*; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry*; D018260:Gelsolin / Q000737:chemistry*; D006801:Humans; D008264:Macrophages / Q000382:microbiology; D013058:Mass Spectrometry; D056004:Molecular Dynamics Simulation; D009154:Mutation; D010587:Phagocytosis; D010766:Phosphorylation; D000072417:Protein Domains; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011721:Pyrenes / Q000737:chemistry; D012694:Serine / Q000737:chemistry; D013912:Threonine / Q000737:chemistry; D015007:Yersinia / Q000378:metabolism*
chemical_list: D000199:Actins; D001426:Bacterial Proteins; D018260:Gelsolin; D011721:Pyrenes; D013912:Threonine; D012694:Serine; C030984:pyrene; C415206:YopO protein, Yersinia enterocolitica; D017346:Protein Serine-Threonine Kinases; D002118:Calcium
keywords: Yersinia; actin; gelsolin; infectious disease; kinase; mass spectrometry (MS); phagocytosis; phosphorylation