title: Mechanisms of Yersinia YopO kinase substrate specificity.
abstract: Yersinia bacteria cause a range of human diseases, including yersiniosis, Far East scarlet-like fever and the plague. Yersiniae modulate and evade host immune defences through injection of Yersinia outer proteins (Yops) into phagocytic cells. One of the Yops, YopO (also known as YpkA) obstructs phagocytosis through disrupting actin filament regulation processes - inhibiting polymerization-promoting signaling through sequestration of Rac/Rho family GTPases and by using monomeric actin as bait to recruit and phosphorylate host actin-regulating proteins. Here we set out to identify mechanisms of specificity in protein phosphorylation by YopO that would clarify its effects on cytoskeleton disruption. We report the MgADP structure of Yersinia enterocolitica YopO in complex with actin, which reveals its active site architecture. Using a proteome-wide kinase-interacting substrate screening (KISS) method, we identified that YopO phosphorylates a wide range of actin-modulating proteins and located their phosphorylation sites by mass spectrometry. Using artificial substrates we clarified YopO's substrate length requirements and its phosphorylation consensus sequence. These findings provide fresh insight into the mechanism of the YopO kinase and demonstrate that YopO executes a specific strategy targeting actin-modulating proteins, across multiple functionalities, to compete for control of their native phospho-signaling, thus hampering the cytoskeletal processes required for macrophage phagocytosis.
authors.affiliation: Institute of Molecular and Cell Biology, A*STAR (Agency for Science, Technology and Research), Singapore.
authors.forename: Wei Lin
authors.initials: WL
authors.lastname: Lee
authors.affiliation: Institute of Molecular and Cell Biology, A*STAR (Agency for Science, Technology and Research), Singapore.|Department of Biological Sciences, National University of Singapore, Singapore.
authors.forename: Pavithra
authors.initials: P
authors.lastname: Singaravelu
authors.affiliation: Institute of Molecular and Cell Biology, A*STAR (Agency for Science, Technology and Research), Singapore.
authors.forename: Sheena
authors.initials: S
authors.lastname: Wee
authors.affiliation: Institute of Molecular and Cell Biology, A*STAR (Agency for Science, Technology and Research), Singapore.
authors.forename: Bo
authors.initials: B
authors.lastname: Xue
authors.affiliation: Institute of Molecular and Cell Biology, A*STAR (Agency for Science, Technology and Research), Singapore.
authors.forename: Khay Chun
authors.initials: KC
authors.lastname: Ang
authors.affiliation: Institute of Molecular and Cell Biology, A*STAR (Agency for Science, Technology and Research), Singapore.|Department of Anatomy, National University of Singapore, Singapore.
authors.forename: Jayantha
authors.initials: J
authors.lastname: Gunaratne
authors.affiliation: Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, UK.|Diamond Light Source Ltd., UK.
authors.forename: Jonathan M
authors.initials: JM
authors.lastname: Grimes
authors.affiliation: Department of Biological Sciences, National University of Singapore, Singapore.
authors.forename: Kunchithapadam
authors.initials: K
authors.lastname: Swaminathan
authors.affiliation: Institute of Molecular and Cell Biology, A*STAR (Agency for Science, Technology and Research), Singapore.|Department of Biochemistry, National University of Singapore, Singapore.|NTU Institute of Structural Biology, Nanyang Technological University, 59 Nanyang Drive, 636921, Singapore.|School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, 637551, Singapore.|Lee Kong Chan School of Medicine, 50 Nanyang Avenue, 639798, Singapore.
authors.forename: Robert C
authors.initials: RC
authors.lastname: Robinson
mesh_terms: D000199:Actins / Q000737:chemistry*; D000244:Adenosine Diphosphate / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry*; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D015008:Yersinia enterocolitica
chemical_list: D000199:Actins; D001426:Bacterial Proteins; D000244:Adenosine Diphosphate; C415206:YopO protein, Yersinia enterocolitica; D017346:Protein Serine-Threonine Kinases