title: Phosphorylation of the eukaryotic translation initiation factor 4E-transporter (4E-T) by c-Jun N-terminal kinase promotes stress-dependent P-body assembly.
abstract: Processing bodies (PBs, or P bodies) are cytoplasmic granules involved in mRNA storage and degradation that participate in the regulation of gene expression. PBs concentrate nontranslated mRNAs and several factors involved in mRNA decay and translational repression, including the eukaryotic translation initiation factor 4E-transporter (4E-T). 4E-T is required for PB assembly, but little is known about the molecular mechanisms that regulate its function. Here, we demonstrate that oxidative stress promotes multisite 4E-T phosphorylation. We show that the c-Jun N-terminal kinase (JNK) is targeted to PBs in response to oxidative stress and promotes the phosphorylation of 4E-T. Quantitative mass spectrometry analysis reveals that JNK phosphorylates 4E-T on six proline-directed sites that are required for the formation of the 4E-T complex upon stress. We have developed an image-based computational method to quantify the size, number, and density of PBs in cells, and we find that while 4E-T is required for steady-state PB assembly, its phosphorylation facilitates the formation of larger PBs upon oxidative stress. Using polysomal mRNA profiling, we assessed global and specific mRNA translation but did not find that 4E-T phosphorylation impacts translational control. Collectively, these data support a model whereby PB assembly is regulated by a two-step mechanism involving a 4E-T-dependent assembly stage in unstressed cells and a 4E-T phosphorylation-dependent aggregation stage in response to stress stimuli.
authors.affiliation: Institute for Research in Immunology and Cancer, Université de Montréal, Montreal, Quebec, Canada.
authors.forename: Marie
authors.initials: M
authors.lastname: Cargnello
authors.forename: Joseph
authors.initials: J
authors.lastname: Tcherkezian
authors.forename: Jonas F
authors.initials: JF
authors.lastname: Dorn
authors.forename: Edward L
authors.initials: EL
authors.lastname: Huttlin
authors.forename: Paul S
authors.initials: PS
authors.lastname: Maddox
authors.forename: Steven P
authors.initials: SP
authors.lastname: Gygi
authors.forename: Philippe P
authors.initials: PP
authors.lastname: Roux
mesh_terms: D002460:Cell Line; D003594:Cytoplasmic Granules / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D007091:Image Processing, Computer-Assisted; D048031:JNK Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism*; D013058:Mass Spectrometry; D057054:Molecular Imaging; D029543:Nucleocytoplasmic Transport Proteins / Q000235:genetics / Q000378:metabolism*; D018384:Oxidative Stress; D010766:Phosphorylation; D010957:Plasmids; D011392:Proline / Q000378:metabolism; D014176:Protein Biosynthesis; D021381:Protein Transport; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D015398:Signal Transduction / Q000235:genetics; D014162:Transfection
chemical_list: C411571:EIF4ENIF1 protein, human; D029543:Nucleocytoplasmic Transport Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D011392:Proline; D048031:JNK Mitogen-Activated Protein Kinases