title: Analysis of the posttranslational modifications of the influenza virus M2 protein.
abstract: The sites of posttranslational modifications of the influenza A virus M2 protein were examined, and the effect of these modifications on the M2 protein ion channel activity was analyzed. Cysteine residues 17 and 19 in the M2 protein ectodomain form disulfide bonds. The cytoplasmic tail is posttranslationally modified by palmitoylation, and mutagenic studies support the view that cysteine residue 50 is the site for fatty acylation. In addition, the cytoplasmic tail of the M2 protein was found to be posttranslationally modified by the addition of phosphate to specific serine residues. Site-directed mutagenesis of serine residues in the M2 protein cytoplasmic tail, combined with phosphoamino acid analysis, indicated that serine residue 64 is the predominant site for phosphorylation but that serine residues 82, 89, and 93 were also phosphorylated but to much lesser extents. Disulfide-bond formation, palmitoylation, and phosphorylation occurred on M2 protein expressed in mammalian cells infected with influenza virus, in mammalian cells in which the M2 protein was expressed from DNA expression vectors, and when the M2 protein was expressed in oocytes of Xenopus laevis. The membrane currents of oocytes of Xenopus laevis expressing wild-type and site-specifically altered forms of the M2 protein, to ablate posttranslational modifications, indicated that none of the posttranslational modifications significantly affected the ion channel activity of the M2 protein in oocytes. Therefore, these data do not indicate a functional role for posttranslational modifications of the M2 protein in its ion channel activity.
authors.affiliation: Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, Illinois 60208-3500.
authors.forename: L J
authors.initials: LJ
authors.lastname: Holsinger
authors.forename: M A
authors.initials: MA
authors.lastname: Shaughnessy
authors.forename: A
authors.initials: A
authors.lastname: Micko
authors.forename: L H
authors.initials: LH
authors.lastname: Pinto
authors.forename: R A
authors.initials: RA
authors.lastname: Lamb
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003545:Cysteine; D006367:HeLa Cells; D006801:Humans; D009980:Influenza A virus / Q000378:metabolism*; D007473:Ion Channels / Q000502:physiology*; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D012694:Serine / Q000378:metabolism; D014763:Viral Matrix Proteins / Q000378:metabolism*; D014982:Xenopus laevis; D000086082:Viroporin Proteins
chemical_list: D003545:Cysteine; D007473:Ion Channels; D012694:Serine; D014763:Viral Matrix Proteins; C031540:M-protein, influenza virus; C488937:M2 protein, Influenza A virus; D000086082:Viroporin Proteins