title: The bacterial effector Cif interferes with SCF ubiquitin ligase function by inhibiting deneddylation of Cullin1.
abstract: Cycle inhibiting factor (Cif) is one of the effectors delivered into epithelial cells by enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic Escherichia coli (EHEC) via the type III secretion system (TTSS). Cif family proteins, which inhibit host cell-cycle progression via mechanisms not yet precisely understood, are highly conserved among EPEC, EHEC, Yersinia pseudotuberculosis, Photorhabdus luminescens and Burkholderia pseudomallei. Levels of several proteins relevant to cell-cycle progression are modulated by Cullin-RING ligases (CRLs), which in turn are activated by conjugation and deconjugation of NEDD8 to Cullins. Here we show that Cif interacts with NEDD8 and interferes with SCF (Skp1-Cullin1-F-box protein) complex ubiquitin ligase function. We found that neddylated Cullin family proteins accumulated and ubiquitination of p27 decreased in cells infected with EPEC. Consequently, Cif stabilized SCF substrates such as CyclinD1, Cdt1, and p27, and caused G1 cell-cycle arrest. Using time-lapse-imaging of fluorescent ubiquitination-based cell-cycle indicator (Fucci)-expressing cells, we were able to monitor cell-cycle progression during EPEC infection and confirmed the arrest of infected cells at G1. Our in vitro and in vivo data show that Cif-NEDD8 interaction inhibits deneddylation of Cullins, suppresses CRL activity and induces G1 arrest. We thus conclude that the bacterial effector Cif interferes with neddylation-mediated cell-cycle control.
authors.affiliation: Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.
authors.forename: Hanako
authors.initials: H
authors.lastname: Morikawa
authors.forename: Minsoo
authors.initials: M
authors.lastname: Kim
authors.forename: Hitomi
authors.initials: H
authors.lastname: Mimuro
authors.forename: Claire
authors.initials: C
authors.lastname: Punginelli
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Koyama
authors.forename: Shinya
authors.initials: S
authors.lastname: Nagai
authors.forename: Atsushi
authors.initials: A
authors.lastname: Miyawaki
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Iwai
authors.forename: Chihiro
authors.initials: C
authors.lastname: Sasakawa
mesh_terms: D002460:Cell Line; D044842:Cullin Proteins / Q000378:metabolism*; D054308:Enteropathogenic Escherichia coli / Q000378:metabolism*; D004927:Escherichia coli Infections / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D016193:G1 Phase; D006367:HeLa Cells; D006801:Humans; D000075687:NEDD8 Protein; D044843:SKP Cullin F-Box Protein Ligases / Q000037:antagonists & inhibitors*; D014452:Ubiquitins / Q000378:metabolism*
chemical_list: C481998:Cif protein, E coli; C100318:Cullin 1; D044842:Cullin Proteins; D029968:Escherichia coli Proteins; D000075687:NEDD8 Protein; C082860:NEDD8 protein, human; D014452:Ubiquitins; D044843:SKP Cullin F-Box Protein Ligases